RESEARCH ARTICLE | MEDICAL SCIENCES

CHEMISTRY

Lipid nanoparticle-mediated lymph node–targeting delivery of

mRNA cancer vaccine elicits robust CD8+ T cell response
Jinjin Chena,b,1, Zhongfeng Yea,1 , Changfeng Huanga, Min Qiua, Donghui Songa , Yamin Lia, and Qiaobing Xua,2

Edited by Daniel Anderson, Massachusetts Institute of Technology, Cambridge, MA; received May 5, 2022; accepted July 19, 2022 by
Editorial Board Member Chad A. Mirkin

The targeted delivery of messenger RNA (mRNA) to desired organs remains a great chal-
lenge for in vivo applications of mRNA technology. For mRNA vaccines, the targeted Significance
delivery to the lymph node (LN) is predicted to reduce side effects and increase the
immune response. In this study, we explored an endogenously LN-targeting lipid nano- Current messenger RNA (mRNA)
particle (LNP) without the modification of any active targeting ligands for developing an vaccines in the clinic were
mRNA cancer vaccine. The LNP named 113-O12B showed increased and specific expres- reported to induce side effects in
sion in the LN compared with LNP formulated with ALC-0315, a synthetic lipid used in the liver, such as reversible
the COVID-19 vaccine Comirnaty. The targeted delivery of mRNA to the LN increased hepatic damages and T
the CD8+ T cell response to the encoded full-length ovalbumin (OVA) model antigen. cell–dominant immune-mediated
As a result, the protective and therapeutic effect of the OVA-encoding mRNA vaccine hepatitis, which might be caused
on the OVA-antigen–bearing B16F10 melanoma model was also improved. Moreover, by the undesired expression of
113-O12B encapsulated with TRP-2 peptide (TRP2180–188)–encoding mRNA also exhib-
antigens in the liver. Therefore,
ited excellent tumor inhibition, with the complete response of 40% in the regular
exploring a lymphoid-
B16F10 tumor model when combined with anti–programmed death-1 (PD-1) therapy,
revealing broad application of 113-O12B from protein to peptide antigens. All the treated organ–specific mRNA vaccine
mice showed long-term immune memory, hindering the occurrence of tumor metastatic could be a promising strategy for
nodules in the lung in the rechallenging experiments that followed. The enhanced anti- developing next-generation mRNA
tumor efficacy of the LN-targeting LNP system shows great potential as a universal vaccines. Herein, we reported a
platform for the next generation of mRNA vaccines. lymph-node–targeting mRNA
vaccine based on lipid
lipid nanoparticles j lymph node-targeting mRNA delivery j mRNA vaccine j cancer immunotherapy j
nanoparticles named 113-O12B
melanoma
for cancer immunotherapy. The
Messenger RNA (mRNA) vaccines have achieved great success amid the pandemic of targeted delivery of the mRNA
severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), attracting increasing vaccine elicits robust CD8+ T cell
attention to this field (1, 2). Compared with other types of vaccines, mRNA vaccines responses, exhibiting excellent
show advantages in several aspects, including rapid production, safety, and high protective and therapeutic effects
immune response (3). mRNA vaccines only result in the transient expression of tumor on B16F10 melanoma. Notably,
antigens, therefore avoiding possible mutations caused by DNA vaccines (4). Moreover, 113-O12B can efficiently deliver
mRNA cancer vaccines can encode various antigens, including full proteins and pepti- both a full-length protein and a
des, in a similar process, showing the flexibility in integrating all required tumor anti- short-peptide–based, antigens-
gens together (5). Furthermore, compared with traditional inactivated pathogen or
encoded mRNA, thus providing a
protein-based vaccines, mRNA cancer vaccines can induce stronger humoral and cellular
universal platform for mRNA
response, leading to an improved therapeutic outcome (6). Inspired by the superiority
of mRNA vaccines, industries have been expanding the applications of mRNA technol- vaccines.
ogy to cancer treatment. To date, more than 20 mRNA cancer vaccines have enrolled
in clinical trials (7). Author contributions: J.C. and Q.X. designed research;
mRNA and delivery system are the two key factors of the mRNA vaccine. Enor- J.C., Z.Y., C.H., M.Q., and Y.L. performed research; J.C.
and Z.Y. analyzed data; and J.C. and D.S. wrote the
mous efforts have been made on optimizing both mRNA production and various paper.
delivery systems. The major limitation of mRNA is the high immunogenicity, which Competing interest statement: Q.X. and J.C. are
has been addressed and mitigated by modification of nucleic acids (8). The addition inventors on a pending patent related to this work
filed by Tufts University. The pending patent was
of a cap structure and polyA tail further stabilizes the mRNA and facilitates transfec- licensed to Hopewell Therapeutics, of which Q.X. is a
tion. Additionally, the development of novel mRNA delivery systems, especially lipid founder and interim CEO and J.C. is a consultant.
This article is a PNAS Direct Submission. D.A. is a
nanoparticles (LNPs), has significantly improved the stability and transfection effi- Guest Editor invited by the Editorial Board.
ciency of mRNA in humans. The most commonly used LNPs for RNA delivery can Copyright © 2022 the Author(s). Published by PNAS.
be classified into three generations based on their properties (9, 10). The first genera- This article is distributed under Creative Commons
Attribution-NonCommercial-NoDerivatives License 4.0
tion is nondegradable, e.g., 1,2-dioleoyl-3-dimethyaminopropane and 1,2-dilinoley- (CC BY-NC-ND).
loxy-N,N-dimethyl-3-aminopropane, showing modest transfection effect but notable 1
J.C. and Z.Y. contributed equally to this work.
in vivo toxicity (11–13). The second generation, such as 4-(dimethylamino)-butanoic 2
To whom correspondence may be addressed. Email:
qiaobing.xu@tufts.edu.
acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester
This article contains supporting information online at
(DLin-MC3-DMA) with biodegradable ester linkers, effectively delivers small RNAs, http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
such as small interfering RNA (siRNA) to liver, leading to high and durable knock- 2207841119/-/DCSupplemental.

down of targeted serum proteins (14). The third generation, including ALC-0315 Published August 15, 2022.

PNAS 2022 Vol. 119 No. 34 e2207841119 https://doi.org/10.1073/pnas.2207841119 1 of 10

 and SM-102, exhibits high transfection effects of long-chain expanded the library based on structures of the head, tail, and
mRNA in vivo and was used in the production of COVID-19 linker, such as side group in head amines, linker types, tail
mRNA vaccines (15). lengths, and tail combinations (Fig. 1A). The LNPs were for-
Although the rapid development of LNPs brings major mulated with cholesterol (Chol), dioleoylphosphatidylcholine
advancements to mRNA delivery, a majority of the reported (DOPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethy-
LNPs administered intravenously (IV) or intramuscularly (IM) lene glycol-2000 (DMG-PEG) at the weight ratio of 16:4:2:1.
show very strong mRNA expression in the liver. As reported in Luciferase mRNA (mLuc) was used as a model mRNA for
pharmacokinetics data provided by Pfizer to the European tracking the in vivo distribution of the expressed protein. The
Medicines Agency (EMA), the COVID-19 mRNA vaccine total intensity within LNs after SC injection of LNP/mLuc
(BNT162b2) administrated by IM injection distributed mainly mRNA for 6 h was calculated in Fig. 1B, showing the influence
in the liver and injected site, leading to reversible hepatic dam- of the chemical structure on mRNA expression in LNs. First,
ages in animals (16). Moreover, the BNT162b2 mRNA could the importance of the tail length was proven by analyzing deliv-
be reverse-transcribed intracellularly into DNA in human liver ery efficiency of lipids containing different length tails. The
cell line (Huh7) as fast as 6 h by an endogenous reverse tran- shorter length tails, O10B and O12B, exhibited a higher
scriptase, resulting in a great threat to human health (16). expression in the LNs compared with longer tails that showed
More importantly, the vaccination by BNT162b2 was also almost no efficacy at all. Additionally, combining tails of differ-
reported to elicit CD8+ T-cell-dominant hepatitis (17). There- ent lengths also proved the importance of tail length; that is,
fore, the targeting expression of mRNA in vivo can minimize the combination of longer tails in the lipid decreased the deliv-
side effects and improve efficacy, which is considered as the key ery efficacy. Second, the ester linker was proven to play an
point of the next-generation LNPs (18). For mRNA cancer important role in mRNA delivery. The replacement of the ester
vaccines, the targeted delivery and expression of mRNA- bond to an amide bond significantly decreased the transfection
encoding tumor antigens in lymphoid organs are considered to in LNs. Third, replacing the methyl groups of the amine head
be a promising strategy to improve the efficacy and reduce the to hydroxyl, ethyl, or N-(1,2-ethanediyl)acetamide groups also
side effects of mRNA vaccines (19). Though many nanosystems reduced the signal. Resultantly, 113-O12B was selected as the
can deliver cargos to specific organs by introducing active- top lipid for mRNA delivery to LNs in this library. Addition-
targeting ligands, there are still some limitations of their clinic ally, the pKa and size of the LNPs were further analyzed to
applications. First, the targeting ligands increase the complexity explore the in-depth relationship between the properties of
of delivery system, hindering the rapid production of the LNPs and mRNA expression (SI Appendix, Fig. S2). However,
mRNA vaccine. Second, as it is challenging to transfect no obvious correlation was observed.
immune cells in vivo, the successful mRNA delivery and trans- The 113-O12B formulation was further optimized for tar-
fection to lymphoid organs are still rarely reported (20, 21). geted delivery to the LNs. We selected ALC-0315 in Pfizer/
Our group has developed a series of LNPs with targeting spe- BioNTech’s COVID-19 mRNA vaccine Comirnaty as a com-
cificity to the liver, spleen, and lung without the use of targeting parison. As shown in Fig. 1C, the active lipid, Chol, helper
ligands (22, 23). In this work, LNP 113-O12B, with lymph lipid, and DMG-PEG all impacted the mRNA transfection in
node (LN)-targeting specificity, was explored and applied for a LNs. The optimized weight ratio of the above four components
therapeutic mRNA cancer vaccine. Compared with LNPs formu- was determined to be 16:4.8:3:2.4, showing much stronger sig-
lated with ALC-0315, a key component in the US Food and nal in LNs compared with ALC-0315/mLuc. The results of the
Drug Administration (FDA)-approved Comirnaty, 113-O12B bioluminescence distribution in mice after SC injection of
showed significantly reduced mRNA expression in the liver and LNP/mLuc are shown in Fig. 1D. The obvious signal in drain-
higher expression in LNs after subcutaneous (SC) injection. The ing LNs could be observed in both 113-O12B and ALC-0315,
targeted delivery of full-length ovalbumin (OVA)-encoding but ALC-0315 showed significantly higher expression in liver.
mRNA vaccine showed remarkably enhanced CD8+ T cell In Fig. 1E, the radiance of luminescence in the liver after treat-
response and thereby excellent protective and therapeutic effect ment with ALC-0315/mLuc was about four times higher than
against OVA-transduced B16F10 tumor model. Moreover, the that in LNs. In contrast, the intensity in the liver decreased to
mRNA vaccine encoding a tumor-associated peptide antigen only 32% of that in LNs in the 113-O12B group, confirming
TRP2180–188 achieved great therapeutic effect on established the superior LN-targeting ability of 113-O12B. The organ-
B16F10 tumor models, revealing that the 113-O12B platform targeting delivery of mRNA by LNPs might be related to the
can be generalized to multiple antigen types. Notably, the combi- protein corona adsorbed on the LNPs during systemic circula-
nation with anti–programmed death-1 (PD-1) antibody further tion (25). However, the detailed mechanism of the protein
improved the complete response (CR) to these established tumor corona and biodistribution of LNPs still remains unknown.
models. All the surviving mice from the therapeutic experiments To explore which cell types could be transfected by LNP
resisted the rechallenging of lung metastatic model, revealing within LNs, the gene-engineered Ai14 mice were employed due
long-term antitumor immunity generated by our mRNA cancer to the activatable and stable expression of red florescence pro-
vaccine. tein, tdTomato, after Cre mRNA (mCre) expression (Fig. 1F).
Briefly, the gene encoding tdTomato was blocked by a stop
Results gene between two LoxP segments. When the mRNA encoding
Cre mRNA is delivered and expressed in cells, the LoxP gene is
Screening and Optimization of LNPs for LN-Targeting Delivery. cut. Subsequently, the tdTomato gene is activated and the pro-
The lipids used for in vivo screening were synthesized via tein is expressed with red fluorescence, which can be detected
Michael addition between amine-bearing heads and acryloyl by flow cytometry. As shown in Fig. 1G, both 113-O12B and
groups containing aliphatic chains as reported in previous work ALC-0315 successfully delivered Cre mRNA to antigen-
(SI Appendix, Fig. S1) (24). Our group developed a library of presenting cells (APCs), including macrophages and dendritic
reduction-responsive lipids, showing liver-specific mRNA deliv- cells (DCs). 113-O12B/mCre showed positive mRNA expres-
ery in previous work (22). Based on previous results, we further sion in ∼27% DCs and ∼34% macrophages, both higher than

2 of 10 https://doi.org/10.1073/pnas.2207841119 pnas.org
 A Lipid name R0 R1 R2
113-O10B
113-O12B
113-O14B
113-O16B
-CH3
B ✱✱
113-O18B
✱
113-N12B
Radiance x106 (p/sec/cm2/sr)

100 ✱✱
113-O12B3-
80
O14B
60 113-O12B3-
O16B
40 -CH2CH2
113-NEA
NHCOCH3
20
113-Ethyl -CH2CH3
0
113-OH -CH2CH2OH
3- B3 2B

11 yl
B 4B

11 16B

11 EA
3- B

3- B

3- B

3- B
3- 13 8B

6- H
B
11 10

11 12

11 14

11 16

12
h
30 O
1
12 O 1
1

Et
N

3-
-N
O

O
3 -O

3-

3-
-
3-
11

1
11 12
O

O
11

C ✱✱ D E ✱
✱✱
Radiance x106 (p/sec/cm2/sr)

113-O12B/mLuc

Ratio of radiance in Liver/LNs

✱✱✱ 8
✱
60 ✱✱✱
✱✱✱

6
40

5.0

20 4
4.0

0
2
ALC-0315/mLuc

Active lipid 16 16 16 16 16 16 16 16 16 16 3.0

Chol 6 8 4.8 4.8 4.8 4.8 4.8 5 5 4.8 ALC-

×106

0
0315
Helper lipid 3 3 2 4 3 3 3 1 1 3 2.0

c
Lu

Lu
DMG-PEG 2.4 2.4 2.4 2.4 1 1.5 2.4 2 2 2.4

/m

m
5/
B
1.0

31
12

-0
O
DSPC DOPE 3-

C
AL
11
Radiance
(p/sec/cm2/sr)

F G
Percentage of tdTomato+ cells (%)

✱✱
40
UT ✱
LoxP STOP LoxP tdTomato
ALC-0315/mCre
30
Cre recombination 113-O12B PEG1.5/mCre
LNP/mCre
20 113-O12B PEG2.4/mCre
tdTomato
S.C. injecon
10
Ai14 mice

0
tdTomato+ cells T cell B cell NK cell DC Macrophage
Fig. 1. Screening and optimization of LNPs with targeting ability to LNs. (A) The chemical structure of lipids used in this study. (B) The bioluminescence within
inguinal LNs after treatment with LNP/mLuc subcutaneously at the tail base for 6 h. (C) The bioluminescence within inguinal LNs after treatment by LNP/mLuc
with different formulations for 6 h. (D) Representative images of bioluminescence distribution in mice treated with 113-O12B/mLuc and ALC-0315/mLuc for 6 h.
(E) Ratio of radiance in liver and inguinal LNs after SC injection of mLuc for 6 h. (F) Mechanism of subcellular analysis of mRNA expression in Ai14 reporter mice.
(G) Percentage of tdTomato-positive cells in different types of immunocytes after treatment with LNP/mCre subcutaneously at tail base for 48 h. The error bar
around each data point is the SD. Tukey’s multiple comparisons test was used to calculate the statistical significance. *P < 0.05 was considered statistically signif-
icant. **P < 0.01 and ***P < 0.001 were considered highly significant.

those levels resulting from ALC-0315/mCre. The enhanced 113-O12B/mOVA Elicited a Robust CD8+ T Cell Response and
expression of mRNA in APCs is important to the following Protective Effect on the B16F10-OVA Tumor Model. OVA was
activation of adaptive immunity. In addition, we discovered chosen as a model antigen, and OVA-transduced B16F10
that the mRNA expression in all cell types was diminished as (B16F10-OVA) cells were used as the model tumor cells. The
the amount of DMG-PEG in LNP decreased, suggesting the vaccination followed the timeline in Fig. 2A, with the prime
necessity of including PEG in formulations for in vivo delivery. dose on day 0 and the boost dose on day 5. The levels of

PNAS 2022 Vol. 119 No. 34 e2207841119 https://doi.org/10.1073/pnas.2207841119 3 of 10

 A

B C

D E

F G

Fig. 2. T cell response and protective effect after vaccination. (A) Timeline for vaccination and blood withdrawal. (B) Changes of cytokines and chemokines in
the mice treated by blank or OVA mRNA-formulated LNPs for 24 h. (C) OVA-specific antibody titers in the mice treated by 113-O12B/mOVA and ALC-0315/
mOVA on day 12. (D) Representative flow cytometry diagrams of IFN-γ-positive cells within CD3+ CD8+ T cells 7 d after second vaccination. (E) Time-dependent
changes of IFN-γ-positive cells 7, 14, and 28 d after second vaccination. (F) Tumor volumes of B16F10-OVA tumor model. (G) Lungs collected 18 d after the intra-
venous injection of B16F10-OVA cells. UT: Untreteated. The error bar around each data point is the SD. Tukey’s multiple comparisons test was used to calculate
the statistical significance. *P < 0.05 was considered statistically significant. **P < 0.01 and ***P < 0.001 were considered highly significant.

cytokines and chemokines after the treatment of blank LNPs cytokines and chemokines compared with those of ALC-0315/
and LNP/mOVA for 24 h were shown in Fig. 2B. Blank ALC- mOVA, which might be due to the rapid expression of OVA in
0315 showed stronger activation of innate immune system with LNs. Notably, the level of IL-6, which plays an important role
significantly increased levels of proinflammatory cytokines in the proliferation and differentiation of T cells for adaptive
(G-CSF, M-CSF, IFN-γ, and IL-6) and proinflammatory che- immunity, was significantly elevated in 113-O12B/mOVA-
mokines (MCP-1, MIP-1α, and MIP-1β). The formulation treated groups compared with the blank 113-O12B, which
with OVA mRNA (mOVA) further increased the acute inflam- might induced by the strong expression of OVA antigen in
matory response upon the expression of the foreign protein LNs (26).
OVA. Though the activation of the innate immunity induced The antibody levels induced by both mRNA vaccines were
by blank 113-O12B was much weaker than blank ALC-0315, evaluated 14 d after the second dose (Fig. 2C). Three types
113-O12B/mOVA still expressed comparable proinflammatory of immunoglobulin G (IgG), including total IgG, IgG1, and

4 of 10 https://doi.org/10.1073/pnas.2207841119 pnas.org
IgG2c, were measured by enzyme-linked immunosorbent assay (Fig. 3D). After the two vaccinations, both Treg cells in the
(ELISA). 113-O12B/mOVA showed comparable antibody res- 113-O12B/mOVA and ALC-0315/mOVA groups decreased to
ponse of all three antibodies compared with those induced by roughly 10%. Impressively, the anti–PD-1 treatment significantly
ALC-0315/mOVA. However, the highest antibody level was reduced the percentage of Treg cells compared with other groups.
observed in the ALC-0315 group, which might result from the A similar phenomenon was observed in many other works, indi-
capture and presentation of secreted OVA protein expressed in cating that the combination of anti–PD-1 and mRNA cancer vac-
the liver by APCs. The percentage of OVA-specific CD8+ T cine is critical for overcoming tumor immunosuppression (27).
cells was evaluated by intracellular cytokine staining. As shown The polarization of macrophages is also important to antitu-
in Fig. 2 D and E, 14 d after the second vaccination, the per- mor immunity. M1-like macrophage benefits antitumor
centage of IFN-γ+ cells within CD8+ T cells stimulated by response, while M2-like macrophage suppresses adaptive immu-
OVA peptide (SIINFEKL) in 113-O12B group reached about nity (28). We further evaluated the polarization of macrophages
2.57%, which was significantly higher than that of ALC-0315 within tumors by flow cytometry. M1-like macrophages were
group (∼1.55%). Moreover, the percentage of IFN-γ+ cells marked as F4/80+, CD11b+, and CD86+. M2-like macro-
remained above 2% in 113-O12B group after 4 weeks after the phages were marked as F4/80+, CD11b+, and CD163+. As
second vaccination (Fig. 2E), indicating the long-lasting T cell shown in Fig. 3E, less than 50% of macrophages were M1-like
response induced by the mRNA vaccine. macrophages in untreated groups. After two doses of the vacci-
The protection effect of the mRNA vaccine was evaluated in nation, the percentage of M1-like macrophages increased to
B16F10-OVA tumor model. One million tumor cells were more than 80%. The ratio of M1/M2-like macrophages
injected SC at the right flank of the untreated or vaccinated mice increased significantly in all vaccinated mice compared with the
on day 13. As shown in Fig. 2F, the tumor grew rapidly in the untreated group. The driven polarization to M1-like macro-
mice without vaccination, until it reached the humane endpoint phages by vaccination further indicates the generation of a
within 20 d. However, no obvious tumor growth was observed strong antitumor immunity.
in both the 113-O12B/mOVA- and ALC-0315/mOVA-vacci-
nated mice, indicating the superior protection effect of the Therapeutic Effect of 113-O12B/mOVA on the Established
mRNA vaccines. To further confirm these findings, we estab- B16F10-OVA Tumor Model. The therapeutic effect of 113-O12B-
lished a metastatic model by IV injecting 1 million cells into the based mRNA vaccine was firstly evaluated in the B16F10-OVA
control and vaccinated mice. After 18 d of the injection, the mice tumor model. One million B16F10-OVA cells were injected SC
were killed, and the lungs were isolated for comparison. As shown at the right flank of C57/BL6 mice. After the tumor inoculation,
in Fig. 2G, four out of five mice from the nontreated group three groups of mice received the prime and boost dose of the
showed obvious metastatic nodules, while zero mice in 113- mRNA vaccine on days 5 and 12. Anti–PD-1 antibody was
O12B/mOVA-vaccinated group had apparent metastatic nodules. administrated by intraperitoneal injection with or without 113-
All these results demonstrated that the LNP/mOVA has excellent O12B/mOVA on days 7, 11, and 15. The percentages of CD3+
protection to the B16F10-OVA tumor model. CD8+ T cells bearing T cell receptors binding to H-2Kb OVA
tetramer-SIINFEKL within peripheral blood mononuclear cells
113-O12B/mOVA Shifted the Immune Cell Composition in the (PBMCs) were measured on day 19. In Fig. 4A, almost no
Established B16F10-OVA Tumor Model. The superior protection SIINFEKL-specific CD8+ T cells could be detected, while the
effect of the mRNA cancer vaccine encouraged us to further eval- vaccination with ALC-0315/mOVA and 113-O12B/mOVA
uate the therapeutic effect on the established tumors. First, the increased the percentage to 2.2% and 2.5%, respectively, revealing
impact of the mRNA vaccine on immune cell composition of the generation of tumor-killing CD8+ T cells after vaccination.
established tumor was studied in B16F10-OVA tumor model. Moreover, the combination with anti–PD-1 further significantly
One million B16F10-OVA cells were inoculated SC on day 14. increased the percentage of SIINFEKL-specific CD8+ T cells to
Two weeks later, the mice received the prime and boost vaccina- 5.3%, suggesting the important role of checkpoint inhibition ther-
tions on day 0 and day 5, respectively (Fig. 3A). To inhibit apy. The levels of IFN-γ-secreting T cells within PBMCs were
immunosuppression, the check point inhibitor anti–PD-1 anti- evaluated by an ELISpot assay in Fig. 4B. Similar to the percent-
body was injected intraperitoneally on days 2 and 7. Tumors age of SIINFEKL-specific CD8+ T cells, the mice without vacci-
were collected on day 12, that is 1 week after the second dose, nation showed no response to the stimulation of SIINFEKL.
and analyzed by flow cytometry in Fig. 3 B and C. All the However, all the vaccinated groups generated IFN-γ-secreting
vaccinated mice exhibited a significantly increased no. of CD8+ T cells to some extent, indicating the robust T cells response gen-
T cells within tumors compared with the untreated group, while erated by the mRNA vaccine.
there was no significant difference in CD4+ T cells (Fig. 3B). The tumor volumes monitored posttreatment are demon-
Interestingly, 113-O12B/mOVA group showed a greater increase strated in Fig. 4C. The untreated mice exhibited rapid growth
in infiltration of macrophages and activated DCs within the of the tumor, reaching an endpoint within 25 d (CR = 0/5).
tumor compared with the ALC-0315 group, suggesting an Single administration of anti–PD-1 antibody failed to inhibit
enhanced therapeutic effect (Fig. 3C). The promoted migration tumor growth (CR = 0/5). However, in the group treated with
of both CD8+ T cells and APCs is important to the therapeutic ALC-0315/mOVA, the eradication of tumor was observed in
outcome of the mRNA vaccine. Moreover, the combination of one mouse, whereas rapid growth of tumor was still observed
anti–PD-1 did not result in a significant difference in the no. of in two out of five mice. 113-O12B/mOVA exhibited a more-
T cells and APCs compared with those in the 113-O12B group. effective tumor inhibition compared with that of ALC-0315.
To further understand the subcellular types of the infiltrated All the mice treated by 113-O12B/mOVA survived longer than
T cells, the CD4+ T cells were stained with FoxP3, which distin- 35 d, and one of the mice showed no tumor growth during the
guishes regulatory T (Treg) cells and conventional T helper whole treatment. The combination of anti–PD-1 did not sig-
cells (Fig. 3D). Roughly 18% of CD4+ cells were determined nificantly improve the overall tumor inhibition but increased
to be Treg cells in the tumors of untreated mice, indicating a the CR to 2/5, which might be caused by the individual varia-
strong immunosuppressive environment of B16F10-OVA tumor tion in response to the anti–PD-1 therapy. To further evaluate

PNAS 2022 Vol. 119 No. 34 e2207841119 https://doi.org/10.1073/pnas.2207841119 5 of 10

 A Tumor 1st 2nd Tumor
inoculation Vaccination Vaccination collection

Anti-PD-1 Anti-PD-1

✱
B 8000 ✱✱
C 100000 ✱ ✱✱
UT

Cell counts (per 1 M cells)

Cell counts (per 1 M cells)

✱✱ ✱✱
✱ 80000 ALC-0315/mOVA
6000 ✱✱
113-O12B/mOVA
60000
113-O12B/mOVA
4000 +anti-PD-1
40000

2000
20000

0 0
CD4+ T cells CD8+ T cells Macrophages MHC II+ DCs

30 ✱✱

FOXP3+ of CD4+ (%)

D 113-O12B/mOVA ✱

UT ALC-0315/mOVA 113-O12B/mOVA +anti-PD-1 ✱

20

10
FoxP3+

CD4+

✱✱

E
113-O12B/mOVA F 20
UT ALC-0315/mOVA 113-O12B/mOVA +anti-PD-1
15
M1/M2 ratio

10

5
CD86+

CD11b+

Fig. 3. Changes of the immune cell composition in established B16F10-OVA tumor after vaccination. (A) Timeline for tumor inoculation and vaccination.
(B and C) Changes of T cells and APCs within the tumor 7 d after the second vaccination. (D) Representative flow cytometry diagrams and percentages of
FoxP3+ cells within CD4+ T cells 7 d after the second vaccination. (E) Typical flow cytometry results of CD86+ cells within CD11b+ F4/80+ cells. (F) Ratio of M1
to M2 macrophages after vaccination. The error bar around each data point is the SD. Tukey’s multiple comparisons test was used to calculate the statistical
significance. *P < 0.05 was considered statistically significant. **P < 0.01 and ***P < 0.001 were considered highly significant.

the long-term immune memory of the mRNA vaccine, the adaptive immune response generated by the mRNA vaccine
mice that did not develop tumors after 30 d were injected with encoding the OVA antigen. However, the therapeutic effect on
1 million B16F10-OVA cells IV. Eighteen days after this injec- regular B16F10 is more meaningful for future clinical applica-
tion, the lungs of the mice were collected and imaged; the pho- tions. Tyrosinase-related protein-2 (TRP2) is a tumor-associated
tograph of the lungs is shown in Fig. 4D. Obvious metastatic antigen overexpressed in murine and human melanomas with a
tumor nodules were found in the lungs of the mice without the weak immunogenicity. Therefore, induction of a strong antibody
vaccination. However, all the mice that survived the therapeutic response and T cell immunity to TRP2 is necessary to generate
experiment showed no metastatic tumors, suggesting the gener- a strong anticancer immune response in the TRP2-based cancer
ation of long-term immune response. vaccine (29). Herein, we chose the TRP2180–188 peptide
(SVYDFFVWL), a major histocompatibility complex (MHC)
Therapeutic Effect of 113-O12B/TRP2180–188 on the Established class I H-2Kb restricted epitope in mice, as the model peptide
B16F10 Tumor Model. The engineered tumor B16F10-OVA antigen for the design of the mRNA cancer vaccine against regu-
expressed OVA antigens, making it easier to be recognized by the lar melanoma (30). N1-methylpseudouridine (N1mψ)-modified

6 of 10 https://doi.org/10.1073/pnas.2207841119 pnas.org
 anti-PD-1 ALC-0315/mOVA
A
✱✱✱
✱✱✱
10

OVA tetramer+ of CD3+ CD8+

✱
✱
8

113-O12B/mOVA 4
UT 113-O12B/mOVA +anti-PD-1
2
OVA tetramer

1
1

15

an B
T

PD
D
U

B+ 12
3
i-P

-0

12 3-O

ti-
t

C
An

AL

11
O
3-
CD8

11
B ✱✱✱

IFN γ+ Spots per 1 x 104 cells

UT 400 ✱✱✱
✱
300 ✱
Anti-PD-1

200
ALC-0315
/mOVA 100
113-O12B
/mOVA 0

1
AL D1

11 315

an B
T

PD
U

B+ 12
113-O12B/mOVA

i-P

-0

-O

ti-
t

C
+anti-PD-1

An

O
12 3
3-
C

11
2000 UT CR: 0/5 D
UT
Tumor volumes (mm3)

Anti-PD-1 CR: 0/5

✱✱✱

24.0% 26.8%

1500 ALC-0315/mOVA CR: 1/5

113-O12B/mOVA CR: 1/5

Tumor rechallenging

ALC-0315 113-O12B
1000 113-O12B/mOVA CR: 2/5 /mOVA /mOVA
+anti-PD-1 0% 0%

500

113-O12B/mOVA
+anti-PD-1
0 0% 0%

0 10 20 30 40
Days post-inoculation
Fig. 4. Therapeutic effect on B16F10-OVA tumor after vaccination by mRNA cancer vaccine. (A) Representative flow cytometry diagrams and percentages of
CD3+ CD8+ T cells bearing T cell receptors binding to H-2Kb OVA tetramer-SIINFEKL within PBMCs 7 d after the second vaccination. (B) ELISpot images and
spot nos. of IFN-γ-secreting T cells within PBMCs of vaccinated mice. (C) Tumor volumes of B16F10-OVA model posttreatment. (D) Lungs collected 18 d after
the intravenous injection of B16F10-OVA cells. The error bar around each data point is the SD. Tukey’s multiple comparisons test was used to calculate the
statistical significance. *P < 0.05 was considered statistically significant. **P < 0.01 and ***P < 0.001 were considered highly significant.

TRP2180–188 mRNA (mTRP2) was synthesized by in vitro tran- The tumor inhibition is correlated to the T cell response (Fig.
scription (IVT) with an ARCA cap and 120 nt poly(A) tail 5C). The mice without treatment reached the endpoint within
(Fig. 5A). 28 d, while the mRNA vaccine extended the endpoint to more
The mice were inoculated with one million B16F10 cells at than 36 d. Notably, the two mice showing strongest T cell
the right flank on day 0. Afterward, two groups of mice response exhibited CR in the group combined with anti–PD-1
received two doses of the mRNA vaccine on days 5 and 12. therapy, indicating the excellent therapeutic effect of the
Anti–PD-1 therapy was also dosed in one group on days 7, 11, mRNA vaccine in combination with the check point inhibitor.
and 15. First, the percentage of IFN-γ+ cells within CD8+ The long-term antitumor immunity was also evaluated using
T cells in PBMCs stimulated by TRP2180–188 peptide was eval- the B16F10 metastatic model. Similarly, one million B16F10
uated and is shown in Fig. 5B. The vaccination with the cells were IV injected into the untreated and surviving mice on
mRNA vaccine significantly increased the percentage of IFN- day 30. After 18 d, the lungs were collected and photographed.
γ-secreting cells 7 d after the second vaccination. Although the As shown in Fig. 5D, metastatic nodules appeared in almost all
administration with anti–PD-1 antibody did not result in a sig- the lungs of mice without treatment, even reaching up to more
nificant difference in the IFN-γ+ cells 7 d after the second vac- than 95% of the lung area. In vaccinated mice, the growth of
cination, two mice achieved relatively higher responses than the tumor nodules was not observed as extensively. More impor-
mice treated with 113-O12B/mTRP2, suggesting the individ- tantly, no obvious metastatic nodules were observed in the
ual variation in the response to anti–PD-1 therapy (Fig. 5B). mice with a CR.

PNAS 2022 Vol. 119 No. 34 e2207841119 https://doi.org/10.1073/pnas.2207841119 7 of 10

 A

IFN γ+ among CD3+ CD8+ cells (%)

B 113-O12B/mTRP2 1.5
✱
UT 113-O12B/mTRP2 +anti-PD-1

1.0

0.5
IFN-γ+

0.0

1
2
T

D
P
U
CD8+

P2 TR

i-P
nt
m
+a
TR
m
C 2000 UT
113-O12B/mTRP2
D
Tumor volumes (mm )

Tumor rechallenging
3

✱
1500 113-O12B/mTRP2
+anti-PD-1 69.3% 92.5% 95.8%

1000 UT
7.4%

500
113-O12B/mTRP2
13.4%
113-O12B/mTRP2
0 +anti-PD-1
0 12 20 28 36 Complete response
Days post-inoculation mice (0%)

Fig. 5. Therapeutic effect on normal B16F10 tumor model after vaccination by the mRNA cancer vaccine. (A) IVT of TRP2180–188 mRNA. (B) Representative
flow cytometry diagrams and percentages of IFN-γ-positive cells within CD3+ CD8+ T cells in PBMCs 7 d after the second vaccination. (C) Tumor volumes of
B16F10 model during the experiments. (D) Lungs collected 18 d after the intravenous injection of B16F10 cells. The error bar around each data point is the
SD. Tukey’s multiple comparisons test was used to calculate the statistical significance. *P < 0.05 was considered statistically significant. **P < 0.01 and
***P < 0.001 were considered highly significant.

Discussion mRNA expression in the LNs compared with ALC-0315.

Notably, ALC-0315/mLuc exhibited a strong signal in the
Although the development of the COVID-19 mRNA vaccines liver, while 113-O12B/mLuc delivered mRNA more specifi-
has shown great success in the protection against SARS-COV-2 cally to the LNs. The expression of mRNA in liver was also
and the advancement of mRNA vaccines, there currently are no observed in other vaccines by IM injection (23). As the unde-
cancer vaccines approved for clinical use. This may be due to the sired transfection of mRNA in the liver might induce side
weak immunogenicity of tumor antigens that requires stronger effects, 113-O12B showed great advantages regarding the safety
and more specific activation of the immune system or the unde- for in vivo applications. 113-O12B also promoted mRNA
sired expression of tumor antigens in other nonlymphoid organs, expression in APCs within the LNs compared with ALC-0315,
such as liver, increases the risk of mRNA cancer vaccine. There- owing to the superior LN-targeting ability. Compared with
fore, the targeted delivery of mRNA in lymphoid organs might other targeting systems with active targeting ligands, 113-O12B
not only improve the antitumor immunity but also reduce unde- with endogenous LN-targeting ability is more practical for clin-
sired side effects, providing a promising strategy for the develop- ical applications.
ment of next-generation mRNA cancer vaccines. In this work, we Administration of blank ALC-0315 showed significantly
developed a LN-targeting lipid, 113-O12B, which was used as a up-regulated cytokines and chemokines related to proinflamma-
delivery vehicle in the therapeutic mRNA cancer vaccine against tion, indicating the highly inflammatory effect of blank ALC-
a melanoma mouse model. We used ALC-0315 used in Comir- 0315. It was reported that LNPs were capable of inducing the
naty as our chosen standard for comparison. inflammatory cell death (31). Then the inflammatory response
First, we evaluated the influence of the lipid structure on induced by blank LNPs is caused by the damage-associated
mRNA expression in the LNs. The tail length, linker bond, molecular patterns released from these cells. The exact role of
and amine head all impacted the delivery efficiency to the LNs. the inherent immunogenicity of mRNA or LNPs in the genera-
The lipids with shorter tails (≤ 12 carbons) exhibit greater effi- tion of adaptive immunity is still unknown (32, 33). On one
cacy compared with the lipids with longer tails (> 12 carbons). hand, the immunogenicity of the mRNA or LNPs activates the
Additionally, the lipids with an ester bond linker proved to innate immune response, benefiting the subsequent activation
have greater efficiency than those with an amide bond linker. of the adaptive immunity. On the other hand, the high immu-
Furthermore, replacing the methyl group in the head amine nogenicity of the mRNA or LNPs hinders the expression of
also significantly decreased the mRNA expression in the LNs. mRNA-encoding antigens, thereby weakening the generation
Second, the optimized formulation of 113-O12B/mLuc was of the adaptive immunity. The LNPs encapsulating OVA
obtained by changing the weight ratio of different components mRNA (LNP/mOVA) showed increased secretion of proin-
and replacing the helper lipids, to resultantly exhibit a better flammatory factors, indicating the successful activation of the

8 of 10 https://doi.org/10.1073/pnas.2207841119 pnas.org
innate and adaptive immunity after the expression of the anti- therapy significantly suppresses and even eradicates the established
gen. The vaccination with both 113-O12B/mOVA and ALC- B16F10 tumor. Finally, all mice surviving from the therapeutic
0315/mOVA showed a strong antibody response. Moreover, experiment show no growth of metastatic nodules, indicating that
113-O12B/mOVA elicits stronger CD8+ T cell response com- the mRNA cancer vaccine shows great promise in providing long-
pared with ALC-0315/mOVA and is still maintained at a high term antitumor efficacy.
level 4 weeks after the second vaccination. The vaccinations
with 113-O12B/mOVA and ALC-0315/mOVA both exhibit a Materials and Methods
full protection effect from the B16F10-OVA tumor over 40 d,
confirming the generation of a strong antitumor immunity. Synthesis and Formulation of LNPs. Lipids were synthesized by Michael
One advantage of LN-targeting 113-O12B/mOVA is the addition between amine-bearing head and acryloyl group containing aliphatic
shift of the immune cell composition, which is confirmed by chain. The head and tail were mixed in the molar ratio of 1:4.8 and reacted at
the up-regulated infiltration of APCs compared with that of 70 °C for 3 d. Then the mixture was purified by flash chromatography (Combi-
ALC-0315/mOVA. The mRNA vaccine reduces the population Flash, USA). The lipids were further characterized by electrospray ionization-mass
of Treg cells by activating the adaptive immunity. More impor- spectrometry.
tantly, the combination of anti–PD-1 therapy significantly The LNPs were prepared by dropwise adding the ethanol solution containing
the mixture of active lipid, Chol, helper lipid, and DMG-PEG at the defined
decreases the percentage of Treg cells to 2.6%, suggesting the
weight ratio to 25 mM sodium acetate solution. Then the mixture was dialyzed
importance of the check point inhibitor. The macrophages within
with Slide-A-Lyzer MINI Dialysis Device (3.5K molecular weight cutoff, Thermo
the tumor of the vaccinated mice also exhibited M1 polarization.
Scientific, USA). The LNP/mRNA was prepared by simply mixing blank LNP with
All these results show that the vaccination significantly changed mRNA at the weight ratio of 10:1 in aqueous solution.
the immune cell composition to inflammatory types.
The therapeutic efficacy of 113-O12B was evaluated in two Synthesis of TRP2180–188 mRNA. The coding sequence for TRP2180–188 was
tumor models, including OVA-engineered B16F10-OVA and amplified by PCR and introduced into a pMRNAxp vector (System Biosciences,
regular B16F10 tumor model. Although the vaccination by USA) using primers A109/A110 (SI Appendix, Table S1). The pMRNA-TRP2180–188
113-O12B/mOVA and ALC-0315/mOVA both achieve similar plasmid was used as templates for gene polyadenylation using the Tail PCR
T cell responses, the 113-O12B/mOVA elicits a prolonged sur- Primer Mix (System Biosciences, USA), of which reverse primer contains 120 oli-
vival time compared with ALC-0315/mOVA. Notably, the godT. The Tail PCR (50 μL) was performed in a Phusion High-Fidelity DNA Poly-
integration of the mRNA vaccine and anti–PD-1 antibody merase Kit following the manufacturer’s protocol (New England Biolab Inc.,
eradicate the tumor in two of five mice. The improved thera- USA). The reaction was then applied to a PCR program: 98 °C 3 min, 98 °C 30 s,
peutic outcome may be attributed to the activation of cytotox- 64 °C 30 s, 72 °C 10 s, 72 °C 10 min, and 4 °C hold for 30 cycles. The PCR
icity T cells and the inhibition of Treg cells. mixture was further treated with Proteinase K and purified with a GeneJET PCR
There are two major challenges of regular B16F10 tumor Purification Kit (Thermo Scientific, USA). N1mψ-modified TRP2180–188 mRNA was
model compared with the model antigen OVA-engineered cell synthesized through IVT reaction. The reaction mixture was treated with DNase I
line, including low immunogenicity of tumor-associated anti- and Antarctic Phosphatase (New England Biolabs, USA) and purified using Mega-
gens and down-regulation of the antigens on the tumor surface Clear Kit (Life Technologies, USA). N1mψ was incorporated to completely substi-
(34). TRP2 is proven to be an effective tumor-associated anti- tute the natural counterparts in TRP2180–188 mRNA synthesis.
gen and TRP2180–188 peptide, which is why it was chosen in In Vivo Expression of Luc mRNA. BALB/c mice (4–6 wk old) were injected
this study. Different from the full protein antigen, the with LNPs containing 5 μg mRNA and 50 μg active lipid SC at the tail base. Six
LN-targeting delivery of peptide antigens might be superior to hours after the injection, 100 μL of luciferin, at a concentration of 15 mg/mL,
that of the untargeted ones. The LN-targeted delivery of was intraperitoneally injected into the mice. After 10 min, the mice were imaged
TRP2180–188 mRNA to APCs in the LNs might lead to the using the In Vivo Imaging System (PerkinElmer).
higher presentation of TRP2180–188 peptide on MHC class I
molecules, subsequently generating more TRP2180–188-specific Delivery of Cre mRNA to LNs in Ai14 Reporter Mice. Ai14 mice were
tumor-killing T cells. When combined with anti–PD-1 ther- injected with LNPs/mCre containing 10 μg mRNA and 100 μg active lipid SC at
apy, 113-O12B/mTRP2180–188 vaccine shows significant tumor tail base. Forty-eight hours after the injection, the mice were killed and inguinal
inhibition with a 40% rate of CR. The long-term memory of LNs were collected. The cell suspensions were prepared by grinding and filtrating
mRNA-based vaccines is also evaluated in the metastatic tumor through a 70-μm strainer. Then 2 × 106 cells were incubated in 100 μL
model. In all the protection and therapeutic experiments, meta- flow cytometry staining buffer (eBioscience) containing fluorophore-conjugated
static nodule was not observed in all mice with CR, implying antibody of interest listed in SI Appendix, Table S2 at the recommended con-
the long-term efficacy of the mRNA vaccine. centration at 4 °C for 1 h. Then the cells were kept at 4 °C for analysis after wash-
In summary, 113-O12B LNP, an LN-targeting LNP delivery ing twice with staining buffer. Data were collected by LSR-II flow cytometer
system, is developed for a mRNA cancer vaccine. The 113- (BD Biosciences) and analyzed by FlowJo-v10. Gating information is shown in SI
O12B/mRNA shows enhanced expression in APCs compared Appendix, Fig. S3.
with ALC-0315/mRNA, indicating the LN-specific targeting ELISA for Antibody Titer. The antibody titer was measured by indirect ELISA.
ability. The vaccination with 113-O12B/mOVA elicits a compa- The high binding ELISA plates (Greiner Bio-One, USA) were covered with 50 μL of
rable antibody response and CD8+ T cell response compared OVA at 20 μg mL1 in sodium carbonate solution (pH 8.0) at 4 °C overnight. The
with ALC-0315/mOVA. Moreover, 113-O12B/mOVA induces plates were then washed with PBS containing 0.5% Tween-20 and blocked by 5%
greater infiltration of APCs to the tumor site, leading to bovine serum albumin solution (Sigma-Aldrich). The serum collected from immu-
improved therapeutic efficacy on the established tumor model nized mice was initially diluted in 1:100. After performing a serial dilution in trip-
compared with ALC-0315/mOVA. In addition to the full OVA licate, the diluted serum was added into the plates for 2 h at room temperature.
antigen, the mRNA encoding a peptide epitope TRP2180–188 is Then the plates were washed and incubated with horseradish-1:10,000-diluted-
also successfully delivered by 113-O12B, suggesting that the peroxidase-conjugated anti-IgG, IgG1, and IgG2c antibodies for 1 h. The plates
LNP/mRNA system may provide a universal platform for proc- were washed and incubated with 100 μL of 3,30 ,5,50 -tetramethylbenzidine sub-
essing multiple types of tumor antigens. The vaccination with strate (Sigma-Aldrich). The reaction was stopped by 0.16 M sulfuric acid solution.
113-O12B/mTRP2180–188 in combination with anti–PD-1 The optical density at 450 nm was measured by BioTex microplate reader. The

PNAS 2022 Vol. 119 No. 34 e2207841119 https://doi.org/10.1073/pnas.2207841119 9 of 10

 endpoint titer is defined as the reciprocal of the highest dilution of a serum that anti–IFN-γ antibody and then streptavidin-alkaline phosphatase conjugate
gives a reading above the cutoff (four times the PBS group). according to the manufacturer’s protocol. The pictures were taken, and spot nos.
of each mouse were calculated automatically.
Intracellular Cytokine Staining. A total of 2 × 106 spleen cells or PBMCs
were isolated and suspended in 200 μL RPMI-1640 medium containing 10% fetal Immunization and Tumor Therapy. To establish B16F10-OVA tumor model,
bovine serum. GolgiPlug protein transport inhibitor (BD Biosciences) was added to 1 × 106 B16F10-OVA cells were injected SC at the right flank of C57/BL6 mice
inhibit the intracellular trafficking of cytokines. The cells were then stimulated with (4–6 wk old, n = 5) on day 0. The mice received two doses of vaccination at the
respective peptides at 2 μg/mL for 6 h. Then the cells were washed by flow cytom- dose equivalent to 50 μg active lipid and 5 μg OVA mRNA on days 5 and 12.
etry staining buffer (eBioscience) and then incubated with fluorescent antibodies Mice without any treatment are used as control group. For one group vaccinated
against surface markers at 4 °C for 1 h. The cells were washed and fixed by Fixa- by 113-O12B/mOVA, the mice were also treated with anti–PD-1 on days 7, 11,
tion Buffer (Biolegend) in the dark for 20 min at room temperature. The fixed cells and 15. The length (L) and width (W) of the tumors were measured every other
were washed with Intracellular Staining Perm Wash Buffer (Biolegend) twice and day. The tumor volumes (Vs) were calculated by the equation: V = L × W2/2. On
labeled with an optimum concentration of fluorophore-conjugated antibodies of day 30, the mice without obvious tumors were rechallenged with 1 × 106
interest (e.g., APC anti–IFN-γ and APC anti-FoxP3) for 20 min in the dark at room B16F10-OVA cells by IV injection. A control group (n = 5) was also treated with
temperature. After washing two more times, the cells were measured by Attune 1 × 106 B16F10-OVA cells by IV injection. On day 48, all the mice were killed
NxT Flow Cytometer. Data were analyzed by FlowJo-10. Gating information is and the lungs were collected for photograph. For regular B16F10 tumor model,
shown in SI Appendix, Fig. S4. all the protocols were the same as those of B16F10-OVA model except for the
does, which was equivalent to 30 μg active lipid and 3 μg TRP2180–188 mRNA.
Tumor Immune Cell Composition Experiment. C57/BL6 mice (4–6 wk old)
were inoculated with 1 × 106 B16F10-OVA cells at the right flank on day 14. Statistics. Statistical analysis was performed using Tukey’s multiple compari-
On days 0 and 5, LNPs formulated containing 50 μg active lipid and 5 μg OVA sons test in GraphPad Prism version 9.0.0 (GraphPad Software). *P < 0.05 was
mRNA were injected SC at tail base as the prime and boost vaccination. More- considered statistically significant. **P < 0.01 and ***P < 0.001 were consid-
over, one of the 113-O12B/mOVA-vaccinated groups was treated with anti–PD-1 ered highly significant.
antibody on days 2 and 7. Tumors were collected on day 12 and suspended into
cells using 70 μm cell strainer (Corning, USA). Then 2 × 106 cells were stained Data, Materials, and Software Availability. All study data are included in
with fluorophore-conjugated antibodies listed in SI Appendix, Table S2 at 4 °C the article and/or SI Appendix.
for 1 h. Then the cells were washed and analyzed by Attune NxT Flow Cytometer.
ACKNOWLEDGMENTS. We acknowledge support from NIH Grant R01 EB027170-
Gating information is shown in SI Appendix, Fig. S5.
01. We acknowledge Jennifer M. Khirallah for reviewing the manuscript.
ELISpot Assay. One week after the second vaccination, PBMCs were isolated
and suspended in 200 μL RPMI-1640 medium containing 10% fetal bovine
serum. The ELISpot assay was conducted using the Mouse Interferon gamma ELI- Author affiliations: aDepartment of Biomedical Engineering, Tufts University, Medford,
SPOT Kit (ab64029, Abcam, USA). Then 2 × 104 PBMCs cells were incubated in MA 02155; and bGuangdong Provincial Key Laboratory of Malignant Tumor Epigenetics
complete RPMI-1640 medium with or without 2 μg/mL of SIINFEKL peptide at and Gene Regulation, Guangdong–Hong Kong Joint Laboratory for RNA Medicine,
Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University,
37 °C for 12 h. Then the plates were washed and incubated with biotinylated Guangzhou 510120, China

1. C. B. Creech, S. C. Walker, R. J. Samuels, SARS-CoV-2 vaccines. JAMA 325, 1318–1320 (2021). 18. D. Loughrey, J. E. Dahlman, Non-liver mRNA delivery. Acc. Chem. Res. 55, 13–23 (2021).
2. Z. Zhao et al., New insights from chemical biology: Molecular basis of transmission, diagnosis, and 19. H. Jiang, Q. Wang, X. Sun, Lymph node targeting strategies to improve vaccination efficacy.
therapy of SARS-CoV-2. CCS Chemistry 3, 1501–1528 (2021). J. Control. Release 267, 47–56 (2017).
3. N. Pardi, M. J. Hogan, D. Weissman, Recent advances in mRNA vaccine technology. Curr. Opin. 20. K. Thapa Magar, G. F. Boafo, X. Li, Z. Chen, W. He, Liposome-based delivery of biological drugs.
Immunol. 65, 14–20 (2020). Chin. Chem. Lett. 33, 587–596 (2022).
4. Z. Jahanafrooz et al., Comparison of DNA and mRNA vaccines against cancer. Drug Discov. Today 21. D. Yin, M. Zhang, J. Chen, Y. Huang, D. Liang, Shear-responsive peptide/siRNA complexes as
25, 552–560 (2020). lung-targeting gene vectors. Chin. Chem. Lett. 32, 1731–1736 (2021).
5. C. Fan et al., Cancer/testis antigens: From serology to mRNA cancer vaccine. Semin. Cancer Biol. 22. M. Qiu et al., Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves
76, 218–231 (2021). liver-specific in vivo genome editing of Angptl3. Proc. Natl. Acad. Sci. U.S.A. 118, e2020401118
6. N. Pardi, M. J. Hogan, F. W. Porter, D. Weissman, mRNA vaccines—A new era in vaccinology. Nat. (2021).
Rev. Drug Discov. 17, 261–279 (2018). 23. N.-N. Zhang et al., A thermostable mRNA vaccine against COVID-19. Cell 182, 1271–1283.e16
7. J. Chen, J. Chen, Q. Xu, Current developments and challenges of mRNA vaccines. Annu. Rev. (2020).
Biomed. Eng. 24, 85–109 (2022). 24. M. Wang et al., Efficient delivery of genome-editing proteins using bioreducible lipid
8. K. Kariko et al., Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 113, 2868–2873 (2016).
with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008). 25. M. Qiu et al., Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of
9. A. Wittrup, J. Lieberman, Knocking down disease: A progress report on siRNA therapeutics. Nat. pulmonary lymphangioleiomyomatosis. Proc. Natl. Acad. Sci. U.S.A. 119, e2116271119 (2022).
Rev. Genet. 16, 543–552 (2015). 26. O. Dienz, M. Rincon, The effects of IL-6 on CD4 T cell responses. Clin. Immunol. 130, 27–33
10. K. H. Moss, P. Popova, S. R. Hadrup, K. Astakhova, M. Taskova, Lipid nanoparticles for delivery of (2009).
therapeutic RNA oligonucleotides. Mol. Pharm. 16, 2265–2277 (2019). 27. K. Yoshida et al., Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells. BMC Cancer
11. M. A. Maier et al., Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for 20, 25 (2020).
systemic delivery of RNAi therapeutics. Mol. Ther. 21, 1570–1578 (2013). 28. C. Yunna, H. Mengru, W. Lei, C. Weidong, Macrophage M1/M2 polarization. Eur. J. Pharmacol.
12. K. J. Hassett et al., Optimization of lipid nanoparticles for intramuscular administration of mRNA 877, 173090 (2020).
vaccines. Mol. Ther. Nucleic Acids 15, 1–11 (2019). 29. T. Yamano, Y. Kaneda, S. Huang, S. H. Hiramatsu, D. S. B. Hoon, Enhancement of immunity
13. D. Zhi et al., Transfection efficiency of cationic lipids with different hydrophobic domains in gene by a DNA melanoma vaccine against TRP2 with CCL21 as an adjuvant. Mol. Ther. 13, 194–202
delivery. Bioconjug. Chem. 21, 563–577 (2010). (2006).
14. K. Fitzgerald et al., Effect of an RNA interference drug on the synthesis of proprotein convertase 30. E. A. Vasievich, S. Ramishetti, Y. Zhang, L. Huang, Trp2 peptide vaccine adjuvanted with (R)-DOTAP
subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy inhibits tumor growth in an advanced melanoma model. Mol. Pharm. 9, 261–268 (2012).
volunteers: A randomised, single-blind, placebo-controlled, phase 1 trial. Lancet 383, 60–68 31. Y. Li et al., Multifunctional oncolytic nanoparticles deliver self-replicating IL-12 RNA to eliminate
(2014). established tumors and prime systemic immunity. Nat. Can. 1, 882–893 (2020).
15. X. Hou, T. Zaks, R. Langer, Y. Dong, Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6, 32. K. J. Kauffman et al., Efficacy and immunogenicity of unmodified and pseudouridine-modified
1078–1094 (2021). mRNA delivered systemically with lipid nanoparticles in vivo. Biomaterials 109, 78–87 (2016).
16. M. Alden et al., Intracellular reverse transcription of Pfizer BioNTech COVID-19 mRNA vaccine 33. S. Linares-Fernandez, C. Lacroix, J.-Y. Exposito, B. Verrier, Tailoring mRNA vaccine to balance
BNT162b2 in vitro in human liver cell line. Curr. Issues Mol. Biol. 44, 1115–1126 (2022). innate/adaptive immune response. Trends Mol. Med. 26, 311–323 (2020).
17. T. Boettler et al., SARS-CoV-2 vaccination can elicit a CD8 T-cell dominant hepatitis. J. Hepatol. 34. R. G. Majzner, C. L. Mackall, Tumor antigen escape from CAR T-cell therapy. Cancer Discov. 8,
10.1016/j.jhep.2022.03.040. (2022). 1219–1226 (2018).

10 of 10 https://doi.org/10.1073/pnas.2207841119 pnas.org