Poster X5 - IL-12 mRNA mediated cancer immunotherapy augmented by IFNγ-IDO axis inhibiting lipid nanoparticle

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

HP

Heewon Park, PhD

Postdoctoral Researcher
KAIST
Daejeon, Republic of Korea

Co-Author(s)

IK

In Kang

Ph.D course
KAIST
Daejeon, Republic of Korea
Susam Lee, PhD

postdoctoral researcher
Korea Advanced Institute of Science and Technology (KAIST)
Daehak-ro, Yuseong-gu, United States
SK

Seungcheol Kim

Graduated student
KAIST, United States
MP

Minsa Park, MS (she/her/hers)

Researcher
Gyeongsang National University
Jinju, Kyongsang-namdo, Republic of Korea
HN

Hoyeon Nam

Ph.D. course
KAIST
Daejeon, Republic of Korea
KL

Kyuri Lee

Professor
Gyeongsang National University
Jinju, Republic of Korea
YL

Yong-kyu Lee

Professor
Korea National University of Transportation, United States
HL

Heung Kyu Lee, PhD

Professor
KAIST
Daejeon, Republic of Korea

Primary Investigator(s)

YK

Yeu-Chun Kim

Professor
KAIST, United States

Introduction: Administration of recombinant cytokines, specifically interleukin-12 (IL-12), holds promising clinical potential at anticancer research field. Sustained intra-tumoral delivery of IL-12 can restore tumor resident CD8+ effector T cells and can induce priming of antitumor CD8+ effector T cells. However, these CD8+ T cell dependent anticancer efficacy is usually transient and accompany immune suppressive CD4+Foxp3+ T regulatory cells. Underlying mechanism of T regulatory cell expansion in IL-12 therapy is IFNγ dependent indoleamine 2,3-dioxygenase (IDO) expression. Due to this negative feedback, combinatorial treatments should be considered to enhance efficacy of IL-12 mediated therapy. Herein, we designed lipid nanoparticle (LNP) system which can successfully deliver IDO inhibitor indoximod (IND) prodrug and IL-12 encoding mRNA. Incorporation molar ratio of IND prodrug toward LNP was optimized by in vitro and in vivo transfection ability. Optimized LNP and IND prodrug encapsulating LNP (IDOi LNP) successfully transfected tumor cells and macrophage, resulting secretion of IL-12 cytokine. With IL-12 transfection, macrophage upregulated T cell co-stimulation factor and released TNF alpha cytokine, indicating tumor microenvironment can be changed from cold tumor to hot tumor for immunotherapy. Also, by elevated IL-12 cytokine, T cell released high level of IFNγ, which act as central role in IL-12 mediated therapy. Furthermore, internalized IDOi LNP release IND prodrug in tumor cell, resulting successful inhibition of IDO pathway. This co-delivery system presents a promising strategy to overcome the limitations of single IL-12 mediated therapy by simultaneously promoting antitumor immune responses and inhibiting immunosuppressive mechanisms, thereby enhancing the overall efficacy of cancer immunotherapy.

Materials and

Methods: (1) Synthesis of Indoximod prodrug
Carboxylic acid functional group of indoximod was covalently conjugated with cholesterol via ester linkage. Chemical synthesis was confirmed through 1H-NMR and HR-MS analysis.
(2) Preparation of IDO inhibiting lipid nanoparticle
Indoximod-cholesterol conjugates was incorporated into lipid nanoparticle via altering cholesterol compound. Incorporation molar ratio was optimized by in vitro & in vivo luciferase transfection experiment.
(3) In vitro IL-12 mRNA transfection efficiency evaluation
Transfection of IL-12 mRNA encapsulating LNP was verified at several cell line (B16F10, MC38 and RAW264.7). By intracellular cytokine staining, IL-12 p40 positive cells were analyzed by flow cytometry. Also secreted IL-12 p70 protein was quantitatively analyzed by ELISA method.
(4) In vitro IL-12 mediated immune modulation evaluation
Activation of macrophages (RAW264.7) and T cells (Spleenocyte) were measured after transfection of IL-12 mRNA encapsulating lipid nanoparticle. Upregulation of CD40, CD80 and CD86 were evaluated by flow cytometry analysis. Spleenocytes were freshly isolated, followed by stimulation with LNP treated tumoral supernatant. After 24 hr stimulation, IFN gamma level in supernatant was detected by ELISA.
(5) In vivo antitumor efficacy test at B16F10 model.
B16F10 xenograft tumor model was established by inoculation of B16F10 cell line at C57BL/6 right flank. When tumor reaches 70~100 mm3, LNP experimental groups which carrying 4 ug of mRNA were intratumorally injected for 4 times. Tumor volume was monitored every other day and survival curve was obtained upon 40 days. For immune analysis, tumor tissue was isolated, homogenized and analyzed by flow cytometry in terms with macrophages T cells.

Results, Conclusions, and Discussions: This study successfully demonstrated the synthesis and characterization of the indoximod (IND) prodrug, Cholesterol-Indoximod, and its incorporation into lipid nanoparticles (LNPs). The prodrug, confirmed through HR-MS and 1H-NMR analysis, exhibited enhanced solubility in ethanol, facilitating its encapsulation into LNPs using a microfluidic device. LNP formulations with varying cholesterol and IND prodrug ratios were prepared, showing favorable biocompatibility and transfection efficiency. The IND prodrug-containing LNPs (IDOi LNP) exhibited enhanced mRNA transfection efficiency in vitro, likely due to improved endo-lysosomal escape mechanisms. In vivo studies revealed that while the IND prodrug enhanced transfection efficiency in tumor cells, its effectiveness varied in tumor tissues, suggesting stability issues post-injection. Subsequent experiments demonstrated that the IDOi LNP platform effectively delivered IL-12 mRNA to both tumor cells and macrophages, inducing significant cytokine production and immune cell activation. IL-12 transfection promoted the reprogramming of macrophages from an M2 to an M1 phenotype, and stimulated IFN-gamma production from splenocytes, which is crucial for anti-cancer immunity. Furthermore, in vivo studies in a B16F10 xenograft model confirmed the antitumor efficacy of IL-12 mRNA-encapsulating IDOi LNPs, showing reduced tumor growth and increased survival rates. The synergistic effect of IL-12 mRNA delivery and IDO pathway inhibition was validated through phenotypic analysis of immune cells within the tumor microenvironment. Enhanced CD8+ T cell activation and increased cytokine production were observed, highlighting the potential of the IDOi LNP platform for effective cancer immunotherapy. This study underscores the versatility and clinical relevance of the developed HLNP platform for delivering therapeutic mRNA in cancer treatment.

Acknowledgements (Optional):