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Background : Anti-PD-1 immunotherapy, although efficient in several cancer indications; has shown some limitations due to the appearance of resistance mechanisms. More specifically, the overexpression of A2A receptor (adenosine receptor) on the surface of infiltrated CD8 T cells following anti-PD-1 treatment blunts the immune response in an adenosine-rich tumor microenvironment (TME). In addition, increased evidence of immune escape has been described through gene hypermethylation processes. Erymethionase (methionine-gamma-lyase encapsulated into red blood cells) is an innovative experimental therapy designed to directly reduce systemic levels of L-methionine, an amino-acid nutrient needed for tumor growth and metastasis. Erymethionase is expected to indirectly decrease adenosine levels in TME and to reduce hypermethylation reactions. This bimodal action makes of erymethionase a promising agent to combine with immune checkpoint inhibitors. The benefit of such combination (erymethionase and PD-1 blockade agent) was investigated in a TNBC-like syngeneic mouse model. Methods : Mice bearing orthotopic EMT-6 syngeneic breast carcinoma were intravenously injected once weekly for 4 consecutive weeks with vehicle or mouse erymethionase at 30 or 60 U/kg alone or in combination with anti-mouse PD-1 antibody (RMP1-14 clone, intraperitoneal route, 10 mg/kg, twice weekly for 3 consecutive weeks) from D7 (D0 referring to injection of tumor cells). Erymethionase treatment was associated to daily oral vitamin B6 (pyridoxine, PN) uptake to maintain enzyme activity. The body weight, the length and width of the tumor were measured twice a week. Animal behavior was checked every day. Satellite animals receiving 60 U/kg of erymethionase or vehicle were sacrificed to collect 500-1000 m3 size-tumors to perform immunophenotyping, measure metabolites and/or assess biomarkers. Results : Analysis of health parameters revealed that all treatments were well tolerated along the experiment. A delayed start of exponential growth was reported for the combination at the highest dose of erymethionase vs single agents leading to a significant tumor growth inhibition and an increased survival (median survival time of 35 days for the combination vs 23 days for anti-PD-1 or erymethionase 60 U/kg alone; statistically significant). The antitumor effects were less pronounced at the lower dose level of erymethionase in combination with anti-PD1. Analysis of collected tumors to investigate on the mechanism(s) of action is ongoing. Conclusion : To our knowledge, this is the first in vivo demonstration of anti-PD-1 therapy potentiation using a L-methionine-restricting agent. The data are supportive of the planned erymethionase first-in-human study. Citation Format: Karine Sénéchal, Sylvie Maubant, Marie Leblanc, Séverine Ciré, Fanny Gallix, Aurély Andrivon, Olivier Duchamp, Fabrice Viviani, Françoise Horand, Alexander Scheer, Vanessa BOURGEAUX. Erymethionase (methionine-gamma-lyase encapsulated into red blood cells) potentiates anti-PD-1 therapy in TNBC syngeneic mouse model [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 2258.

L-asparaginase (ASNase), an enzyme currently used in combination with chemotherapy for Acute Lymphoblastic Leukemia (ALL) treatment, hydrolyzes L-asparagine (Asn) - a non-essential amino acid - into aspartic acid (Asp) and ammonia, leading to Asn removal from the circulation. Similarly, restriction to exogenous L-methionine (Met) - an essential, sulfur-containing proteinogenic amino acid - blocks cell cycle in the late S or G2 phase in Met-dependent cancer (Hoffman and Jacobsen 1980; Guo et al., 1993; Kokkinakis et al., 1997, 2004, 2005 and 2006; Pavillard et al., 2006; Yano et al., 2014). ERYTECH Pharma's strategy was to combine Met depletion, by using Methionine-γ-lyase (MGL) (a pyridoxal-dependent enzyme) with Asn depletion (via ASNase activity), and investigate its in vitro and in vivo therapeutic potential. In vitro sensitivity to MGL combined with ASNase was assessed on 7 cell lines representative of different cancers (glioblastoma, AML, gastric and pancreatic adenocarcinoma or carcinoma). Fixed enzyme concentrations (IC50), determined previously in dose-response studies using each enzyme alone, were used in a sequential therapeutic scheme adding MGL first and ASNase 3 or 4 days later. Tumor cells sensitivity was evaluated by measuring cell viability. In vivo combination study, in a human gastric xenograft mouse model, was performed using ASNase and MGL encapsulated into red blood cells, respectively named erymethionase and eryaspase. The scheme of administration consisted in erymethionase treatment on Day 7 and 15 followed by eryaspase treatment on Day 21. Control arms received vehicle or single therapy with 3 repeated administrations. In vitro study results showed enhanced mortality with MGL-ASNase sequential therapy vs single therapies (ASNase and MGL tested alone) in 72% (5/7) of the tested cellular models (AML, glioblastoma, gastric and pancreatic cancers models). Those data were confirmed in vivo with an enhanced tumor inhibition when erymethionase was administered twice before eryaspase, compared to vehicle or erymethionase-alone treatments (respectively 57% and 36% on Day 37). The result significance is supported by a p-value inferior to 0.0001. To conclude, our in vitro results showed promising potential for enzymatic bi-therapy treatment on several cancer models and that intrinsic tumor cells properties determined its efficiency. In vivo data confirmed, in a gastric cancer model, the therapeutic efficacy of this sequential erymethionase-eryaspase treatment. However, this enzymatic bi-therapy still requires further investigation. The identification of involved mechanisms of action should allow to optimize the therapeutic design and select best responders. A preclinical program is currently ongoing on specific biomarkers, investigation on tumor cell synchronization with ASNase therapy and optimal therapeutic enzyme sequence for treatment. Citation Format: Karine Aguera, Fanny Gallix, Fabien Gay, Karine Senechal, Severine Cire, Françoise Horand, Alexander Scheer, Vanessa Bourgeaux. Enzymatic combination investigation in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5827.

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 ( 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.

L-asparaginase (L-ASPA) is included in current chemotherapy regimen for the treatment of acute lymphoblastic leukemia (ALL). The sensitivity of ALL to L-ASPA has been linked to the incapacity of leukemic cells to produce their own asparagine, since they are deficient in a specific enzyme, asparagine synthetase (ASNS). Thus, ASNS-negative leukemic cells rely on plasmatic asparagine and can be starved to death by L-ASPA treatment. Several studies evidenced the potential of asparagine depletion to treat lymphomas. Indeed, L-ASPA is routinely administered to treat canine lymphomas and its adjunction in chemotherapy regimens significantly improves the outcome of patients with NK/T cell lymphoma. Some studies suggest its benefit for the treatment of B and T-cell non-Hodgkin lymphomas (NHL). In this study, we assessed the in vitro sensitivity to L-ASPA of 11 lymphoma cell lines, including diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL) and other NHL cell lines. We analyzed ASNS expression in tissue microarrays and biopsies from 226 cases of lymphomas, including 158 DLBCL, 15 PTCL, as well as other NHL and Hodgkin lymphomas. Sensitivity to L-ASPA (expressed as an IC50) was assessed by measuring the cell viability in the presence of various concentrations of L-ASPA. ASNS expression was assessed with a validated immunohistochemistry method attributing a score based on labeling intensity from 0 (no expression) to 3 (strong expression). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. All lymphoma cell lines were proved to be sensitive to L-ASPA. Their in vitro sensitivity usually exceeded well-known sensitive cell lines (MOLT-4 and HL-60) representative of acute leukemia. ASNS expression was null/low in 171/226 (76%) total cases of lymphomas. 118/158 (75%) DLBCL cases expressed no/low ASNS, whereas ASNS expression was low/null in 2/15 (13%) PTCL cases. Globally, B-cell NHL displayed a lower expression of ASNS than T-cell NHL, Hodgkin lymphoma and non tumoral lymph nodes. The in vitro sensitivity to L-ASPA of all lymphoma cell lines and the low/null expression of ASNS in the majority of the lymphoma tissues tested suggest that L-ASPA may be effective for the treatment of various lymphomas. As suggested by ASNS expression, L-ASPA treatment may be particularly effective in DLBCL and others B-cell NHL, whereas its efficacy in T-cell NHL is probably more limited. However, L-ASPA has only been used scarcely in the treatment of lymphomas despite promising clinical responses. Its well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis…) render its use particularly hazardous in older or frail patients. The development of a new formulation of L-ASPA with better safety profile must be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Anne-Marie Chevrier, Alexandra Traverse-Glehen, Yann Godfrin. Low asparagine synthetase expression and in vitro sensitivity highlights L-asparaginase potential for the treatment of aggressive lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5369. doi:10.1158/1538-7445.AM2015-5369

Due to asparagine synthetase (ASNS) deficiency, tumors cells auxotrophic for L-asparagine lack capability to synthesize this amino acid and depend to circulating L-asparagine for survival. Based on this rational, L-asparaginase (L-Aspa), an enzyme depleting L-asparagine, is an essential drug in the treatment of acute lymphoblastic leukemia (ALL), as these hematological cancer cells express low levels of ASNS. Recent evidence suggests that some solid tumors are also deficient in ASNS (Lorenzi, 2008; Dufour, 2012; Zhang, 2013), providing rationale for testing L-Aspa treatment in other contexts. In this study, we investigated the levels of ASNS expression in tumor tissues from patients with urological cancers (bladder, prostate and kidney) and we investigated the sensitivity to L-Aspa of bladder and prostate cell lines. Material & Method: ASNS expression was evaluated by a validated immunohistochemistry (IHC) method on tissue biopsie microarrays (USBiomax, Rockville, MD) including 384 cases of bladder, 94 cases of prostate and 75 cases of kidney tumors. A score was attributed to each tumor based on ASNS IHC labeling intensity from 0 (no labeling) to 3 (strong labeling). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. Sensitivity to L-Aspa (expressed as an IC50) was assessed in vitro by measuring the cell viability of bladder cancer cell lines (RT-4 and UM-UC-3) and prostate cancer cell line PC-3 in the presence of various concentrations of L-Aspa. ASNS expression in cell lines was evaluated by western-blot. Results: ASNS expression was low/null in 77.3% of bladder tumors, 86.2% of prostate tumors and 72% of kidney tumors. In bladder tumors, no correlation was found between the grade of the tumor and its expression of ASNS. Bladder and prostate cancer cell lines were all sensitive to L-Aspa (IC50 = 0.31, 0.12 and 0.22 IU/mL for RT-4, UM-UC-3 and PC-3 cell lines, respectively) at a level similar to the sensitivity observed with the L-Aspa-sensitive MOLT-4 ALL cell line (IC50=0.19 IU/mL). All cell lines displayed low basal expression of ASNS. Conclusion: The sensitivity of bladder and prostate cancer cell lines to L-Aspa and the low/no expression of ASNS in the majority of urological tumor biopsies suggest that an extensive population of patients with urological tumors may respond to asparagine depletion. However, L-Aspa is accompanied with well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, liver failure) and this drug should be used very carefully especially in older or frail patients. A new safer formulation is requested to make easier the clinical development of L-Aspa in urological cancer. Citation Format: Willy Berlier, Karine Aguera, Fanny Gallix, Fabien Gay, Gautam Borthakur, Yann Godfrin. Potential of asparagine depletion for the treatment of bladder cancer and other urological tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5436. doi:10.1158/1538-7445.AM2014-5436