Abstract
INTRODUCTION: Lack of standard treatment of Primary Central Nervous System Lymphoma (PCNSL) was acknowledged in phase III intergroup study (HOVON 105/ALLG NHL 24). PCNSL incidence is increasing for unknown reasons, particularly among persons over 65 years. One of the major limitations of successful treatment of PCNSL is the blood-brain barrier (BBB), which prevents drug delivery into the brain. METHODS: Polymer nanoconjugate based on biodegradable poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA) was used for treatment of BALB/C mice with intracranially inoculated murine A20 lymphoma. PHPMA-based nanoconjugates have defined synthesis strategy for BBB transcytosis, tumor targeting, and cancer cell killing. Nano immunodrug PHPMA/AP-2/Fab’/c-Myc inhibitor/H6 conjugate contains multiple AP-2 peptides for efficient delivery across the BBB and Fab’ fragments of αCD20 mouse Ab for CD20 receptor crosslinking on PCNSL cells. c-Myc inhibitor bound via a disulfide bond is transferred to the cytoplasm followed by cleavage. The therapeutic efficacy and survival were evaluated together with spectral flow cytometry and RNA-seq bioinformatic analysis of tumor tissues. RESULTS: Nanoconjugates were able to penetrate BBB and accumulate in brain parenchyma. The best survival results were obtained for the group where nanoconjugate with αCD20 Fab’ causing tumor cell apoptosis and c-Myc antisense inhibitor was combined with αPD-1 checkpoint inhibitor. Survival compared to PBS-treated controls was significant (P=0.0006 by ANOVA). Ex vivo analysis of A20 brain lymphoma tissue after treatment with nanoconjugates demonstrated reduction of Tregs, associated with downregulation of IL-10 and IL-1β in plasma. Spectral flow cytometry revealed activation of tumor infiltrating T lymphocytes and M1 macrophages in lead nanodrug treated groups. RNA-seq data correlated with flow cytometry results indicating the activation of genes linked to IFN-γ pathway important for anti-tumor response and M1 macrophage activation. Immunostaining confirmed a marked increase of M1-specific iNOS expression in the nanodrug-treated tumors. Support: NIH grants: R01 CA188743, R01 CA206220, R01 CA209921