In cancer patients, visual identification of sentinel lymph nodes (LNs) is achieved by the injection of dyes that bind avidly to endogenous albumin, targeting these compounds to LNs, where they are efficiently filtered by resident phagocytes1,2. Here we translate this ‘albumin hitchhiking’ approach to molecular vaccines, through the synthesis of amphiphiles (amph-vaccines) comprising an antigen or adjuvant cargo linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain. Administration of structurally optimized CpG-DNA/peptide amph-vaccines in mice resulted in marked increases in LN accumulation and decreased systemic dissemination relative to their parent compounds, leading to 30-fold increases in T-cell priming and enhanced anti-tumour efficacy while greatly reducing systemic toxicity. Amph-vaccines provide a simple, broadly applicable strategy to simultaneously increase the potency and safety of subunit vaccines.
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This work was supported in part by the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute, the National Institutes of Health (grants AI091693, AI104715 and AI095109), the Department of Defense (W911NF-13-D-0001 and W911NF-07-D-0004, T.O. 8) and the Ragon Institute of Massachusetts General Hospital, the Massachusetts Institute of Technology and Harvard. D.J.I. is an investigator of the Howard Hughes Medical Institute. We thank T. C. Wu for kindly providing the TC-1 tumour cells. We thank the Koch Institute Swanson Biotechnology Center for technical support, specifically the applied therapeutics and whole animal imaging core facility, histology and flow cytometry core facility. The authors acknowledge the service to the MIT community of the late Sean Collier.
A patent application for amphiphile vaccines has been filed, with D.J.I. and H.L. as inventors.
Extended data figures and tables
Extended Data Figure 1 Interaction between albumin and amph-CpGs.
a, SEC of FBS, albumin and fluorescein-labelled amph-CpGs. FBS and bovine serum albumin (BSA) were monitored using absorptions at 280 nm, whereas CpG oligonucleotides were monitored at 480 nm (fluorescein peak). b, Lipo-CpG, but not CpG, interacts with serum albumin as shown by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) after protein pull-down assays: FBS was incubated with 3′-biotin-labelled CpG (CpG-biotin), lipo-CpG (lipo-CpG-biotin) or PBS for 1 h at 37 °C. Streptavidin-conjugated magnetic beads were added to capture biotinylated CpGs and any associated proteins, separated by a magnet, boiled to release bound CpG/proteins, and subjected to SDS–PAGE analysis. Lane 1: protein MW ladder; lane 2: purified BSA; lane 3: FBS (100× dilution, 10 μl loading); lane 4: pull-down control, FBS incubated with streptavidin-magnetic beads; lane 5: pull-down with CpG-biotin, FBS was incubated with CpG-biotin and streptavidin magnetic beads; lane 6: pull-down with lipo-CpG-biotin, FBS incubated with lipo-CpG-biotin and streptavidin magnetic beads; lane 7: FBS (100× dilution, 5 μl loading). c, Fluorescein-labelled CpG or lipo-CpG was incubated with albumin-conjugated agarose resin for 1 h at 37 °C, and the resin was separated by filtration. The filtrate and recovered agarose were visualized by a gel imager and quantified by fluorescence measurements. d, Bio-layer interferometry measurements of lipo-CpG and CpG binding to immobilized BSA. Albumin-conjugated BLI probes were immersed in solutions of lipo-CpG, and wavelength shifts (Δλ) of the interferometry pattern association and dissociation curves were followed over time to determine affinity constants of binding at 25 °C. Shown are apparent kon, koff and KD values from fits to the data. e, FRET between FAM-labelled lipo-CpG and rhodamine-conjugated albumin (BSA-Rh) assessed by fluorescence spectroscopy. CpG-F or lipo-CpG-F (1.65 µM) alone or mixed with BSA-Rh (1.5 µM) in PBS were excited at 488 nm and emission was recorded from 500–650 nm.
Extended Data Figure 2 Construction and characterization of G-quadruplex-stabilized CpG adjuvants.
a, G-quadruplex-stabilized CpG micelles are self-assembled from amphiphiles composed of three distinct segments: an immunostimulatory CpG sequence, a central repeat block containing n = 1–10 G-quartet-forming guanines followed by (10 − n) non-interacting thymidines, and a diacyl lipid tail. In aqueous solutions, these amphiphiles self-assembled into three-dimensional spherical micelles with a CpG corona and a lipid core. In the presence of K+, neighbouring guanine repeats in the oligonucleotide corona form G-quadruplex structures via Hoogsteen hydrogen bonds and stabilize the micelle structure. The stability of the oligonucleotide micelles in the presence of serum was programmed by altering the length of the guanine repeat. b, Parallel G-quartet formation among DNA strands within the micelles was detected by circular dichroism (CD) spectroscopy, as manifested by the shifting of positive peaks from 278 nm towards 262 nm and troughs at 245 nm as the number of guanines in the structure increased. c, Pyrene excimer fluorescence was used to assay the stabilities of G-quadruplex micelles in the presence of albumin: pyrene dye incorporated in stabilized CpG micelles (n > 2) retained excimer fluorescence in the presence of high concentrations of albumin. By contrast, albumin binds to the lipid moiety of unstabilized micelles (n ≤ 2) and disrupts the micelle structures, leading to loss of excimer fluorescence in an albumin-concentration-dependent manner as the protein disrupts the micelles into albumin-bound unimers. Shown below the schematic is the fraction of amph-CpG remaining in the micellar state as a function of albumin concentration as reported by excimer fluorescence. Arrow indicates the plasma concentration of albumin. d, Stability profiles of G-quadruplex CpG micelles as measured by SEC in the presence of FBS. Fluorescein-labelled CpG micelles were incubated with 20% FBS in PBS in the presence of 10 mM Mg2+ and 20 mM K+ at 37 °C for 2 h, then analysed by SEC. FBS and BSA were monitored using absorptions at 280 nm, whereas lipo-Gn-CpG amphiphiles were monitored at 480 nm (fluorescein peak). Lipo-Gn-CpG with n = 0 or 2 partitioned to co-migrate with albumin, whereas amphiphiles with n > 2 showed increasing fractions of the amphiphiles migrating as intact micelles in the presence of serum with increasing n. e, Fluorescein-labelled lipo-G6-CpG (5 µM) and Alexa Fluor 647-labelled BSA (5 µM) were incubated for 2 h at 37 °C in PBS plus 20 mM KCl, and then analysed by SEC. Spectra were monitored at 480 nm (ODN channel, green line, fluorescein) and 640 nm (protein channel, red line, Alexa Fluor 647). The majority of BSA and CpG micellar aggregates eluted separately. f, Size of amph-CpG micelles as determined by dynamic light scattering. All data are mean ± s.e.m. Statistical analysis was performed by one-way ANOVA with Bonferroni post-tests.
Extended Data Figure 3 LN localization of amph-CpGs with macrophages and dendritic cells.
a, Immunofluorescent images of inguinal LN section 24 h after injection of 3.3 nmol lipo-CpG or lipo-G2-CpG, showing dendritic cells (CD11c, blue), macrophages (F4/80, red) and CpG (green). b–d, Mice (n = 3 per group) were injected subcutaneously with 3.3 nmol of fluorescein-labelled CpG formulations. After 24 h, LNs were digested and LN cells stained with 4′,6-diamidino-2-phenylindole (DAPI) and antibodies against F4/80, CD11c and CD207. b, c, Shown are representative flow cytometry plots of F4/80 staining (b) and CD11c staining (c) versus CpG fluorescence in viable (DAPI−) cells. d, percentages of CpG+ cells in the LNs determined by flow cytometry at 24 h. ***P < 0.001; all data are mean ± s.e.m. Statistical analysis was performed by unpaired Student’s t-test.
Extended Data Figure 4 CpG-albumin conjugates accumulate in LNs.
a, b, C57BL/6 mice (n = 4 LNs per group) were injected subcutaneously at the tail base with 3.3 nmol fluorescein-labelled free CpG, mouse albumin-CpG conjugates (MSA-CpG) or lipo-CpG. Inguinal LNs and axillary LNs were isolated 24 h after injection and imaged (a) and quantified (b) by IVIS optical imaging. All data are mean ± s.e.m. **P < 0.01 by one-way ANOVA with Bonferroni post-test.
Extended Data Figure 5 In vitro characterization of amph-CpG.
a, Rhodamine-labelled CpG or amph-CpG (1 μM) was incubated with murine bone-marrow-derived dendritic cells at 37 °C for 4 h with LysoTracker (Life Technologies) and imaged using a Zeiss LSM 510 confocal microscope. b, Rhodamine-labelled CpG or amph-CpG (1 μM) was incubated for 30 min, 2 h, 6 h and 24 h with the murine dendritic cell line DC2.4. Cells were stained with DAPI and uptake was quantified by flow cytometry using the mean fluorescence intensity (MFI) of viable (DAPI−) cells. c, Amph-CpG or PAM2CSK4 (a strong TLR2 agonist) was incubated for 24 h with the InvivoGen HEK-Blue murine TLR2 reporter cell line, a secreted embryonic alkaline phosphatase (SEAP) reporter system. SEAP levels were quantified by incubating supernatant with QuantiBlue substrate for 1 h and reading absorption at 620 nm. d, Amph-CpG, CpG or control amph-GpC (1 μM) were incubated with InvivoGen RAW-Blue mouse macrophage reporter cells, which secrete SEAP upon TLR, NOD or Dectin-1 stimulation. SEAP levels were quantified by incubating supernatant with QuantiBlue substrate for 1 h and reading absorption at 620 nm. e, Bone-marrow-derived immature dendritic cells were incubated overnight with indicated concentrations of OVA and maturation stimuli (or medium alone). Dendritic cells were washed three times with PBS and 30,000 CFSE-labelled OT-I CD8+ T cells were then added to each well. Cells were collected after 2 days of co-culture, and stained and gated for DAPI− (viable) CD8+ T cells using Flowjo v.7.6.5 (Treestar). The extent of proliferation was quantified by determining the percentage of cells that had undergone division by determining the percentage of viable CD8+ T cells that had diluted CFSE using T cells alone as a control for the no division/dilution peak. Shown are mean ± s.e.m. b–d, ***P < 0.001 by one-way ANOVA with Bonferroni post-test. Bars in e represent medians and whiskers represent range (n = 2 wells per condition).
Extended Data Figure 6 Albumin-binding lipo-CpGs elicit robust expansion of antigen-specific CD8+ T-cells when combined with soluble protein.
a–h, Groups of C57BL/6 mice (n = 4–8 per group) were immunized subcutaneously on day 0 and day 14 with 10 µg OVA and 1.24 nmol CpG formulations as indicated. Six days after the final immunization, mice were bled and peripheral blood mononuclear cells were evaluated by SIINFEKL-tetramer staining and intracellular cytokine staining. a, Representative flow cytometric dot plots of H-2Kb/SIINFEKL tetramer staining of CD8+ cells. b, Representative flow dot plots of intracellular staining on CD8+ cells for IFN-γ and TNF-α after 6 h ex vivo restimulation with SIINFEKL peptide. c, Serum samples were collected and assayed by enzyme-linked immunosorbent assay (ELISA) for anti-OVA IgG (day 34). d, Mice were immunized on day 0 and day 14 with 1.24 nmol lipo-G2-CpG mixed with 10 µg SIV Gag protein, blood samples were collected and analysed by peptide-MHC tetramer staining for CD8+ T cells recognizing the immunodominant AL11 epitope of Gag. e, f, Anti-MSA and anti-OVA IgG (e, day 20) or IgM (f, day 20) were measured by ELISA. g, Groups of C57BL/6 mice (n = 4 per group) were immunized with 10 µg OVA alone or mixed with 1.24 nmol of a non-TLR agonist lipo-GpC, the same diacyl lipid tail conjugated to PEG (lipo-PEG, 48 EG units), or DSPE-PEG2000. Mice were boosted with the same formulation on day 14, and OVA tetramer+ CD8+ T cells in peripheral blood were assayed by flow cytometry on day 20. ***P < 0.001. h, TLR2 knockout or wild-type mice were immunized as described in a, and OVA tetramer+ CD8+ T cells were assayed as previously. All data are mean ± s.e.m. *P < 0.05. Statistical analysis was performed by unpaired Student’s t-test.
Extended Data Figure 7 Albumin-binding CpG induces local lymphadenopathy but reduces systemic toxicity compared with soluble CpG adjuvant.
a, C57BL/6 mice (n = 3 per group) were injected with 1.24 nmol CpGs subcutaneously on day 0 and 2.48 nmol CpGs on days 2 and 4. On day 6 mice were killed and LNs were isolated and photographed with a digital camera. b, Bead-based flow analysis of proinflammatory cytokines elicited in peripheral blood of mice injected with a single dose (6.2 nmol) of different CpG formulations. Blood samples were collected at different time intervals and analysed for TNF-α, as per manufacturer’s instructions. All data are mean ± s.e.m. **P < 0.01, *P < 0.05. Statistical analysis was performed by one-way ANOVA with Bonferroni post-test.
Extended Data Figure 8 Hydrophilic block length of amphiphiles determines cell membrane insertion and LN accumulation.
a, Amphiphiles with varying hydrophilic PEG lengths were prepared by solid phase synthesis of diacyl tails coupled to 1–8 hexa-ethylene glycol phosphorothioate units. Fluorescein was incorporated either at the 3′ terminal (for membrane insertion analyses) or adjacent to the lipid moiety (for albumin-binding analyses). b, Splenocytes from C57BL/6 mice (5 × 107cells per ml) were incubated with lipo-(PEG)n-fluorescein (1.67 μM) and albumin (100 μM) at 37 °C for 1 h. Shown is a representative image of membrane insertion observed by confocal microcopy for lipo-(PEG)1-fluorescein. c, Equilibrium partitioning measurements shown as a function of albumin concentration at 37 °C. Lipo-fluorescein-(PEG)n (5 μM) was incubated with varying concentrations of BSA and fluorescence intensities were monitored by fluorescence spectroscopy. BSA binding disrupted the micellar structure and decreased the self-quenching of fluorescein. All samples reached maximum fluorescence intensities at around 10 μM BSA, indicating 100% micelle breakup at this concentration (at higher BSA concentrations, fluorescence decreases due to solution turbidity). Arrow indicates plasma concentration of albumin. d, e, Lipo-Tn-FAM amphiphiles (n = 5, 10, 15, 20) were injected subcutaneously in C57BL/6 mice (n = 4 LNs per group), and excised LNs were imaged after 24 h (d). Mean LN fluorescence from groups of mice are plotted in e. All data are mean ± s.e.m.
Extended Data Figure 9 Lipo-PEG-peptide amphiphiles exhibit greatly enhanced LN accumulation compared with unmodified peptides.
a, Peptides with amino-terminal cysteines were conjugated to maleimide-PEG2000-DSPE. b, c, FAM-labelled immunodominant peptide derived from the HPV-16 E7 protein (FAM-FTVINYHARC, synthesized in reverse sequence order using d-amino acids to obtain the same chiral organization of side chains as the typical l-amino acid sequence in a protease-resistant peptide) was injected subcutaneously at the tail base as a free peptide (D-E7) or as a PEG-DSPE conjugate (amph-D-E7). Shown are IVIS images of draining LNs 24 h after injection (b) and fluorescence quantifications (c). All data are mean ± s.e.m. **P < 0.01. Statistical analysis was performed by unpaired Student’s t-test.
Extended Data Figure 10 Long-peptide amphiphiles, when combined with amph-CpG, elicit a potent antigen-specific CD8+ T-cell response with therapeutic benefits, as compared to soluble formulation.
a, C57BL/6 mice were primed on day 0 and boosted on day 14 with amph-E7long (HPV-16 E743–62, 10 μg peptide) and amph-CpG (lipo-G2-CpG, 1.24 nmol), or equivalent soluble peptide/CpG vaccines. Six days after the boost, mice were bled and analysed for tetramer-positive CD8+ T cells in peripheral blood. b–e, C57BL/6 mice (n = 8 per group) were inoculated with 3 × 105 TC-1 tumour cells subcutaneously in the flank and left untreated or immunized with soluble or amphiphile long-peptide vaccines on days 6 (10 µg peptide, 1.24 nmol CpG), 13 (20 µg peptide, 1.24 nmol CpG) and 19 (20 µg peptide, 1.24 nmol CpG). b–d, Shown are individual tumour growth curves for no treatment (b), immunization with soluble E7long and CpG (c), or immunization with amph-E7long plus amph-CpG (d). Kaplan–Meier survival curves of eight mice per group are shown in e. f, Long-peptide amphiphiles also elicit potent immune responses when combined with non-CpG, non-LN-targeting alternative adjuvants. C57BL/6 mice (n = 4 per group) were immunized as before, using amph-E7long peptide (10 μg) combined with monophosphoryl lipid A (MPLA, 10 μg) or polyinosinic:polycytidylic acid (poly I:C, 50 μg). The frequencies of E7 tetramer+ CD8+ T cells in peripheral blood were assayed on day 20. All data are mean ± s.e.m. ***P < 0.001, **P < 0.01, *P < 0.05 by unpaired Student’s t-test.
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Liu, H., Moynihan, K., Zheng, Y. et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 519–522 (2014). https://doi.org/10.1038/nature12978
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