CD33 Antibody-Modified Nanoparticles Encapsulated IL-15 Induce ISG15 to Promote MHC I Expression in Leukemia Cells Via Innate Immunity Pathway Inducing Graft-Versus-Leukemia without Aggravating Graft-Versus-Host Disease
Authors: Wei Ruowen , Zhang Ao , Wang Moran , Cao Xie-Na , Zhu Ruiqi , Ma Shengling , Xia Linghui , Shi Wei 
Published: December 2024

Background
Disease recurrence is a major complication after allogeneic hematopoietic stem cell transplantation in AML. Inducing graft-versus-leukemia (GVL) effect can control recurrence but often aggravates graft-versus-host disease (GVHD). Previous studies showed high IL-15 expression in AML cells relates to GVL, but the mechanism remains unclear. We aimed to develop an AML cell-targeted IL-15 delivery system to induce GVL without exacerbating GVHD.
Methods
We constructed PEG-PLGA-COOH nanoparticles encapsulating human recombinant IL-15 protein (IL15NP) and conjugated anti-CD33 antibodies to their surface (CD33-IL15NP) using EDC-NHS coupling. Nanoparticle uptake, IL-15 expression, and HLA-ABC changes in AML cell lines (OCI-AML3, MOM-13, HL60, MV4-11) were assessed by laser scanning confocal microscopy and flow cytometry. Proteomic and transcriptomic sequencing identified key genes and pathways involved in MHC class I upregulation. We created ISG15 knockdown cell lines using lentivirus and monitored proteasome subunit activities with different substrates. Specific proteasome subunit inhibitors (PR924, ML604440) were used to confirm regulatory roles. A humanized GVL NSG mouse model evaluated the nanoparticles' therapeutic potential in vivo, using 2Gy irradiation and transfusion of healthy donor peripheral blood stem cells with OCI-AML3 cells (NC or ISG15sh).
Results
IL15NP uptake by leukemia cells was concentration and time-dependent. IL-15 expression peaked at 12 hours post-administration, normalizing at 24 hours. AML cell lines showed significant HLA-ABC upregulation after 24-72h IL15NP co-culture, unlike with extracellular IL-15 protein. Proteomics revealed a strong correlation between MHC class I and ISG15, a ubiquitin-like protein linked to the ubiquitin-proteasome system. HLA-ABC upregulation was reversed in ISG15 knockdown cells across four AML cell lines. Transcriptomics showed upregulation of innate immune pathways (mTOR signaling, C-type lectin receptor signaling) related to ISG15 upregulation triggered by targeted IL-15 delivery.
Proteasome activity assays indicated significantly enhanced immunoproteasome activity (β1i, β5i) regulated by ISG15. Only combined use of β5i inhibitor (PR924) and β1i inhibitor (ML604440) reversed IL15NP-induced HLA-ABC upregulation. High concentrations of proteasome inhibitors showed strong anti-tumor effects but significantly downregulated MHC I molecules, suggesting excessive proteasome inhibition may lead to immune evasion due to MHC I loss.
In vivo imaging 21 days post-transplantation showed that for NSG mice with NC-OCI-AML3, systemic tumor progression in the IL15pro group (IL-15 protein-given) was comparable to the NS group (saline-given). IL15NP and CD33-IL15NP groups showed significantly reduced leukemia cells compared to NS and IL15pro groups. However, the IL15NP group had inferior survival due to severe GVHD. For NSG mice with ISG15sh-OCI-AML3, the IL15NP group showed significant tumor reduction compared to NS and CD33-IL15NP groups, but with severe systemic GVHD. Tumor progression in the CD33-IL15NP group was similar to NS despite targeted nanoparticle administration under situation of ISG15 knockdown.
Conclusion
Targeted IL-15 delivery induces ISG15 expression through innate immune pathway activation, enhancing immunoproteasome activity and upregulating MHC I molecules on AML cells. This strategy increases leukemia cell surface IL-15 and HLA-ABC expression, triggering GVL without severely increasing GVHD. Our study provides a novel approach for post-transplantation relapse treatment and establishes a foundation for clinical translation, suggesting that despite their malignant origin, leukemia cells retain innate immunity qualities.