Abstract 4812: Arginine deiminase loaded in erythrocytes: a promising formulation for L-arginine deprivation therapy in cancers
Authors: Gay Fabien , Aguera Karine , Senechal Karine , Bes Julie , Chevrier Anne-Marie , Gallix Fanny , Guicher Christine , Lorenzi Philip , Bourgeaux Vanessa , Berlier Willy , Horand Françoise , Godfrin Yann 
Published: July 2016

Based on the asparaginase paradigm, several arginine-catabolizing enzymes have been developed for the treatment of arginine-dependent cancers (Wheatley, 2004). The arginine deiminase (ADI) enzyme catalyzes the hydrolysis of arginine (Arg) (Sugimura, 1990). ADI purified from Mycoplasma has a short half-life (≈4h) in the circulation and was found to be highly immunogenic (Holstberg, 2002). In order to increase half-life and to limit immunogenicity, a pegylated form of the enzyme (ADI-PEG-20) was developed. Phase I/II clinical studies in hepatocarcinoma and in advanced melanoma concluded on its limited efficacy at the tested doses. Notably, reduction of the duration of Arg depletion was linked to the emergence of ADI-PEG-20 antibodies (Ascierto, 2006). Encapsulation of ADI into red blood cells (RBC) is a promising alternative to improve the half-life and reduce the immunogenicity of the protein. Argininosuccinate synthase (ASS1) is the key enzyme in arginine biosynthesis (Haines, 2011). ASS1 expression varies in tumors and ASS1 loss is associated with poor prognosis in different cancers (Qiu, 2015). All these data strengthen the importance of selecting ASS1-negative patients for Arg-depletion based enzymatic therapy.

Using a scalable, standardized production process, we synthesized an ADI enzyme from an optimized M. arginini coding sequence. ADI was encapsulated into RBC by hypotonic dialysis (ERY-ADI) and PK-PD parameters were evaluated in CD1 mice, in comparison with free ADI. Sensitivity to ADI was assessed in vitro by measuring the cell viability of 3 cancer cell lines with different ASS1 expression levels. ASS1 expression was screened by immunohistochemistry (IHC) in a large panel of tissue microarrays (TMA) from 16 human cancer types.

Administration of ERY-ADI (5.5IU/mL) reduced mouse plasmatic Arg level to 30% of control values and led to a maintained depletion for 5 days. The same dose of free ADI strongly depleted Arg (&lt;5μM) for 24h but concentration returned to baseline by 2.5 days. ERY-ADI at higher dose (10.4IU/mL) resulted in the complete depletion of plasmatic Arg over the 5-day period of the study. No problem of tolerability were noticed. In cell lines, sensitivity to ADI was linked to ASS1 expression: AGS cell line (ASS1-negative) was highly sensitive to ADI treatment (IC50 = 0.4mIU/mL; 100% cell mortality) whereas NCI-N87 (ASS1-high) was strongly resistant (no mortality at the highest tested dose) and HL-60 (ASS1-low) was moderately sensitive (IC50 = 0.3mIU/mL; 70% cell mortality). ASS1 screening in TMA revealed several cancers of interest with down-regulated ASS1 expression.

All these results highlight that arginine depletion through ADI treatment is effective against ASS1-negative cancer cells. ERY-ADI represents an innovative product with an improved efficacy for sustained Arg depletion and suitable for the treatment of ASS1 deficient cancers.

Citation Format: Fabien Gay, Karine Aguera, Karine Senechal, Julie Bes, Anne-Marie Chevrier, Fanny Gallix, Christine Guicher, Philip Lorenzi, Vanessa Bourgeaux, Willy Berlier, Françoise Horand, Yann Godfrin. Arginine deiminase loaded in erythrocytes: a promising formulation for L-arginine deprivation therapy in cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4812.