Abstract 2869: Nano-immuno combination therapy to treat brain primary central nervous system lymphoma
Authors: Sun Tao , Shatalova Ekaterina , Braubach Oliver , Klymyshyn Dmytro , Holler Eggehard , Wang Jiawei , Li Lian , Yang Jiyuan , Kopecek Jindrich , Ljubimova Julia Y. 
Published: September 2020

Abstract
Purpose: 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 of age. The major limitation of successful treatment of PCNSL is the blood-brain barrier (BBB), which prevents drug delivery into the brain. Our novel treatment employs nano immunoconjugates (NICs) that cross BBB and activate brain lymphoma immunity. These NICs specifically block c-Myc expression and induce CD20 crosslinking. The NICs, in combination with αPD1, were systemically delivered to the lymphoma mice and this treatment led to a local and systemic broad-spectrum immune response by activation of IFN-γ that is an important anti-tumor M1 macrophage activator.
Methods: Polymer nanoconjugate based on biodegradable poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA)-P 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 P/AP-2/CD20 Fab'or c-Myc inhibitor (anti-sense oligonucleotides)/H6 conjugate contains multiple AP-2 peptides for efficient delivery across the BBB and Fab' fragments of αCD20 Ab (mouse specific) for CD20 receptor crosslinking on PCNSL cells. c-Myc inhibitor bound via a disulfide bond is transferred to the cytoplasm followed by cleavage; this is facilitated by (His)6 peptide-mediated endosome disruption. The nanoconjugate was examined for the ability to cross BBB in vivo by live imaging. The therapeutic efficacy and survival were evaluated together with spectral flow cytometry (FC) and RNA-seq bioinformatic analysis of tumor tissues.
Results: NIC was able to penetrate BBB and accumulate in brain parenchyma. The best survival results were obtained from the combination groups with CD20 Fab' crosslinking CD20 receptors resulting in apoptosis and c-Myc antisense inhibition in combination with αPD-1 checkpoint inhibitor. Survival compared to PBS-treated controls was significant (P=0.0006, P=0.0034, respectively, by ANOVA). FC analysis of A20 brain lymphoma tissue after treatment with NICs 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 the M1 macrophages in lead treatment 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.
Conclusion: The PCNSL treatment results demonstrate for the first time that the BBB delivery of NICs modified brain tumor microenvironment and activated brain local immune system though modulation of T cells and IFN-γ pathway. Support: NIH grants: R01 CA188743, R01 CA206220, R01 CA209921
Citation Format: Tao Sun, Ekaterina Shatalova, Oliver Braubach, Dmytro Klymyshyn, Eggehard Holler, Jiawei Wang, Lian Li, Jiyuan Yang, Jindrich Kopecek, Julia Y. Ljubimova. Nano-immuno combination therapy to treat brain primary central nervous system lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2869.