Developing a Polymeric Nanoparticle Vaccine for Co-delivery of

12 Synergistic Adjuvants and “Mix and Go” Cancer Neoantigens

Jessalyn Baljon1, Daniel Shae2, Mohamed Wehbe2, Lihong Wang-Bishop2, Kyle Becker2, John T. Wilson1,2
1Department of Biomedical Engineering 2Department of Chemical and Biomolecular Engineering

Background and Introduction NanoSTING-vax Activates Dendritic Cells and T Cells “Mix and Go” Platform
A CD86 B CD40 C MHC-II D SIINFEKL-MHC-I E T Cell Activation 80 EE (%)
A B C
• Immune checkpoint blockade (ICB) has revolutionized cancer immunotherapy but is 8500
****
1500
****
7500 10000
****
0.40 Trp2 15 Empty NP

MFI (SIINFEKL-H-2Kb)
MFI (MHC-II APC/Cy7)
**** **** *** **** 60 Reps1 NP + 5 mol% lipid-peptide

MFI (CD86-PE/Cy7)
not effective in all patients. **** **** **** ****

Intensity (%)
M30

MFI (CD40-FITC)
6500 8000 **** NP + 10 mol% lipid-peptide
**** ** **** ** **** Adpgk 10
**** 40

A570 (AU)
** ns
**** ns NP + 20 mol% lipid-peptide
• Patients with tumors that have minimal T cell infiltration often do not respond to ICB. 4500 1000 4000 ns
****
6000 0.25
***
M27 20 OVA 5

• Cancer vaccines can increase the number of cancer-specific T cells and make ICB 2500 4000 R=0.9429 0
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

10

00

00
work better.

0.

10

00
500 500 500 2000 0.10 Hydrophobicity

10
Diameter (nm)
There are 2 main components to a cancer vaccine: nanoSTING-vax NP-cGAMP + Peptide NP-Peptide cGAMP + Peptide Peptide Untreated Figure 7. Lipidated peptides can insert into the nanoparticle. (A) Hydrophobic neoantigen peptides are
encapsulated with higher efficiency into the nanoparticle, motivating the lipidation and insertion of all peptides.
Neoantigen: Tumor specific proteins that arise from tumor mutations. Not expressed Figure 3. In vitro dendritic cell and T cell activation. Treatment with nanoSTING-vax enhances the expression of co-stimulatory markers CD86
(B) Overview of lipid insertion. (C) Amount of peptide inserted does not affect nanoparticle size.
(A) and CD40 (B), as well as MHC-II (C) on bone marrow derived dendritic cells (BMDCs). NanoSTING-vax also enhances the presentation of
on healthy cells. the antigen SIINFEKL on MHC-I (D) and the ability of BMDCs to activate T cells via a B3Z assay (E).
Adjuvant: Activates an innate immune response to provide dendritic cell co-stimulation
and cytokine secretion. We will be using cGAMP which activates the stimulator of
A B 50 C D Reps1 Adpgk
Using Adjuvant Combinations

% Tetramer+ CD8+ T cells

*** 15 **** 5

% IFNγ+TNFα+ CD8+ T cells

40 ** ****

% IFNγ+TNFα+ CD8+ T cells

***
interferon genes (STING) pathway. p=0.059 p=0.15
4 *
** A CD86 Expression B 2000000
Interferon Activation
0.3
NF-κB Activation

Relative Luminescence
30 25000

Relative Absorbance
10
These components need both need to delivered into the cytosol, using a pH responsive 20
3 20000 1500000
0.2

polymersome: 2
15000 1000000

MFI
10 5 10000 0.1
500000
A B 1
5000
0
0 0.0
nanoSTING-vax NP-cGAMP+Peptide 0 0 0 cGAMP: 10 100 1000 10000 100000 cGAMP: 10 100 1000 10000 100000
MPLA: 5.73 57.3 573 5730 57300 MPLA: 5.73 57.3 573 5730 57300
cGAMP + Peptide Peptide NT cGAMP MPLA cGAMP+MPLA
nanoSTING-vax NP-cGAMP + Peptide cGAMP + Peptide Peptide Dose (ng/mL) Dose (ng/mL)
CpG cGAMP+CpG R848 cGAMP+R848
Figure 4. NanoSTING-vax increases the number of antigen-specific T cells in vivo. (A) Dosing scheme. (B) NanoSTING-vax increases the cGAMP Polymersomes MPLA Polymersomes cGAMP + MPLA Polymersomes
number of SIINFEKL-specific CD8+ T cells measured using tetramer staining. (C) Dosing Scheme. (D) NanoSTING-vax increases the number of C D E F
1500 100
Reps1 and Apgdk activated T cells measured using intracellular cytokine staining. 25

Tumor volume(mm3)
*

Percent survival
% Tetramer Positive
75
20 1000
*

NanoSTING-vax Improves Response to ICB

15 50

10 500
25
5 **
0 0
A B C D 0 0 4 8 12 16 20 24 0 10 20 30 40 50
1500 100 100 Days After Inoculation Days After Inoculation
** PBS NP-cGAMP-Pep NP-cGAMP-MPLA-Pep

Tumor Volume (mm3)

80
75 **

Percent survival
Percent survival
1000 Figure 8. Multiple adjuvants enhance the immune response. (A) Using multiple adjuvants enhance CD86
60 expression on BMDCs. (B) Nanoparticles loaded with both cGAMP and MPLA have enhanced interferon and NF-κB
50
* activation. (C) Dosing scheme for mice vaccinated with nanoSTING-vax with or without MPLA. Mice vaccinated with
40
500 nanoSTING-vax that also has MPLA loaded have more SIINFEKL-specific CD8+ T cells (D), delayed B16.OVA tumor
25 growth (E), and improved survival (F).
20
Figure 1. (A) cGAMP and tumor neoantigens are encapsulated in a pH-responsive polymeric nanoparticle, known as nanoSTING-vax.
(B) NanoSTING-vax is endocytosed. In the low pH of the endosome the cGAMP and peptide are delivered to the cytosol where the 0 0 0
cGAMP activates STING and the peptide is processed and presented on MHC class I molecules.
10 15 20 25
Days Post-Inoculation
30 20 30 40 50
Days Post-Inoculation
0 10 20 30 40 50
Days Post-Rechallenge Conclusions
PBS cGAMP+Peptide αPD-1 cGAMP+Peptide+αPD-1 Naïve mice
• Dual delivery of cGAMP and neoantigen peptides generates CD8+ T cell
NanoSTING-vax Improves Lymph Node Accumulation
NP-cGAMP+αPD-1 nanoSTING-vax+αPD-1 Rechallenged mice
nanoSTING-vax
responses that improves response to immune checkpoint blockade.
Figure 5. MC38 Tumor Challenge. (A) Dosing scheme of MC38 tumor challenge. (B) Tumor growth curve shows nanoSTING-vax + ICB (αPD-1) • Incorporating “mix and go” technologies and synergistic adjuvants will allow
inhibits tumor growth the most. (C) Survival curve shows 5/7 complete responders with nanoSTING-vax + ICB. (D) Survival of mice re-challenged
A B C with MC38 tumors shows immunological memory.
for a more effective and translatable personal cancer vaccine.
A B C
Acknowledgements
1500 100 nanoSTING-vax + αPD-1/αCTLA-4
nanoSTING-vax
Tumor Volume (mm3)

nanoSTING-vax

Percent survival
1000
cGAMP+Peptide NP-cGAMP + αPD-1/αCTLA-4
50
cGAMP + Peptide Kyle Becker, Carcia Discovery Grant
Program

500 ***
cGAMP + Peptide + αPD-1/αCTLA-4
Carson, Jessalyn Baljon,
PBS *
αPD-1/αCTLA-4
Lihong Bishop, Moe
0 0
Wehbe, Kyle Garland,
PBS
0 5 10 15 20 25 30
Days Post-Inoculation
0 20 40 60
Days Post-Inoculation
80
Christian Palmer, Lucinda
Figure 2. NanoSTING-vax size allows for increased lymph node accumulation. (A) Dynamic light scattering of nanoSTING-vax indicates
Pastora, Taylor Sheehy
Figure 6. B16.F10 Tumor Challenge. (A) Dosing scheme of B16.F10 tumor challenge. (B) Tumor growth curve shows nanoSTING-vax + ICB
the nanoparticles are ~100-200nm in diameter. (B) NanoSTING-vax increases the lymph node accumulation of peptide and cGAMP injected
(αPD-1/αCTLA-4) inhibits tumor growth the most. (C) Survival curve shows 2/6 complete responders with nanoSTING-vax + ICB.
subcutaneously, as measured by IVIS. (C) Quantification of the IVIS images in B. Shae, D*; Baljon, JJ*; et al. Co-delivery of Peptide Neoantigens and Stimulator of Interferon Genes Agonists
Enhances Response to Cancer Vaccines. ACS Nano, 2020, 14, 9904-9916.