Plasma asparagine depletion by RBC-entrapped L-asparaginase in selected patients having no low or no ASNS may be a promising therapeutic approach for pancreatic cancer.

Erymet is a new therapy resulting from the encapsulation of a methionine gamma‐lyase (MGL; EC number 4.4.1.11) in red blood cells (RBC). The aim of this study was to evaluate erymet potential efficacy in methionine (Met)‐dependent cancers. We produced a highly purified MGL using a cGMP process, determined the pharmacokinetics/pharmacodynamics (PK/PD) properties of erymet in mice, and assessed its efficacy on tumor growth prevention. Cytotoxicity of purified MGL was tested in six cancer cell lines. CD1 mice were injected with single erymet product supplemented or not with vitamin B6 vitamer pyridoxine (PN; a precursor of PLP cofactor). NMRI nude mice were xenografted in the flank with U‐87 MG‐luc2 glioblastoma cells for tumor growth study following five intravenous (IV) injections of erymet with daily PN oral administration. Endpoints included efficacy and event‐free survival (EFS). Finally, a repeated dose toxicity study of erymet combined with PN cofactor was conducted in CD1 mice. Recombinant MGL was cytotoxic on 4/6 cell lines tested. MGL half‐life was increased from <24 h to 9–12 days when encapsulated in RBC. Conversion of PN into PLP by RBC was demonstrated. Combined erymet + PN treatment led to a sustained Met depletion in plasma for several days with a 85% reduction of tumor volume after 45 days following cells implantation, and a significant EFS prolongation for treated mice. Repeated injections in mice exhibited a very good tolerability with only minor impact on clinical state (piloerection, lean aspect) and a slight decrease in hemoglobin and triglyceride concentrations. This study demonstrated that encapsulation of methioninase inside erythrocyte greatly enhanced pharmacokinetics properties of the enzyme and is efficacy against tumor growth. The perspective on these results is the clinical evaluation of the erymet product in patients with Met starvation‐sensitive tumors.

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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.

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