(T1430-04-23) Boosting Checkpoint Immunotherapy with 2nd Generation HPMA Polymer Conjugates with Synchronous PD-L1 Attenuation

Purpose: Over recent decades, immune checkpoint inhibitors (ICIs) have revolutionized the treatment landscape for various cancers, and became a standard component of oncological protocol. However, a significant portion of cancer patients exhibit poor responses to ICI therapy. In patients with "hot" tumors—characterized by an immune-inflamed cancer environment—there is generally a more favorable response to ICIs, marked by significant lymphocyte infiltration within the tumor environment. In contrast, "cold" tumors typically elicit a poor response to ICIs, with these tumors being distinguished by low levels of T-cell infiltration and other immune-active cells [1]. Previously, we demonstrated that the 2^nd generation long-circulating N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-epirubicin conjugate (KT-1) that elicits immunogenic cell death to “heat up“ immunologically cold tumors, in combination with a multivalent polymer-peptide anti-PD-L1 antagonist (MPPA) which utilizes receptor crosslinking as a molecular switch and shunts PD-L1 from recycling to lysosomal degradation, enhanced 4T-1 murine triple-negative breast cancer (TNBC) model and other ‘cold‘  tumor microenvironment responsiveness [2]. This study aims to refine the KT-1/MPPA chemo-immunotherapy dosing regimen to optimize treatment efficacy. Furthermore, we employ single cell mRNA sequencing to dissect the intricate changes within the tumor microenvironment, providing a more detailed understanding of the cellular and molecular mechanisms influenced by our therapeutic strategy.

Methods: KT-1 and MPPA were synthesized as previously described [2]; their structures are shown in Figure 1A,B. Our study primarily utilized the B16F10 melanoma model in C57BL/6 mice, alongside with the 4T1. Both KT-1 and MPPA were administered either simultaneously or sequentially via intravenous injection, and their efficacy was evaluated according to National Cancer Institute standards [3]. To assess KT-1's impact on the tumor microenvironment, tumors were harvested at specific intervals for single-cell suspension and analysis. Calreticulin (CRT), high mobility group box 1 protein (HMGB1), and PD-L1 levels were measured using commercial assays and flow cytometry, with PD-L1 further validated by Western blot. Following the final treatment, mice were euthanized three days later, and tumor tissues were dissociated using the Miltenyi Biotec Mouse Tumor Dissociation Kit for single-cell suspensions. These cells were stained and analyzed by flow cytometry. Additionally, for single-cell mRNA sequencing, CD45-positive cells were isolated and sequenced using 10x Genomics technology.

Results: In the B16F10 melanoma mouse model, we evaluated various dosing strategies with treatments administered every two days. The concurrent KT-1 and MPPA regimen demonstrated the most favorable outcomes, significantly reducing tumor volumes (Figure 2A,B). Additionally, surface CRT levels, an immunogenic cell death marker, remained elevated up to 10 days post-treatment, indicating prolonged circulation of KT-1 (Figure 2C). A significant increase of HMGB1 release was detected following KT-1 treatment, with no marked difference between chemotherapy alone and the combination with immunotherapy (Figure 2D). Interestingly, KT-1 treatment significantly elevated PD-L1 expression, but administering MPPA concurrently or consecutively reduced PD-L1 levels (Figure 2E-F). Analysis of activated dendritic cells highlighted the concurrent regimen as most effective, prompting its selection for further studies due to minimal tumor volume (Figure 2G). Treatments led to a notable increase in CD45+ cells, particularly in the KT-1 and MPPA combination group (Figure 3A). CD8+ cytotoxic subtypes, were significantly enhanced in the treated groups, suggesting effective recruitment of tumor-infiltrating T cells (Figure 3C-E). However, variations in the myeloid lineage, including dendritic cells, macrophages, and myeloid-derived suppressor cells (MDSCs), precluded definitive conclusions (Figure 3F-H). Single-cell mRNA sequencing further corroborated these findings, showing a significant increase in activated CD8 T cells in the combination group, aligning with flow cytometry data. The MDSC population notably decreased, indicating a shift from a tumor-suppressive to an immune-activating environment, particularly in the combination group (Figure 3I). Finally, gene expression analysis indicated upregulation of CD8-related genetic markers and downregulation of tumor-migration-related genetic markers in the combination group, contrasting with the cold tumor microenvironment maintained in the saline group (Figure 3J).

Conclusion: Our study has successfully identified an efficacious treatment regimen for the B16F10 melanoma mouse model, and corroborated findings previously observed in our 4T1 TNBC model. In both models, the combination treatment of KT-1 and MPPA significantly improved outcomes. The initial treatment with KT-1 alone led to the recruitment of tumor-infiltrating T cells to the tumor sites, decelerating tumor growth and "heating" the tumor microenvironment—a crucial step towards transforming these typically unresponsive tumors. The combination of KT-1 and MPPA significantly boosted the activity of T cells, thereby enhancing the overall treatment efficacy. This dual approach not only reactivates cold tumors but also markedly improves the effectiveness of immune checkpoint inhibitors, offering a promising pathway for future therapeutic strategies.

References: [1] P. Ouyang et al., Front. Immunol. 15 (2024) 1344272.
[2] L. Lian et al., Adv. Funct. Mater. 30 (2020) 1908961.
[3] J. Deckert, Clin. Cancer Res. 20 (2014) 4574.

Acknowledgements: Supported by Department of Defense grant W81XWH-20-1-0573 and Huntsman Cancer Institute PCD Program Award (to JY).


Figure 1. Combination chemotherapy and immunotherapy for cancer treatment using a backbone-degradable N-(2-hydroxypropyl)methacrylamide (HPMA) – epirubicin conjugate (KT-1) and multivalent HPMA copolymer – peptide PD-L1 antagonist (MPPA). (A) Structure of KT-1 conjugate. (B) Structure of MPPA. (C) Schematic illustration comparing cold and hot tumors, and depiction of the role of conjugates in modulating the tumor microenvironment.


Figure 2. Anti-tumor effects of KT-1 and MPPA in B16F10-bearing C57BL/6 mice. (A) Tumor volume over time in mice treated with KT-1 alone, KT-1 + MPPA concurrently, and KT-1 + MPPA sequentially. (B) Statistical analysis of tumor growth inhibition using the T/C ratio. (C) Surface CRT levels in melanoma tumors after KT-1 treatment. (D) HMGB1 levels in melanoma tumors following treatment with KT-1 alone and in combination with MPPA. (E) PD-L1 levels in melanoma tumors post-treatment with KT-1 and the combination of KT-1 and MPPA. (F) Validation of PD-L1 levels in melanoma tumors by Western blot analysis. (G) Analysis of activated dendritic cells by flow cytometry.


Figure 3. Analysis of the tumor microenvironment using flow cytometry and single-cell mRNA sequencing. (A) CD45+ immune cell population. (B) Tumor-infiltrating B cells. (C) CD3+ T cells. (D) CD8+ cytotoxic T cells. (E) CD4+ helper T cells. (F) Dendritic cells. (G) Macrophages. (H) Myeloid-derived suppressor cells (MDSCs). (I) UMAP plot showing activated CD8 T cells and MDSCs across various treatment groups. (J) Expression levels of specific marker genes.