Abstract 977: Nano immunotherapeutics crossing blood-brain barrier to activate local brain tumor immune system
Authors: Galstyan Anna , Chiechi Antonella , Sun Tao , Shatalova Ekaterina S. , Patil Rameshwar , Black Keith L. , Holler Eggehard , Ljubimov Alexander V. , Ding Hui , Ljubimova Julia Y. 
Published: August 2019

Abstract
Introduction: Glioblastoma multiforme (GBM) has limited treatment options. Checkpoint inhibitors anti-CTLA-4 and/or anti-PD-1 antibodies (Abs) cannot activate anti-tumor immune response in the brain because of their inability to cross blood-brain barrier (BBB) and modulate brain privileged immunity. A new generation of nano immunotherapeutics (NIT) that pass BBB and activate general and local brain tumor immune systems was developed.
Experimental procedures: BBB-crossing NIT were synthesized, based on the poly (β-L-malic acid) polymer (P), P/a-CTLA-4 Ab and P/a-PD-1 Ab, and a-mouse transferring receptor (a-msTfR) Ab or AP-2 peptide for delivery through BBB. Syngeneic GL261 glioma cells were intracranially inoculated into C57/BL mice. Six treatment groups received 5 I.V. injections of PBS, anti-PD1 and anti-CTLA4 Abs (controls), and polymers conjugated with anti-PD-1(P/a-PD-1), anti-CTLA-4 (P/a-CTLA-4) and a combination (P/a-CTLA-4+P/a-PD-1) at 10 mg/kg.
Summary of the data: Single NIT treatment (P/a-CTLA-4 or P/a-PD-1) improved brain tumor bearing mice survival vs. free a-CTLA-4 and a-PD-1 (p=0.0076 and 0.0017, respectively). The combination group (P/a-CTLA-4 + P/a-PD-1) showed the best efficacy for survival (p=0.0001). Flow cytometry (FC) analysis of T cell populations in the brain tumor revealed a reduction of total CD4+ T-cells in animals treated with P/anti-PD-1 and P/a-CTLA-4 + P/a-PD-1 combination vs. free anti-PD-1 Ab. The fraction of Tregs (CD4+FOXP3+) was reduced by all polymer treatments compared to free Abs. Activation of CD8+ T cells (CD8+IFN-γ+ and CD8+CD69+) was significantly increased by P/a-CTLA-4 or P/a-PD-1 and combination therapy, vs. free a-CTLA-4 and a-PD-1 Abs. To independently confirm FC data, the local immune response was investigated for CD8 and CD4+FoxP3 positive T-cells, and IFN-γ, iNOS markers for M1 macrophages by immunofluorescence staining using FIJI software. In P/a-CTLA-4 + P/a-PD-1 group vs. PBS the percentage of CD8+ T cells was significantly increased (p=0.0005), together with reduction of CD4+FoxP3+ T cells (p=0.0006) and increase of iNOS-positive macrophages (p=0.0001). FC and histology results confirmed that the NIT treatment stimulated local brain immune system. FC analysis of T-cell population in blood and spleen also demonstrated significant activation of systemic immune response after combination therapy, based on cytokines level: IL-1β, IL-2, IL-10, IL-4, IL-5, TNF-α, IL-6, IL-12, and IFN-γ after treatment.
Conclusion: GBM treatment with BBB-crossing polymer-conjugated antibodies a-PD-1 and a-CTLA-4 was able to increase tumor-bearing animal survival, which was supported by immunological results indicating the activation of T cell population systemically and locally in the brain. Support: NIH grants R01 CA206220, R01CA230858
Citation Format: Anna Galstyan, Antonella Chiechi, Tao Sun, Ekaterina S. Shatalova, Rameshwar Patil, Keith L. Black, Eggehard Holler, Alexander V. Ljubimov, Hui Ding, Julia Y. Ljubimova. Nano immunotherapeutics crossing blood-brain barrier to activate local brain tumor immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 977.