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Lack of preclinical patient-derived xenograft (PDX) cancer models in which to conduct large scale molecular studies seriously impairs the development of effective personalized therapies. We report here on an in vivo concept consisting of implanting human tumor cells in targeted tissues of an avian embryo, delivering therapeutics, evaluating their efficacy by measuring tumors using light sheet confocal microscopy, and conducting large scale RNAseq analysis to characterize therapeutic-induced changes in gene expression. The model was established to recapitulate triple negative breast cancer (TNBC) and validated using TNBC standards of care (SOCs) and an investigational therapeutic agent.

Objectives In this study, our aim was to test whether asparagine synthetase (ASNS) deficiency in pancreatic malignant cells can lead to sensitivity to asparagine starvation. We also investigated, in tumor-bearing mice, the efficacy of L-asparaginase entrapped in red blood cells (RBCs), a safe formulation, to induce asparagine depletion. Methods First, ASNS expression was evaluated by immunohistochemistry in sporadic pancreatic ductal adenocarcinoma. Then, 4 pancreatic carcinoma cell lines were examined by Western blot, immunocytochemistry, and cytotoxicity assay to L-asparaginase and in asparagine-free or reduced-asparagine media. Finally, mice bearing the most in vitro sensitive cell line received RBC-entrapped L-asparaginase to investigate the anticancer efficacy of serum asparagine depletion in vivo. Results Approximately 52% of pancreatic adenocarcinomas expressed no or low ASNS. The highest in vitro cytotoxicity to L-asparaginase or to reduced asparagine medium was observed with SW1990 line when ASNS expression was the lowest. In vivo sensitivity was confirmed for this cell line. Conclusions 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.

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.

International audience; Actin depolymerizing factor (ADF)/cofilin family proteins are key regulators of actin filament turnover and cytoskeleton reorganization. The role of cofilin-1 in cell motility has been demonstrated in several cell types but remained poorly documented in the case of colon cancer. In addition, the putative function of destrin (also known as ADF) had not been explored in this context despite the fact that it is expressed in all colon cancer cell lines examined. We were therefore prompted to evaluate the respective contributions of these proteins to the invasive properties of the human colon cancer Isreco1 cell line, which expresses a comparatively high destrin/cofilin ratio. Reduction of cofilin-1 or destrin expression in Isreco1 cells using RNA interference led to an increase of the number of multinucleated cells and altered polarized lamellipodium protrusion and distribution of paxillin-containing adhesions. Both cofilin-1 and destrin silencing enhanced cell adhesion to extracellular matrix components. However, only destrin appeared to be required for cell migration on collagen I and for cell invasion through Matrigel in response to the proinvasive neuroendocrine peptide bombesin. This differential functional involvement was supported by a destrin-dependent, cofilin-independent phosphorylation of p130Crk-associated substrate (p130Cas) upon cell adhesion to collagen I or Matrigel. Taken together, our results suggest that destrin is a significant regulator of various processes important for invasive phenotype of human colon cancer Isreco1 cells whereas cofilin-1 may be involved in only a subset of them.

Asparaginase encapsulated in erythrocytes (ERY-ASP) is a potentially effective drug in patients with pancreatic adenocarcinoma (PAC) with null/low asparagine synthetase (ASNS) expression. Our aims were to assess ASNS expression in PAC from a large cohort and its prognostic and/or predictive value and to conduct a phase I trial with ERY-ASP in patients with metastatic PAC.Asparagine synthetase expression was evaluated using immunohistochemistry in resected PAC (471 patients) and in pairs of primary tumor and metastases (55 patients). Twelve patients were included in the phase I trial and received a single administration of ERY-ASP (25-150 IU/kg).Null/low ASNS expression was found in 79.4% of the resected PAC with a high concordance between primary tumor and metastases. Asparagine synthetase expression was significantly correlated with sex and CXCR4 expression. In the phase I trial, ERY-ASP was well tolerated by patients with metastatic PAC. No patient had DLTs, and 6 patients had at least 1 ERY-ASP causally related adverse event out of the 12 adverse events reported.Given the high rate of PAC with null/low ASNS expression and the good tolerability profile of ERY-ASP, ERY-ASP should be evaluated in further clinical studies in metastatic PAC.

Methionine (Met) dependence is a cancer-specific metabolic defect that has emerged as a target during the last two decades. The use of methionine gamma-lyase (MGL; EC number 4.4.1.11), a bacterial Met-catabolizing enzyme, is a promising strategy for treatment of Met-dependent cancers. However, one challenge is that MGL has a very short half-life (~2 hours), resulting in a short-term Met depletion in vivo. Additionally, its cofactor, pyridoxal 5’-phosphate (PLP) is rapidly eliminated from plasma (Yang et al., 2004). PEGylation extends the MGL half-life in mice to up to 38 hours. Nevertheless, frequent injections are still necessary for maintaining an effective Met depletion over time (Sun et al., 2003). In addition, the low bioavailability of PLP remains a major hurdle due to both scavenging by plasma proteins and very short half-life (< 15 minutes) in the blood stream (Zempleni, 1995). Studies showed that pyridoxine (PN), a vitamin B6 vitamer, can easily cross red blood cells (RBCs) membrane to be rapidly converted to PLP via an enzymatic reactions cascade (Anderson et al., 1971). Considering those particular characteristics of RBCs, we propose that RBC-encapsulated MGL (ERY-MET) will protect the enzyme from degradation/immune reactions and therefore overcome the pharmacodynamic limitations for its use in the treatment of Met-dependent cancers. Pharmacokinetics, pharmacodynamics, and safety parameters of MGL-loaded RBCs were evaluated in healthy mice. In parallel, the role of RBC in PLP biosynthesis from exogenous uptake of PN was investigated in vivo. Finally, we investigated the antitumoral effect of repeated injections of MGL-loaded RBCs combined with daily intragastric administration of PN in two subcutaneous xenografted mouse models for human gastric and glioblastoma tumors. The MGL half-life increased from < 24 hours to ~ 10 days when encapsulated in RBC, with no toxicity reported after one injection of ERY-MET. Following intragastric administration (3.2 mg/kg), PN was rapidly (15 minutes) converted into PLP within the RBC, resulting in an increase of MGL activity (holoenzyme). Combining a weekly single intravenous injection of ERY-MET for 5 days, in association with daily PN supplementation by gavage led to a sustained Met depletion in plasma and induced a 85% inhibition of tumor growth 45 days following implantation of glioblastoma cells. In the gastric mouse model, tumor growth was inhibited by 72% at the same time point (45 days).This study clearly demonstrated that encapsulation of MGL in erythrocytes both strongly improved the half-life and contributed to provide active cofactor. In parallel, repeated injections of ERY-MET were effective against tumor growth in mouse models. Thus, due to the RBC intrinsic characteristics, ERY-MET represents a new promising treatment against a broad scope of cancers that rely on Met metabolism. Citation Format: Fabien Gay, Karine Aguera, Karine Senechal, Philip Lorenzi, Alexander Scheer, Françoise Horand, Vanessa Bourgeaux. Use of methionine gamma-lyase-loaded erythrocytes to induce effective methionine depletion in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2134. doi:10.1158/1538-7445.AM2017-2134

78 Background: Methionine (Met) requirement is a cancer specific–metabolic defect that seems a promising target, especially in gastric cancers. Methionine gamma–lyase (MGL), a pyridoxal–5′–phosphate (PLP)–dependent enzyme, is an emerging approach consisting in tumors Met starvation via systemic Met depletion. ERY-MET is a new therapeutic product overcoming the short in vivo half-life of free MGL by its encapsulation into Red Blood Cells (RBCs). Indeed, ERY-MET works as a bioreactor degrading Met that passively diffuses inside the RBC. In addition, entrapped MGL activity can be controlled by supplying Vitamin B6 (PN), the precursor of MGL’s cofactor (PLP), converted inside RBCs. ERY-MET anti-tumor activity was evaluated in vivo in NMRI nudemice bearing subcutaneous gastric carcinoma. Methods: First, in vitro sensitivity of NCI-N87 and AGS human gastric cell lines to free MGL was assessed by IC50 determination using CCK–8 assay. MGL encapsulated into mouse RBCs by hypotonic dialysis was injected once in CD1 mice to determine PK-PD parameters with or without PN supplementation. The anti-tumor activity of weekly ERY-MET injections (x5) at 116 IU/kg ± 25% was assessed with or without PN supplementation in female NMRI nudemice (n = 10/group) xenografted with NCI-N87 cells. Met depletion was determined 6 days after each cumulative injection while tumor growth was followed twice a week by caliper measurement. Results: In vitro studies showed that NCI-N87 as well as AGS cell lines displayed a sensitivity to free MGL with IC50 of 0.35 ± 0.01 and 0.12 ± 0.02 IU/mL, respectively. ERY-MET with daily PN supplementation significantly increased active MGL half-life in vivo (from < 24h to 8–9 days). ERY-MET induced 80% inhibition of tumor growth at day 45 (p < 0.0001). Response rate obtained was 76% of treated mice (15/20). Besides, PN supplementation induced a slow-down of tumor growth during the supplementation period and improved ERY-MET efficacy. Conclusions: Theses results suggest that ERY-MET can induce tumor growth inhibition in mice bearing human gastric adenocarcinoma and that its effect can be regulated by PN supplementation. As such, ERY-MET seems a promising anti-tumor drug to treat gastric cancers.

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.

Several studies reported a complete dependence of different tumor cells on L-methionine (Met), an essential amino acid, for maintaining their high proliferation rate while normal cells keep their ability to grow on Met-deprived medium (Cavuoto and Fenech, 2012). Met restriction appearing as an important strategy for cancer treatment, different therapeutic approaches using either Met restricted regimens or Met-catabolizing enzymes were investigated. Phase I and II clinical trials associating dietary Met restriction with chemotherapy were conducted with cancer patients (Thivat et al., 2009; Durando et al, 2010). A 40% reduction of plasmatic methionine was achieved with a 1-day Met-free diet per month. Interesting long-duration of disease stabilization was observed in some patients. However this diet is extremely difficult to maintain. In order to obtain longer Met depletion in plasma, enzyme therapy using Methionine gamma-lyase (MGL), a pyridoxal-dependent enzyme that specifically degrades Met into alpha-ketobutyrate, ammonia and thiols, was explored. MGL enzyme isolated from Pseudomonas putida was selected as preferred candidate for the development of therapy. However, preclinical studies conducted in primates showed a very short half-life of the active form of MGL enzyme (2h) due to a rapid in vivo dissociation of the pyridoxal (PLP) cofactor (Yang et al., 2004). To overcome this problem of PLP-dissociation, we encapsulated the active MGL into erythrocytes, natural pool of PLP in the body and evaluated PK-PD parameters of product over a 5-day period in a mouse model. Once entrapped into erythrocytes, the MGL enzyme maintained more than 30% of its specific activity after 5 days in circulation. A 60-70% Met depletion was achieved 15 min post-injection and then depletion was stabilized at 40% over the 5 day-period of the study. In comparison, administration of the free MGL at similar doses led to a rapid loss of the active form in several hours. Met was totally deprived 15 min post-injection but plasmatic Met level returned to baseline by 24h. Increasing two-fold the concentration of free methioninase did not improved the in vivo Met depletion. In conclusion, we report that methioninase-loaded-erythrocytes can lower Met concentration in mouse plasma for at least 5 days and may be an innovative strategy to kill tumors by met-starvation. Citation Format: Fabien Gay, Julie Bes, Vanessa Bourgeaux, Yann Godfrin. Methioninase-loaded erythrocytes: a promising drug for L-methionine restriction therapy in cancer. [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 5330. doi:10.1158/1538-7445.AM2015-5330

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

L-Asparaginase (L-Asp) depletes L-Asn in the plasma and is commonly used in the treatment of acute lymphoblastic leukemia (ALL) for over 40 years. Its indication is motivated by the fact that L-asparagine synthetase deficient leukemic cells are unable to synthesize L-Asn to meet metabolic demands and therefore depend on circulating L-asparagine (L-Asn) to survive and to proliferate. Unfortunately, its use in patients with solid tumours failed due to high toxicity. L-Asp entrapped inside red blood cells (GRASPA®) significantly improves its therapeutic index, thus allowing its use with acceptable tolerance in patients with solid tumors. There is strong rational for plasmatic L-Asn depletion in patients with negative or low-expressing L-asparagine synthetase (ASNS) cancers. Indeed ASNS catalyzes the biosynthesis of L-Asn from L-Aspartate and L-Glutamine and its overexpression appears to be crucial in L-Asp resistance in an ALL cell line model (Aslanian, 2001). In the same way, an inverse causal relationship was observed between ASNS expression (at the protein level) and L-Asp activity in a panel of human ovarian cancer cell lines, and a weak expression of ASNS by immunohistochemistry for 15% of the 55 ovarian cancers tested (Lorenzi, 2006). To demonstrate the implication of ASNS in solid tumors, we performed a large screening of ASNS expression by tissue microarray in several cancer types (bladder, brain, breast, colon, head & neck, kidney, liver, lung, lymphoma, melanoma, myeloma, ovary, pancreas and prostate). We first focused on pancreatic tumors and studied ASNS expression on 4 different tissue microarrays (n=172) and on a large series of standard pancreatic tumors slides (n=124). By this indirect immunohistochemistry method, we determine the proportion of low-expressing tumors in a representative subset of human pancreatic ductal adenocarcinomas. In parallel, we evaluate the IC50 of L-Asp in SW1990 (low ASNS expression), PANC-1 and MIA-PaCa-2 (ASNS positive) pancreatic cancer cell lines. Our results indicate that: - ASNS expression was strongly decreased in human ductal adenocarcinoma (percentage of negative or low-positive tumors > 65%) as compared to the high-expressing adjacent healthy pancreas tissue (< 10%); - In vitro, The ASNS low-expressing cell line SW1990 was more sensitive to L-Asp than the PANC-1 and MIA-PaCa-2 ASNS positive cell lines (IC50 (IU/mL L-Asp): 0.07±0.04 versus 0.18±0.05 and 0.24±0.06, respectively). In conclusion, we demonstrate that ASNS expression is strongly down-regulated in human pancreatic cancer. We also highlight in vitro causality between ASNS status and sensitivity to L-Asp in 3 pancreatic cancer cell lines. According to these results, ASNS was identified as a candidate biomarker to screen patients in solid tumors, especially for pancreatic carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4137. doi:10.1158/1538-7445.AM2011-4137