WO2023067200A1

WO2023067200A1 - Asparaginase encapsulated in red blood cells for the treatment of pancreatic cancer

Info

Publication number: WO2023067200A1
Application number: PCT/EP2022/079630
Authority: WO
Inventors: Iman Ahmed Zakaria EL-HARIRY; John Francis Hoke
Original assignee: Erytech Pharma
Priority date: 2021-10-24
Filing date: 2022-10-24
Publication date: 2023-04-27

Abstract

The present invention relates to the therapeutic treatment of pancreatic cancer using eryaspase, which comprises red blood cells encapsulating asparaginase (ASNase), in combination with chemotherapeutic agents.

Description

Asparaginase Encapsulated in Red Blood Cells for the Treatment of Pancreatic Cancer

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the therapeutic treatment of pancreatic cancer using eryaspase, which comprises red blood cells encapsulating asparaginase (ASNase), in combination with a combination of chemotherapeutic agents.

SUMMARY OF THE INVENTION

The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Pancreatic adenocarcinoma (PAC) is one of the most aggressive and fatal malignancies, and is ranked as the seventh leading cause of cancer death. [2] The overall prognosis for PAC is poor, with a 5- year survival rate of 7%. [3] This is attributable to the difficulty of diagnosing pancreaticcancer in the early stages, poor tumor resectability, and poor response to chemotherapy.[4] Indeed, most pancreatic cancer patients progress to either metastatic or locally advanced disease in the asymptomatic phase. [5]

Current treatment strategies for non-resectable, metastatic disease focus on conventional cytotoxic therapies. Established first-line treatment options include gemcitabine-based chemotherapy, either alone or in combination with nab-paclitaxel, or the FOLFIRINOX regimen (a combination of leucovorin [LV], 5-fluorouracil [5-FU], irinotecan, and oxaliplatin). [5] Gemcitabine treatment results in only modest improvements in overall survival (OS) and qualityof life (QoL).[6] While the FOLFIRINOX regimen has demonstrated a robust clinical benefit compared with gemcitabine, [7] this regimen should only be considered for patients with good performance status (Eastern Cooperative Oncology Group Performance Status [ECOG PS] 0 or 1) due to considerable toxicity. [5-8]

In the second-line setting, there remains a lack of consensus regarding the standard of care. Treatment options are dependent on the risk-benefit balance for the patient and the treatment received in the first line. [5] In 2017, treatment guidelines were updated to recommend that the combination of nanoliposomal irinotecan (Onivyde®) with fluoropyrimidine regimens (i.e. 5-FU with LV) can be considered an active and tolerable treatment option in fit patients (ECOG >2) previously treated with gemcitabinebased therapy.[8-9] This combination extended survival by 1.9 months compared with patients treated with 5-FU in combination with LV.[10]

No formal recommendations are available for patients who have progressed on first-line FOLFIRINOX; although gemcitabine is widely used in this setting, disease control is only achieved in one in 5 patients. [11] The limited efficacy provided by cytotoxic therapies is driving the need to identify novel targets for which to develop more effective therapeutic strategies. [4]

In our previous work, we demonstrated the potential for eryaspase (ASNase encapsulated in red blood cells) to augment the efficacy of the aforementioned pancreatic cancer treatment regimens.

In the present application, we disclose the detailed Phase 3 clinical trial protocol and the results produced by the conducting of same. Unexpectedly, and contrary to the Phase 2 results, asparaginase appeared to improve the survival of patients receiving the irinotecan-based chemotherapy to a greater degree than in patients receiving the non-irinotecan-based chemotherapy. Moreover, this effect was stable across various subgroups and eryaspase did not significantly increase toxicity when compared with either chemotherapy regimen alone. As such, these results appear to demonstrate— for the first time— that asparaginase enhances the efficacy of irinotecan-based chemotherapy against a solid tumor.

Accordingly, the disclosure provide a method for treating pancreatic cancer comprising the steps of administering an effective amount of red blood cells encapsulating asparaginase to a patient in need thereof. A sufficient amount is one that increases a patient's overall survival and/or progression free survival.

The present application comprises and encompasses the inventions as defined in the attached claims. All characteristics disclosed herein, including in the attached claims, such as those characteristics regarding the active principles, their combinations within a treatment regimen, the status of the patient with respect to cancer and its treatments, the effects thereof, do apply to the various inventive objects encompassed by the present invention, including the methods of treatment, the combination of active principles for use, the compositions or kits for use, and the use of the active principle(s) for the manufacture of medicaments.

BRIEF DESCRIPTION OF THE FIGURES Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

FIG. 1 is a Kaplan-Meier plot showing Overall survival - ITT.

FIG. 2 is a Kaplan-Meier plot showing Overall survival - Per Protocol.

FIG. 3 is a summary of Overall Survival by Treatment Group backbone chemotherapy - ITT.

FIG. 4 is a Kaplan-Meier plot showing Overall survival - gemcitabine-based chemotherapy - PP.

FIG. 5 is a Kaplan-Meier plot showing Overall survival - Irinotecan-based chemotherapy - PP.

FIG. 6 is a Forest plot of OS hazard ratios in subgroups - PP, part 1.

FIG. 7 is a Forest plot of OS hazard ratios in subgroups - PP, part 2.

FIG. 8 is a Forest plot of PFS hazard ratios in subgroups - ITT, part 1.

FIG. 9 is a Forest plot of PFS hazard ratios in subgroups - ITT, part 2.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference m their entirety as though fully set forth. Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4^th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

As used herein the term "about" when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. For example, the language "about 50%" covers the range of 45% to 55%. In various embodiments, the term "about" when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1 %, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

As used herein, a "subject" means a human or animal. Typically, the animal is a vertebrate such as a primate, rodent, or domestic animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog. The terms, "patient", "individual" and "subject" are used interchangeably herein. In an embodiment, the subject is mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, cow, or other mammal. In some embodiments, the subject is a human.

A subject may be one who has been previously diagnosed with or identified as suffering from or having a disease, disorder or condition in need of treatment or one or more complications related to the disease, disorder, or condition, and optionally, have already undergone treatment for the disease, disorder, or condition or the one or more complications related to the disease, disorder, or condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a disease, disorder, or condition or one or more complications related to the disease, disorder, or condition. For example, a subject may be one who exhibits one or more risk factors for a disease, disorder, or condition or one or more complications related to the disease, disorder, or condition or a subject who does not exhibit risk factors. A "subject in need" of treatment for a particular disease, disorder, or condition may be a subject diagnosed as having that disease, disorder, or condition. In some embodiments, the subject has already been treated only once prior to treatment with the compositions and methods disclosed herein.

"Therapeutically effective amount" as used herein refers to that amount which is capable of achieving at least one beneficial result in a patient with pancreatic cancer. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the physiological characteristics of the mammal, the type of delivery system or therapeutic technique used and the time of administration relative to the progression of the disease.

"Treatment" and "treating" as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down and/or lessen the disease even if the treatment is ultimately unsuccessful.

"Disease progression" as used herein refers to clinical or radiographic disease progression, as defined by RECIST 1.1 criteria.

"Progression-free survival" (PFS) as used herein refers to the interval from randomization until disease progression, including clinical progression, as per RECIST version 1.1, or death.

"Overall survival" (OS) as used herein refers to the interval from randomization until death.

"Duration of response" (DoR) as used herein refers to the duration of time from first documentation of an objective response to the earliest date disease progression is documented or death from any cause.

"Time to response" as used herein refers to the duration of time from the first administration of the inventive therapy described herein to the first documentation of an objective response.

There is currently no "standard of care" (SOC) for locally advanced or metastatic pancreatic cancer that has progressed following either FOLFIRINOX or a gemcitabine-based regimen. And while there are potential options, there is no proven benefit for any regimen, and treatment choice is generally an extrapolation from front-line studies. Based on systematic reviews and National Comprehensive Cancer Network (NCCN) guidelines, gemcitabine-based therapy may be given to those previously treated with fluoropyrimidine-based chemotherapy. Fluoropyrimidine-based chemotherapy regimens are acceptable second-line options for patients who received prior gemcitabine-based therapy. Based the recent Phase 3 NAPOLI-1 trial [36], Onivyde was approved by the FDA and EMA in combination with 5-FU/leucovorin for use following gemcitabine-based therapy.

Asparaginase (ASNase) is a key component of multi-agent chemotherapy used in the treatment of childhood acute lymphoblastic leukemia (ALL). In ALL, as leukemic cells lack ASNS, ASNase exerts its anti-tumor effect through depletion of serum asparagine by catalyzing the deamination of asparagine to aspartic acid. [20] This starves susceptible cells of asparagine required for protein synthesis, which results in cell cycle arrest and, ultimately, apoptosis. [20-23] As glutamine is also metabolized by the enzyme [24], ASNase depletes serum glutamine levels, contributing further to cell growth inhibition. [20]

And while ASNase has shown efficacy against solid tumor cells in vitro, clinical studies have been limited due to the challenge of a narrow therapeutic index for ASNase. Excessive toxicity of native and pegylated ASNase (peg-ASNase) formulations has been observed in clinical studies of solid tumors (pancreatic, ovarian) and multiple myeloma. [28-30] Indeed, a recent phase 1 study evaluating peg-ASNase in combination with gemcitabine in patients with metastatic solid tumors and lymphoma was terminated early due to toxicity.[31] In contrast to the foregoing presentations of ASNase, encapsulation of ASNase within RBCs through a proprietary process provides a novel approach to the delivery of ASNase at biologically effective doses with reduced propensity for toxic effects. Various Erytech publications describe the gentle encapsulation process, especially US 8,617,840, US 10,273,444, US 10,286,008, US 8,974,802, US 10,046,009, and US 10,780,126, which are herein incorporated by reference in their entireties. The encapsulated ASNase remains biologically active, with a half-life of approximately two weeks. [32] It has been demonstrated that encapsulation has no effect on the permeability of RBCs to asparagine. [33] Thus, asparagine is actively transported across a concentration gradient into the RBC where it is de-aminated into aspartic acid and ammonia, depleting the serum of asparagine. It is postulated that the same process occurs with glutamine. Encapsulation prevents the rapid degradation of ASNase in the blood, thus prolonging activity, and limits exposure to the immune system, thereby minimizing undesirable effects, especially including hypersensitivity reactions. [32] To date, five clinical trials have been conducted with eryaspase in pediatric and adult patients with ALL and acute myeloid leukemia (AML), and two clinical trials have been conducted in pancreatic cancer.

The Phase 2 pancreatic cancer trial was a multicenter, open-label study investigating eryaspase in combination with gemcitabine or mFOLFOX6 (modified FOLinic acid-Fluorouracil-Oxaliplatin-6 regimen) as second-line treatment in patients with metastatic PAC (see Hamel et al., EJC 124:2020, 91-101, which is incorporated by reference herein in its entirety). Patients were randomized in a 2:1 ratio to either eryaspase plus chemotherapy or chemotherapy alone, and were stratified according to chemotherapy regimen. The co-primary endpoints were progression-free survival (PFS) and overall survival (OS) in patients with low or no ASNS expression (ASNS 0/1+). Secondary endpoints included PFS and OS in the Intent to Treat (ITT) and high-ASNS expression (ASNS 2+/3+) populations, objective response, and toxicity. It should be noted that the study was not powered for the co-primary endpoints of PFS and OS. A total of 141 patients were randomized, 95 to eryaspase plus chemotherapy and 46 to chemotherapy alone. Both co-primary endpoints were met in the ASNS 0/1+ subgroup (eryaspase, n = 66; control, n = 32), with HRs of 0.67 for PFS and 0.63 for OS, Table 1.

Table 1. Key Efficacy Outcomes in the ITT Population and ASNS Subpopulations in the Phase 2b study

Despite the encouraging findings of the Phase 2 trial, it still remained to conduct a pivotal study to definitively establish the safety and efficacy of eryaspase against pancreatic cancer. Moreover, it remained to be determined which subgroups of patients would respond best to eryaspase. As such, a pivotal Phase 3 trial was developed and conducted as disclosed below. In some embodiments, eryaspase plus chemotherapy provides significant improvements in one, or more, or all of the following: Overall Survival (OS), Progression-free Survival (PFS), Objective Response Rate (ORR), Duration of Response (DoR), Disease Control Rate (DCR), Pharmacokinetics (PK) and Pharmacodynamics (PD) induction of Anti-ASNase antibodies (Abs), Adverse events (AEs), Quality of life (QoL), or Biomarkers. As regards biomarkers, based upon Applicant's in vitro data related to ASNase- resistant pancreatic cancer cells, it is envisioned that tumors having higher and/or inducible levels of glutamine synthetase (GS) may be less susceptible to E. coli ASNase. Accordingly, GS levels may be used to inform treatment (e.g. in cases of high tumor GS levels, a glutaminase inhibitor may be added).

A first object of the invention is a suspension of red blood cells (RBCs) encapsulating asparaginase as a medicament for treating pancreatic cancer.

A second object of the invention is a therapeutic composition or a medicament intended for the treatment of pancreatic cancer, comprising an effective quantity of a suspension of RBCs encapsulating asparaginase. In some embodiments, the composition further comprises free asparaginase in an amount effective to elicit in a patient an initial pulse of reduction of asparagine and/or glutamine, followed by a delayed increase to about baseline level (s). By "delayed increase" it is meant that the asparagine and/or glutamine levels increase to about baseline level(s) more slowly than if the same amount of free asparaginase had been administered without the red cell-encapsulated asparaginase. Accordingly, in embodiments where some portion of the administered asparaginase is free and some portion is encapsulated, the presence of the encapsulated asparaginase reduces the rate at which the level(s) of asparagine and/or glutamine recover from the initial pulse(s) of depletion.

In yet other embodiments, other forms of asparaginase may be usefully combined with chemotherapeutic agents in the practice of the invention. For example, covalently or otherwise modified forms of asparaginase may be used to achieve desired magnitudes and durations of asparagine and glutamine reduction. In particular, PEGylated, or PASylated, or otherwise modified asparaginases having desirable in vivo half-lives may be used in the practice of the invention.

In some embodiments, the asparaginase may be RYLAZE™ (asparaginase erwinia chrysanthemi (recombinant)-rywn), as defined at the time of this filing. In some embodiments, the asparaginase may also be crisantaspase or pegaspargase.

A further object of the invention is the use of red cells encapsulating asparaginase or a suspension of such cells for the preparation of a medicament intended for the treatment of a pancreatic cancer. In some embodiments, the pancreatic cancer is second line (2L). The invention relates to the treatment of patients whatever the stage of development of the pancreatic cancer, the histological form taken by the cancer and the likelihood of pancreatitis of greater or lesser severity. In some embodiments, the cancer is in a late stage, and has progressed despite the administration of at least one prior therapeutic regimen. Accordingly, it is envisioned that the disclosed compositions and methods would be useful for 3L pancreatic cancer in addition to 2L.

In some embodiments, the invention provides a method of treating a patient having one or more of the following: a primary tumor of the pancreas; local adenopathy, with or without affected local lymphatic ganglia; pancreatic cancer with remote metastases; cancer of the head of the pancreas; pancreatic cancer with ductal adenocarcinoma; pancreatic cancer with mucinous cystadenocarcinoma; pancreatic cancer with mucinous intraductal carcinoma; pancreatic carcinoma with acinar adenocarcinoma; pancreatic cancer with cystic tumors including cystadenocarcinoma; pancreatic cancer with tumor of the excretory canals of the pancreas; cancer of the endocrine pancreas; and/or a partial or total resection of the pancreas.

In some embodiments, the addition of red cells encapsulating asparaginase to a chemotherapeutic regimen increases the survival and/or progression-free survival of a patient suffering from pancreatic cancer. In some embodiments, administration of the red cells encapsulating ASNase (eryaspase) enhances the efficacy of one or more of the non-asparaginase therapeutic agents.

In some embodiments, red cells encapsulating asparaginase may be administered by intravenous or intra-arterial infusion and/or injection.

In some embodiments, a dose of eryaspase may be from about 150 to about 350 ml of suspension.

In some embodiments, the amount of encapsulated asparaginase may be from about 30 to about 300 III per ml of encapsulated red cell suspension.

In some embodiments, the suspension of red cells encapsulating asparaginase further comprises free asparaginase. In some embodiments, the ratio of free asparaginase to red cell-encapsulated asparaginase may be about 0:100, from about 1:99 to about 10:90, about 2:98 to about 9:91, about 3:97 to about 8:92, about 4:96 to about 7:93, about 5:95 to about 6:94, or about 6:94. In some embodiment, repeated doses of a combination of free and encapsulated asparaginase are administered at about 7 to about 21 day intervals. In some embodiments, the interval may be about 10 to about 18 days, about 11 to about 17 days, about 12 to about 16 days, about 13 to about 15 days, or about 14 days.

A treatment may comprise the administration of one dose or of several doses according to the protocol decided. Treatment may be administered at monthly, biweekly or weekly intervals, over the recommended duration of the treatment.

In some embodiments, treatment comprises administration of about 50 to 150 III asparaginase encapsulated in red cells per kg per dose.

The term "asparaginase" includes asparaginase (ASNase) of any origin, naturally-occurring or recombinant, including modifications thereof (e.g. PEGylated ASNase), and active fragments retaining L- asparaginase activity.

Example 1. A Randomized, Phase 3 Study of Eryaspase in Combination with Chemotherapy versus Chemotherapy Alone as Second-Line Treatment in Patients with Pancreatic Adenocarcinoma

Primary Objective. To determine whether the addition of eryaspase to chemotherapy improves overall survival (OS) in second-line treatment of pancreatic adenocarcinoma (PAC) compared to chemotherapy alone.

Secondary Objectives. 1) To compare PFS, ORR, DoR, and DCR, between the two treatment arms. 2) To evaluate the safety and tolerability of eryaspase in combination with chemotherapy versus chemotherapy alone. 3) To assess the effect of eryaspase on quality of life using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ- C30). 4) To determine the pharmacokinetics (PK) of eryaspase. 5) To assess the immunogenicity of eryaspase in terms of the induction of anti-ASNase antibodies and neutralizing antibodies. 6) To evaluate the relationship of clinical outcome with relevant biomarkers and genetic changes present in tumor tissues and blood and/or serum samples. 7) To investigate the predictive relationship between the patient's deoxyribonucleic acid (DNA) sequence variation, e.g. exploratory single nucleotide polymorphism (SNP) genotyping in select candidate genes, and their response to combination treatment in terms of safety and tolerability (pharmacogenetics [PGx]).

Primary and Secondary Endpoints. Primary: Overall Survival (OS) in the ITT population. Secondary: Progression-free Survival (PFS); Objective Response Rate (ORR); Duration of Response (DoR), Disease Control Rate (DCR), Pharmacokinetics (PK) and Pharmacodynamics (PD) induction of Anti-ASNase antibodies (Abs), Adverse events (AEs), Quality of life (QoL), Biomarkers, and the like.

Study Design and Methodology. This is an open-label, multicenter, randomized, Phase 3 study in patients with adenocarcinoma of the pancreas who have failed only one prior line of systemic anti-cancer therapy for advanced pancreatic cancer and have measurable disease as defined by the modified Response Evaluation Criteria in Solid Tumors (RECIST 1.1) criteria. After provision of informed consent and conduct of screening assessments, patients who meet all inclusion and no exclusion criteria will be randomized in a 1:1 ratio to one of the following treatment arms: Arm A (investigational arm): eryaspase in combination with either gemcitabine/Abraxane or irinotecan-based therapy (FOLFIRI [FOLinic acid- Fluorouracil-IRInotecan regimen] or Onivyde®/5-fluorouracil/leucovorin); Arm B (control arm): gemcitabine/Abraxane or irinotecan-based therapy (FOLFIRI or Onivyde/5-fluorouracil/leucovorin). Eryaspase will be administered at 100 U/Kg given on Days 1 and 15 of each 4-week cycle and each chemotherapeutic agent will be administered according to its label and standard of practice.

Study Phases. 1) Screening (eligibility screening assessments within 3 weeks of randomization). 2) Randomization (after patient determined eligible). 3) Treatment (first chemotherapy dose administered within 3 days after randomization). Treatment continues until objective disease progression, unacceptable toxicity, or patient withdrawal of consent. Unacceptable toxicity determined at Investigator's discretion, but as a guidance, unacceptable toxicity could consist of prolonged Grade 3 or 4 toxicity lasting more than 2 weeks. 4) Follow-up (patients will be monitored for survival at 8-week intervals). Patients who discontinue treatment for reasons other than DP will continue to be assessed radiologically every 8 weeks until DP, or until withdrawal from the study, or death. A patient is considered to have "completed the study" if he or she has completed all phases of the study including the last visit or the last scheduled procedure shown in the Schedule of Events (SOE), Table 2. The "end of the study" is defined as completion of the last patient last visit.

In the investigational treatment arm (Arm A), eryaspase will be administered on Day 1 and Day 15 of each 4-week cycle in combination with chemotherapy. Eryaspase will be administered by intravenous (IV) infusion over approximately 60 minutes, followed by one hour of rest and then followed by chemotherapy infusion. Chemotherapy will be one of the following two treatment regimens: Gemcitabine and Abraxane combination chemotherapy or Irinotecan-based therapy: FOLFIRI (FOLinic acid- Fluorouracil-IRInotecan regimen) or Onivyde/5-fluorouracil (5-FU)/leucovorin (LV).

The choice of the chemotherapy regimen for a particular patient will be determined by the prior treatment received in the first-line setting. If a patient received prior gemcitabine/Abraxane in the first- line setting, then on disease progression, the patient will be assigned to FOLFIRI (or Onivyde/5-FU/LV) in the current study. If a patient received prior irinotecan-based therapy (FOLinic acid-Fluorouracil- IRInotecan-Oxaliplatin; FOLFIRINOX), then on disease progression, the patient will be assigned to gemcitabine/Abraxane in the current study. Patients will be randomized in a 1:1 ratio to chemotherapy with or without eryaspase. Randomization will be stratified according to the following factors: 1) ECOG performance status (0 or 1); 2) Chemotherapy regimen in this study (gemcitabine/Abraxane or irinotecan-based treatment [FOLFIRI or its equivalent Onivyde/5-FU/LV]); and 3) Time interval since initial diagnosis of advanced disease to date of randomization in the study (<6 months or >6 months). An Independent Data Monitoring Committee (IDMC) was established to review safety and efficacy data at regular intervals, including the interim and final analyses.

Safety Evaluations. Clinical and laboratory parameters and adverse events will be assessed in all patients to evaluate disease status and toxicity. Patients will have safety assessments (laboratory tests, physical exams, vital signs including temperature, heart rate, and blood pressure, electrocardiograms [ECGs; to be performed during screening and then as clinically indicated], and performance status score) performed on Day 1 and Day 15 of each cycle and at the end of treatment. Adverse event reporting will begin at the time of informed consent signature. Adverse events and concomitant medications will be collected until 90 days after the last dose of study treatment, or until start of a new anti-cancer treatment whichever is sooner.

Efficacy Evaluations. Tumor assessments utilizing thoraco-abdominal computed tomography (CT)/MRI) scans will be repeated every 8 weeks, calculated from the date of randomization, until disease progression, or until withdrawal from the study, or death. Every effort should be made to adhere to the assessment schedule. The same imaging technique must be used throughout the study. For patients who discontinue treatment for reasons other than objective disease progression, and patients who start new anti-cancer treatment without evidence for objective disease progression, disease status evaluation will continue to be repeated every 8 weeks, calculated from the date of randomization, until disease progression. All radiological images must be collected in a de-identified manner, quality controlled, stored, and available for future review, including independent radiological review as necessary.

Survival information will be collected by phone, follow-up visit, or medical records review every 8 weeks from the date of the EOT visit until the patient's death, until the patient is lost to follow-up, or until study closure. Survival follow-up information will include collection of any subsequent anticancer therapy received after discontinuation from study medication. Quality of life (QoL) assessment (EORTC QLQ-C30) will be performed, at Day 1 of each cycle prior to dosing, at the end of treatment visit and every 8 weeks during survival follow up.

PK, PD, and Translational Research. Where possible, blood and plasma samples will be collected in the eryaspase arm for PK and PD determination, and for immunogenicity evaluation. Blood/plasma samples for PK and PD assessments will be collected at the following time points of Cycles 1 and 3 of study treatment: Day 1 prior to eryaspase administration, at 5-10 minutes post-eryaspase-infusion, at 5-8 days post-infusion (at the Investigator's discretion), and at Day 15 pre-dose. Samples will be analyzed for whole blood and plasma concentrations of ASNase and amino acids. The sparse PK data will be combined with previous data as part of a Population PK (POP PK) analysis. Samples for assessment of anti-L-ASNase Abs and neutralizing Abs will be collected pre-dose at Cycle 1 Day 1 and Day 15, at Day 1 of every second cycle thereafter, upon determination of disease progression, and at the EOT visit, as specified in the SOE. Exploratory biomarker analyses will examine potential predictive biomarkers correlating with eryaspase activity. Tissue samples will be collected at study start. In addition, blood/plasma samples for biomarker analysis will be collected from all patients at Cycle 1 Day 1 and Day 15, at Day 1 of every second cycle thereafter, upon determination of disease progression, and at the EOT visit. A blood sample for pharmacogenetic (PGx) analysis will be obtained once during the study, preferably during the screening phase, for patients who consent to this optional procedure.

"Intent to Treat" (ITT) population means all patients randomized, irrespective of whether they received study medication.

"Safety population" (SP) means all randomized patients who receive at least one dose of study medication (eryaspase or chemotherapy).

"Per Protocol" (PP) population is a subset of the ITT population, and consists of all randomized patients who meet the major inclusion criteria and none of the major exclusion criteria and who receive at least one cycle of treatment.

The target sample size was 482 patients (though over 500 were ultimately enrolled). With a power of 88.4% and an overall one-sided type I error of 2.5%, and including one interim analysis for efficacy with an O'Brien-Fleming-type stopping rule, a total of 390 deaths are required to detect a treatment effect hazard ratio of 0.725 for eryaspase plus chemotherapy versus chemotherapy alone. The interim analysis for efficacy is planned to take place once 261 (67%) events have been observed. Assuming a recruitment period of 26 months, a median overall survival in the control group of about 6.0 months, an about 10% probability of dropping out during the course of the study, and a minimum follow-up of about 9 months, the study size will be based on the recruitment of 482 patients.

As regards readouts, one interim analysis for superiority after 67% of the initial targeted numbers of events are observed. The primary analysis will be the comparison of OS between the two treatment arms in the ITT population using the one-sided stratified log-rank test, stratified for ECOG status, chemotherapy regimen, and time from diagnosis of advanced disease. Secondary analyses include PFS will be compared between the two treatment arms using the same method of analysis as for OS.

The following efficacy analyses will also be performed in the ITT population: ORR, defined as the proportion of patients who achieve objective tumor response (complete response [CR] or partial response [PR]) per RECIST 1.1. Each patient overall response (BOR) will be summarized (CR, PR, stable disease [SD], progressive disease [PD], or unknown); and DCR (disease control rate), defined as the proportion of patients who achieve CR, PR and SD. DoR will be evaluated in patients who achieve CR/PR. It will be measured from the time CR/PR (whichever is first recorded) is first met to until the first date that recurrence or PD is objectively documented. Extensive evaluation of the consistency of treatment effect for OS and PFS across the population as a whole will be undertaken by providing analyses in subgroups, with displays in forest plots and p-values for interaction. All efficacy analyses will be repeated in the PP population.

Confirmatory testing for the primary and secondary efficacy endpoints will be performed hierarchically (OS->PFS->DCR->ORR) in order to account for multiplicity. Any statistically significant findings occurring below a non-significant result in the hierarchy will be considered as exploratory findings.

Safety and tolerability of eryaspase in combination with chemotherapy versus chemotherapy alone will be assessed by analyzing AEs (incidence, intensity, seriousness, and causal relationship of AEs to the study drug, action taken following AE), drug exposure (duration of treatment), dosing information/compliance, concomitant medications, clinical laboratory results, ECG findings, vital signs, physical examination, body weight, ECOG PS, and treatment and study termination status data.

Table 2. Schedule of Events

Abbreviations: D=Day; Rand=Randomization; BP=b ood pressure; ECG=electrocardiogram; CT=computed tomography; MRI=magnetic resonance imaging;QoL=quality of life; EORTC QLQ-C30= European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30; AE=adverse event; SAE=serious adverse event; IAST=irregular antibody screening test; CA19-9=cancer antigen 19-9; ctDNA=circulating tumor deoxyribonucleic acid; ASNase=asparaginase.

1. There must be a minimum of 10 days between 2 eryaspase injections.

2. Per ECOG/Karnofsky scales (KPS (if required per ECOG score) only during screening and at Cycle 1 Day 1).

3. Performed during screening, and then as clinically indicated.

4. Radiological assessment to be completed within 3 weeks of randomization and then every 8 weeks (±3 days) from time of randomization until disease progression, start of subsequent anticancer therapy or until withdrawal from the study, or death, using the same method throughout. Tumor assessments will continue in patients, who start new cancer treatment without evidence of disease progression Every effort should be made to adhere to the evaluation schedule, irrespective of any treatment delays or modifications. Bone and/or brain scans are to be repeated every 12 weeks if clinically indicated.

5. Randomization performed via interactive web response system (IWRS) within 3 days of 1st chemotherapy dose.

6. During follow-up, questionnaire is to be completed every 8 weeks.

7. A complete RBC phenotype (including D, C, E, c, e, K and other antigens tested as per site practice), ABO blood group status, and Rhesus factor, all assessed on two separate samples (can be collected on the same day), to be done as soon as possible but preferably at least 5 working days before the first eryaspase infusion. Exact instructions are provided in the Investigational Medicinal Product (IMP) Manual. Historical results of IAST can be used at screening if known and available.

8. Prescription form indicating the patient's identifiers as well as his/her most recently collected weight, the Investigator recipient of the product, and the place and time of the delivery must be sent as soon as possible once the eryaspase infusion is scheduled, and at the latest 5 working days prior to Cycle 1, Day 1 and then 5 working days prior to each subsequent eryaspase infusion. Exact instructions are provided in the IMP Manual.

9. IAST must be completed within 72 hours prior to each eryaspase administration. The results (of the previous infusion) will be provided to the Sponsor along with the prescription form prior to subsequent eryaspase infusion.

10. In case of incompatibility, collection of an additional blood sample will be required for further investigation.

11. Laboratory tests to be performed at local laboratory as follows: Hematology: Complete blood count with differential (hemoglobin, hematocrit, RBC count, white blood cell (WBC) count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, and platelet count); Biochemistry: Sodium, potassium, bicarbonate, calcium, chloride, creatinine, albumin, ammonia, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase, glucose, urea, triglycerides, total cholesterol, amylase, lipase and uric acid; Coagulation parameters fibrinogen, antithrombin III; Tumor marker: CA19-9.

12. Baseline labs to be collected within 14 days of randomization.

13. For patients of childbearing potential, serum pregnancy test to be performed during screening period and at End of Treatment and urine pregnancy test to be performed prior to dosing of any chemotherapy agent Day 1 of each cycle. Additional pregnancy testing should be performed in case of delayed menstrual period and is recommended to be performed monthly and until the end of treatment exposure extended by 30 days, in case of sexual activity.

14. Pre-dose Day 15 sample to be collected only during Cycle 1.

15. Where possible, samples to be collected Cycles 1 and 3 of study treatment on Day 1 prior to eryaspase administration, 5-10 minutes post-eryaspase infusion, 5 to 8 days post-infusion, and on Day 15 pre-dose.

16. Survival follow up to be conducted 8 weeks after the patient's End of Treatment visit by phone, visit, or medical records review every 8 weeks (±3 days) from the date of the EOT visit (or date of last dose of study medication if patient is unable to come in due to declining health) until patient's death, lost to follow up, or study closure. Subsequent therapy should be collected during this follow-up. 17. For patients under eryaspase arm, in addition to regular evaluation of liver function tests before treatment administration, patients will be monitored on a weekly basis in the event of occurrence of >Grade 2 elevation of liver enzymes and bilirubin levels.

18. Performed pre-dose Cycle 1 Day 1 and then pre-dose Day 1 of every second cycle thereafter i.e., Cycle 3 Day 1, Cycle 5 Day 1, upon determination of disease progression, or EOT, whichever is sooner.as clinically indicated.

** Additional lab tests may be required (e.g., weekly monitoring per standard practice or label requirements should be followed) and should be reported in the electronic Case Report Form (eCRF) if clinically significant.

Table 3. Abbreviations, Terms

Patient Eligibility

The following inclusion criteria are for purposes of the trial described herein. Performance of the embodiments of the present invention does not limit subject to these inclusion criteria.

1. Must be 18 years of age or older.

2. Must have histologically confirmed PAC.

3. Must have Stage III or IV disease.

4. Must have received one line of systemic chemotherapy in advanced setting with or without targeted agents, immunotherapy, or radiotherapy for treatment of advanced pancreatic adenocarcinoma. NOTE: patients whose disease progresses on, or within 3 months of neo(adjuvant) chemotherapy, may be considered eligible.

5. Must have radiological evidence of disease progression following most recent prior treatment, defined as appearance of any new lesion or increase of >20% of one or more existing lesions.

6. Must have measurable lesion(s) per RECIST version 1.1 by CT scan with contrast (or MRI, if the patient is allergic to CT contrast media). Measurable disease may be in the field of prior irradiation; however, at least 4 weeks must have elapsed between the completion of radiation therapy and the baseline scan documenting disease status. Bone disease is considered radiologically measurable only if there is at least a 50% lytic component. NOTE: Bone disease consisting of blastic lesion only is not measurable.

7. Archival or fresh tumor tissue must be available for evaluating relevant biomarkers. Formalin-fixed paraffin-embedded [FFPE] block preferred, or a minimum of 10 unstained FFPE slides of one archived block is required. NOTE: if archival tissue is unavailable and an elective biopsy cannot be scheduled due to COVID, this will be waived. NOTE: Cytology samples from fine needle aspirates or brushing biopsies are not sufficient.

8. Must have adequate performance status: a. ECOG Performance Status (PS) score of 0, or b. ECOG PS score one and score >80 on Karnofsky Performance Status (KPS) scale. NOTE: Must have body mass index (BMI) >18.5 kg/m² (obtained <14 days prior to randomization.

9. Must have life expectancy of >12 weeks according to the investigator's clinical judgment.

10. Females of childbearing potential must have a negative pregnancy test at screening and additional negative pregnancy test prior to first dose. Males and females of childbearing potential must agree to use a highly effective method of contraception during treatment and for at least 6 months after the last dose of study treatment. These include, but not limited to: a. combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation: i. intravaginal, ii. transdermal b. progestogen-only hormonal contraception associated with inhibition of ovulation: i. injectable, ii. implantable c. intrauterine device (IUD) d. bilateral tubal occlusion e. vasectomized partner f. sexual abstinence (defined as refraining from heterosexual intercourse during the entire period of risk associated with the study treatments) is intended. The true abstinence is when this is in line with the preferred and usual lifestyle of the patient. [Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods) and withdrawal are not acceptable methods of contraception], g. males with partners of childbearing potential must agree to use condoms. NOTE: Since an indirect interaction between components of the oral contraceptives and ASNase cannot be ruled out, oral contraceptives are not considered acceptable as contraceptive methods in the current clinical trial. A method other than oral contraception should be used in women of childbearing potential. NOTE: All chemotherapeutic agents may be teratogenic and excreted in breast milk. Patients who are breast feeding should consider alternative methods.

11. Must have adequate laboratory parameters at baseline (obtained <14 days prior to randomization). Laboratory parameters outside of these ranges that are deemed clinically insignificant should be discussed with the medical monitor: a. Absolute neutrophil count > 1.5 x 10⁹/L. b. Hemoglobin >9 g/dL. Patients with a baseline Hemoglobin 13 g/dL should be discussed with the medical monitor. c. Platelet count > 100,000/mm³ (100 x 10⁹/L). d. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <2.5 x upper limit of normal (ULN) x ULN in presence of liver metastases). e. Total bilirubin < 1.5 x institutional ULN. f. Serum creatinine within normal limits or calculated clearance >60 mL/min/1.73 m² for patients with serum creatinine levels above or below the institutional normal range. g. Acceptable coagulation parameters: plasma antithrombin III >70% and fibrinogen >1.5 g/L. h. Serum albumin >3.0 g/dL.

12. Patients requiring biliary stent placement must have the biliary stent placed >7 days prior to screening and must have normalization of bilirubin level after stenting.

13. Must not be receiving therapy in a concurrent clinical study and must agree not to participate in any other interventional clinical studies during their participation in this trial while on study treatment. Patients taking part in surveys or observational studies are eligible to participate in this study.

14. Must be able to understand and comply with the conditions of the protocol and must have read and understood the consent form and provided written informed consent.

A patient is not eligible to participate in the study if any of the following exclusion criteria are met:

1. Resectable or borderline resectable PAC at the time of signing theinformed consent.

2. Histology other than PAC (e.g., neuroendocrine, adenosquamous, etc.).

3. More than one line of prior treatment in advanced or metastatic setting.

4. Patient has experienced medically significant acute decline in clinical status including a. ECOG PS down to >1 (or KPS <70) between baseline visit and 72 hoursprior to randomization. b. Weight loss of >10% during screening.

5. Presence of active or symptomatic untreated central nervous system (CNS) metastases. NOTE: Patients with asymptomatic or stable CNS metastases are eligible, provided thatthe CNS metastases are radiologically and clinically stable, and the patient is off high- dose steroid treatment for at least one month prior to randomization.

6. Prior radiotherapy to the only area of measurable disease. NOTE: Patients must have completed treatment and recovered from all acute treatment-related toxicities prior to administration of the first dose of eryaspase or chemotherapy.

7. Bone as the only site of metastatic disease from pancreatic cancer (bone-only disease).

8. History of recent clinical pancreatitis, according to revised Atlanta criteria, within3 months of randomization. NOTE: The revised Atlanta classification [1] requires that two or more of the following criteria be met for the diagnosis of acute pancreatitis: (a) abdominal pain suggestive of pancreatitis, (b) serum amylase or lipase level >3 x ULN, or (c) characteristic imaging findings using CT or MRI.

9. Neurosensory neuropathy > Grade 2 at baseline.

10. Pregnancy or breastfeeding.

11. History of infection with human immunodeficiency virus (HIV) and/or active infection with hepatitis B or hepatitis C. NOTE: Patients with unknown status of hepatitis B or C must be tested and declarednegative before randomization.

12. Hypersensitivity to any of the components of the chemotherapy or ASNase. NOTE: Patients known to be homozygous for UGT1A1*28 who are assigned to an irinotecan-containing regimen must have the initial irinotecan dose reduced unless they have previously tolerated full doses of irinotecan. Subjects whose UGT1A1 status is not known but are being considered for irinotecan-based chemotherapy must be screened for UGT1A1*28 allele unless they have previously tolerated full doses of irinotecan before enrollment into the trial and must have the initial irinotecan dose reduced if demonstrated to be homozygous for the UGT1A1*28 allele. NOTE: Patients assigned to the irinotecan/5-FU arms in the study should not have dihydropyridine dehydrogenase deficiency (DPD). Patients whose DPD status is unknown at time of screening should be tested before enrollment in the irinotecan/5-FUarm unless they have previously tolerated full doses of 5-FU.

13. Patients who have received live or live attenuated vaccines within 3 weeks ofrandomization.

14. History of other malignancies. NOTE: Adequately treated non-melanoma skin cancer or curatively treated in-situ cancer of the cervix may be eligible. NOTE: Patients successfully treated for other malignancies and are disease-free for atleast 5 years may be eligible.

15. Any other severe acute or chronic condition/treatments that may increase the risk of study participation including: A) History of abdominal fistula, gastrointestinal perforation, peptic ulcer, or intraabdominal abscess within 6 months prior to randomization. B) Current or history within 6 months prior to randomization of medically significant cardiovascular disease including symptomatic congestive heart failure >New YorkHeart Association (NYHA) Class II, unstable angina pectoris, clinically significantcardiac arrhythmia. C) Patients with pre-existing coagulopathy (e.g. hemophilia). D) Psychiatric illness/social situations or any other serious uncontrolled medical disorders in the opinion of the Investigator that would limit compliance with studyrequirements.

The "study drugs" include all chemotherapy agents used in both treatment arms, as well as the "investigational drug" which is eryaspase ("Investigational Medicinal Product"). The chemotherapy agents used in this study (gemcitabine, Abraxane, irinotecan, and 5-FU/leucovorin) are commercially available and used in accordance with approved labeling. Eryaspase is a dispersion for infusion of allogeneic RBCs encapsulating recombinant E. coli L-ASNase, in a saline preservative solution. Eryaspase is an off-shelf investigational agent. Eryaspase is produced for each individual patient in view blood group and phenotype and dose of ASNase appropriate for body weight.

Gemcitabine is an antineoplastic agent. It is commercially available as a lyophilized powder for solution for IV use. Gemcitabine will be reconstituted/used as per the manufacturer's suggestions and will be administered as per institutional procedure.

Abraxane (protein-bound paclitaxel) is a microtubule inhibitor. It is commercially available as a lyophilized powder in a single-use vial for reconstitution for IV use. Abraxane will be reconstituted/used as per the manufacturer's suggestions and will be administered as per institutional procedure.

Gemcitabine and Abraxane will be sourced locally. Investigators are responsible for ensuring that patients receive supplies of gemcitabine and Abraxane for the entire duration of the study treatment, except in countries where regulatory authorities mandate that the Sponsor must supplyall medications required for study participation.

Irinotecan is an antineoplastic agent of the Topoisomerase I inhibitor class. It is commercially available as an aqueous solution intended for dilution prior to IV infusion. Irinotecan will be reconstituted/used as per the manufacturer's suggestions and will be administered as per institutional procedure.

Onivyde (liposomal irinotecan) is a Topoisomerase I inhibitor. It is commercially available as an opaque liposomal dispersion in a single-dose vial for IV infusion. Onivyde will be reconstituted/used as per the manufacturer's suggestions and will be administered as per institutional procedure.

5-FU is an antineoplastic antimetabolite agent. It is commercially available as an injectable solution for IV use. 5-FU will be administered as per institutional procedure.

Leucovorin is folinic acid (the active metabolite of folic acid). It is an essential coenzyme fornucleic acid synthesis and is used to enhance the cytotoxicity of 5-FU. It is commercially available as a powder for reconstitution for IV use. Leucovorin will be administered as per institutional procedure.

Irinotecan or Onivyde and 5-FU/leucovorin will be sourced locally, investigators are responsible for ensuring that patients receive supplies for the entire duration of the study treatment, except in countries where regulatory authorities mandate that the Sponsor must supply all medications required for study participation.

At the treatment initiation, eryaspase will be prepared and dispatched after all screening assessments have been completed and the results reviewed and after it has been confirmed thatthe patient meets all eligibility criteria.

The dose of eryaspase is 100 U/Kg, to be administered at Days 1 and 15 of each 4-week cycleuntil disease progression, unacceptable toxicity, or withdrawal of consent. Eryaspase pre-administration acceptance criteria include the following: <6% free asparaginase in the bag, [note to Draft: include others, but think about which ones actually might contribute to efficacy]

Eryaspase is administered over approximately 60 minutes per bag, depending on the volume of the bag(s). The entire content of each bag is to be administered, unless otherwise specified. Eryaspase administration must be completed before the expiry time clearly stated on the label ofthe eryaspase bag. Date of administration, start and end times of infusion, and volume administered will berecorded.

Gemcitabine and Abraxane are administered on Days 1, 8, and 15 of each 4-week cycle: Abraxane: 125 mg/m² IV over 30-40 minutes, followed byGemcitabine: 1000 mg/m² IV over 30 minutes.

In more detail, Onivyde (irinotecan nanoliposomal) + 5-FU/leucovorin are to be administered on Days 1 and 15 of each 4-week cycle as follows: Onivyde 70 mg/m² IV over 90 minutes (recommended starting dose of Onivyde inpatients homozygous for UGT1A1*28 is 50 mg/m²). Leucovorin 400 mg/m² IV over 30 minutes, though modifications to the leucovorin dose per sites' standard administration protocol may be allowed in the study following approval from the medical monitor. 5-FU 2400 mg/m² over 46 hours. FOLFIRI (irinotecan, 5-FU, and leucovorin) is to be administered every 2 weeks on Day land Day 15 of each 4-week cycle as follows: Irinotecan 180 mg/m² IV infusion over 90 minutes (recommended starting dose ofirinotecan in patients homozygous for UGT1A1*28 is 150 mg/m²); Leucovorin 400 mg/m² IV infusion over 2 hours, though the leucovorin dose may be modified as discussed immediately supra. 5-FU 400 mg/m² IV bolus injection over 2-4 minutes, immediately following leucovorin infusion, and 5-FU 2400 mg/m² IV continuous infusion over 46 hours, immediately following bolus 5-FU.

Patients will be treated until disease progression, unacceptable toxicity as determined by the Investigator, or withdrawal of consent. Each patient will be evaluated for drug-related toxicity. Ifa patient experiences unacceptable toxicity for more than two weeks, then such patients should be withdrawn from study treatment. Unacceptable toxicity will be determined at the investigator's discretion; however, as a guidance, unacceptable toxicity could consist of prolonged Grade 3 or 4 toxicity not resolving within two weeks. The medical monitor must beconsulted if any patient requires further treatment delays or dose modifications.

Throughout the study, the following toxicity criteria will be used to guide treatment modifications and treatment delays. However, standard practice at sites may also be referred to, particularly for any of the chemotherapy components. Appropriate supportive care will be provided for the management of drug-related toxicities. In the instance of AEs that require course changes, the general guidance is delaying dose as opposed to skipping a dose. Eryaspase dose modification. One 25% dose reduction of eryaspase is permitted at the first occurrence of an eryaspase-related toxicity. Additional dose reduction to 50% is allowed on second instance. If further dose reductions are necessary, then eryaspase should be discontinued. Table 4 presents guidelines for carrying out eryaspase dose reductions when deemed appropriate. Dose reduction approach will depend on whether the drug is at site and ready for infusion (during a cycle) or is planned for a subsequent cycle. Dose reduction during a cycle will follow an empirical estimation of the volume in the eryaspase bag. Dose reduction in subsequent cycles will be reflected in the prescription form and will therefore be carried out by reducing the dose level per unit of body weight to be used in manufacturing the drug for the next cycle(s).

Table 4. Pre-Specified Dose Reductions for Adverse Events Related to Eryaspase

After a dose reduction, the dose of eryaspase may be re-escalated to the previous higher dose level, provided that toxicity resolves to at Grade 1 (or 2 where specified). Treatment with eryaspase may be delayed for up to 2 weeks at the first instance of an eryaspase-related toxicity to allow for resolution of toxicity, defined as a return to <Grade 2, where indicated. The sponsor's Medical Monitor must be consulted if any patient requires further treatment delays or dose modifications. If eryaspase-related AEs do not resolve after 2 weeks, the patient should discontinue eryaspase and should then complete the EOT visit and Follow-up visits per the Schedule of Events (Table 2).

For agents other than eryaspase, the product labeling information and local institutional practice should be referred to regarding hematological and non-hematological AEs attributable to the individual chemotherapy agents used in this study. In addition, it is recommended that patients receive premedication according to respective product labeling information. In case of Abraxane and Onivyde, premedication standard doses of dexamethasone (or an equivalent corticosteroid) together with a 5- hydroxytryptamine 3 (5-HT3) antagonist (or other antiemetic) at least 30 minutes prior to start of chemotherapy infusion. Table 5 presents a summary of dose modifications for gemcitabine, Abraxane, and eryaspase. Patients who experience adverse events will be re-evaluated weekly, or more often according to the Investigator's discretion. Dose delays or reduction for gemcitabine or Abraxane follow the respective prescribing information and are provided in the table below for ensuring consistency across sites. If toxicities do not resolve within two weeks, then the patient will discontinue the study treatment, but will continue to be followed for 90 days or until start of new anti-cancer treatment, whichever is first, for adverse events and serious adverse events. Thereafter, the patient will be followed for radiological disease assessment and survival until withdrawal from study or death.

Table 5. Dose Modifications for Gemcitabine, Abraxane, and Eryaspase

(1) Infusion may be resumed at a lower infusion rate, provided that it is within shelf-life and the infusion iscompleted within the 6-hour window of bringing eryaspase to room temperature.

Table 6 presents a summary of dose modifications for irinotecan, Onivyde, and eryaspase. Patients who experience adverse events will be re-evaluated weekly, or more often according to the investigator's discretion. Dose delays or reductions for irinotecan or Onivyde follow the respective prescribing information, and are provided in the table below for consistency across sites. If toxicities do not resolve to <Grade 2 within two weeks, then the patient will discontinue the study treatment, but will continue to be followed for 90 days or until start of new anti-cancertreatment, whichever is first, for adverse events and serious adverse events. The patient will be followed for radiological disease assessment and must complete an EOT visit and Follow-up visits per the Schedule of Events (Table 2).

Following 5-FU treatment, mucositis, stomatitis, hand-foot syndrome, and diarrhea occur most commonly. Leukopenia is the usual dose-limiting toxicity after IV bolus administration. Patientswith dihydropyrimidine dehydrogenase deficiency (DPD) are at risk of severe life-threatening toxicity with 5- FU. While severe deficiency is rare, 3-4% of the population has some degree of DPD deficiency. Patients with DPD deficiency cannot be enrolled in the 5-FU arm unless they have tolerated full doses of 5-FU previously. In general, dose modifications and delays follow a similar approach to that used with irinotecan. However, in case of mucositis or stomatitis, only 5-FU (and not irinotecan or eryaspase) should be withheld during the cycle, and resumed at fulldose in subsequent cycles.

Table 6. Dose Modifications for Irinotecan, Onivyde, and Eryaspase

(1) For patients starting irinotecan at a reduced dose of 150 mg/m² due to the presence of UGT1A1*28 homozygous allele, dose should be reduced to 120 mg/m² at first occurrence and an additional decrease ofabout 20% at the second occurrence of toxicity.

(2) Onivyde dose reductions are presented based on a starting dose of 70 mg/m2 irinotecan free base (equivalent to 80 mg/m2 of Onivyde).

(3) For patients starting Onivyde at a reduced dose of 50 mg/m2 irinotecan free base due to the presence of UGT1A1*28 homozygous allele, dose should be reduced to 43 mg/m2 at first occurrence and to 35 mg/m2 at the second occurrence.

(4) Treatment with eryaspase can be resumed, provided that it is within shelf-life and the infusion is completed within the 6-hour window of bringing eryaspase to room temperature.

(5) For mucositis or stomatitis, decrease only 5-FU, not irinotecan and not eryaspase.

Hypersensitivity reactions have been reported with Abraxane, irinotecan, Onivyde, and eryaspase. As such, patients may be pretreated with corticosteroids, diphenhydramine, and/or H2 antagonists as per institutional protocols. The risk of allergic reactions to eryaspase is potentially attributed to a) Ab-mediated reactions to ASNase; b) non-Ab-mediated infusion reactions to ASNase; and/or c) the RBC component. And while hypersensitivity reactions to eryaspase are rare, reactions to ASNase range from local rash or flushing to severe systemic reactions with features such as urticaria, bronchospasm, angioedema, and severe anaphylactic shock. The following provides guidance for addressing hypersensitivity reactions.

Grade 2 Symptoms: Stop administration; Administer IV dexamethasone 10 mg and diphenhydramine hydrochloride (HCL) 25 to 50 mg; After recovery of symptoms, treatment may be resumed once the patient is stable provided the product is not expired and the bag has been out of the cold box for less than 6 hours; Use premedication for subsequent infusions of eryaspase.

Grade 3 and 4 Symptoms (such as hypotension, angioedema, or respiratory distress): Stop administration; Administer IV dexamethasone 10 mg and diphenhydramine HCL 25 to 50 mg. Add adrenaline or bronchodilators as indicated; For Grade 4 toxicity, the treatment should be discontinued. For example, an Infusion Premedication Regimen may include: Dexamethasone 12 mg PO and diphenhydramine HCL 25-50 mg PO approximately 6 to 12 hours prior to the next dose of eryaspase. Management of infusion/transfusion reactions. Allogeneic blood transfusions are associated with immune-related effects in the form of alloimmunization due to the exposure to alloantigens, cytokines, or other cellular components. Transfusion reactions are typically classified into the following entities [38]: i) TRALI; ii) volume overload in susceptible patients with cardiovascular problems; iii) bacterial contamination; iv) acute hemolytic reactions, either immune-related with ABO incompatibility or non- immune related (for example, concomitant medications causing RBC hemolysis, incorrect storage of RBCs, or non-validated administration systems); v) non-hemolytic febrile reactions due to cytokines and other allo-proteins; and vi) allergic reactions, mostly related to unidentified allergens in the donor blood. Management of transfusion/infusion reactions follows well-known published guidelines, as summarized in these publications [39-41], Table 7 provides a summary of patient management for transfusion reactions.

Table 7. General Guidelines for Managing Transfusion Reactions

Management of Diarrhea. Diarrhea is commonly reported with irinotecan and Onivyde, which typically has an onset in 24 hours after starting Onivyde (early onset). A late onset (>24 hours) diarrhea has also been reported. When appropriately managed with anti-diarrheal treatment, diarrhea is generally mild to moderate. These broad general management principles are provided as a guideline to proactively avoid serious complications of diarrhea syndrome. These guidelines do not replace sound clinical judgment. In the event of diarrhea, patients should take loperamide at initial 4 mg dose, followed by 2 mg doses every 4 hours until 12 hours have lapsed without symptoms. Patients should be advised to increase fluid intake (water, sports drinks, clear juices, decaffeinated tea), and modify diet (banana, rice, apples, and toast).

If mild to moderate diarrhea persists for more than 12 hours, loperamide should be taken at a dose of 2 mg every 2 hours. If mild to moderate diarrhea persist after 24 hours despite treatment with loperamide, a cocktail of atropine-diphenoxylate (Lomotil) and loperamide may be considered. Loperamide 2 mg may be alternated with one tablet of Lomotil every 3 hours. Loperamide should not be used for more than 48 consecutive hours due to risk of paralytic ileus. Oral antibiotic support (e.g. fluoroquinolone for 7 days) should be also considered. If diarrhea persists for more than 48 hours, loperamide should be stopped, and fluid replacement should be considered. For Grade 3 or 4 diarrhea or complicated Grade 1 or 2 (severe cramping, severe nausea/vomiting, decreased performance status, fever, sepsis, Grade 3 or 4 neutropenia, frank bleeding, dehydration), IV fluids should be used as appropriate, as well as prophylactic antibiotics.

Acquisition. Eryaspase will be dispatched to the site only after receipt of the required information in accordance with Erytech procedures. Information to be provided at study start: RBC phenotype (including D, C, E, c, e, K and other antigens tested as per site practice), ABO blood group status, and Rhesus factor, all assessed on two separate samples, which can be collected on the same day. In addition, provide historical results of an irregular antibody screening test (IAST), if available. This information is required at least 5 working days before the first infusion. Prior to Cycle 1 Day 1 and each eryaspase dose: A prescription form indicating patient identifiers and body weight should be provided at the latest 5 working days prior to Cycle 1 Day 1 (for all patients) and each eryaspase infusion (patients in eryaspase arm). Results of IAST performed for previous eryaspase dose to be provided at the latest 5 working days prior to subsequent eryaspase infusion.

Information required prior to shipping eryaspase. The name and contact details of the recipient, who should be the investigator or designee. The location for delivery (local pharmacy or blood bank). Detailed instructions for completing and providing the phenotype, blood group, and Rhesus factor information, the IAST results (from the previous infusion), and the prescription form are provided in the IMP manual. Eryaspase will be shipped to the Investigator in a qualified container by a specialized carrier, who will ensure that the cold chain is maintained between +2-8 °C (35-46 °F). If a temperature excursion outside the range of +2 °C to +8 °C (35-46 °F) occurs, the product should be quarantined and Erytech Pharma should be contacted to determine the usability of the product. If it is not used immediately after receipt, it is mandatory that eryaspase be stored at temperatures between +2 and +8 °C (35-46 °F). Eryaspase may be stored at room temperature for up to 6 hours prior to administration, including infusion time; it must not be stored at room temperature for more than 6 hours.

Accountability. The Investigator or designee is responsible for IMP accountability, reconciliation, and record maintenance (i.e., records of receipt, reconciliation, and final disposition). Further guidance and information concerning the final disposition of unused IMP are provided in the IMP Manual. Please refer to the IB for details of eryaspase formulation and appearance. Eryaspase is packed in medical-grade polyvinyl chloride (PVC) blood bags according to GMP requirements and place in a photoprotective pocket. The final volume of the eryaspase bag depends on the patient's weight and the dose prescribed. The volume of the bag ranges from 50 mL to 300 mL depending on the individual's dose (multiple bags may be required to achieve the full dose). Three (3) removable segment-tubes are attached to the bag for use in blood compatibility testing before administration.

Label statements are specific to the clinical trial and comply with legal requirements for IMPs. The date and time of eryaspase expiration will be noted on the label. In addition, the label displays specific information necessary for traceability of source cell material and medicinal product (blood bank identifier for original RBCs, phenotype, patient identification number, etc.) to allow verification of the patient identity and blood group before administration.

Preparation. Prior to eryaspase administration, an IAST and a complete compatibility test (crossmatch test) between the patient's blood and eryaspase, using the removable segment tubes, must be performed to confirm compatibility. In case a patient receives multiple bags of eryaspase to achieve the intended prescribed dose, separate cross-match tests must be performed with each eryaspase bag to confirm compatibility. Eryaspase should not be transferred to another container before infusion. Eryaspase should not be mixed or administered simultaneously with any other products, solutions, or medicinal products. After eryaspase administration, partially administered and empty bags may be destroyed locally under the biomedical waste disposal process as approved by the specific institution. However, in case of a product defect or quality issue, Erytech must be contacted for further instruction. Additional details will be described in the IMP Manual, which provides detailed instructions related to eryaspase administration and contact information in case of any issues.

The study is an open-label pivotal trial. Investigators and patients will be unblinded to the treatment due to the nature of the investigational therapy. Patients will be randomized to receive either chemotherapy in combination with eryaspase or chemotherapy alone in a 1:1 ratio, using an IWRS. At screening, the IWRS system will assign a unique patient number that will remain constant over the duration of the study. If a patient is rescreened after initial screen failure, the IWRS will allocate the same number. Treatment arm will be automatically determined by the IWRS system. Detailed instructions will be provided in the IWRS manual. Randomization will be stratified by these factors:ECOG PS score (0 or 1), chemotherapy regimen (gemcitabine plus Abraxane or irinotecan-based therapy[FOLFIRI or Onivyde, 5- FU, and leucovorin]), and time interval since diagnosis of advanced disease to date of randomization in the study (<6 months or >6 months). Eryaspase will be administered by IV infusion under the supervision of study personnel. Compliance with the treatment dose and schedule will be documented in the source documents and recorded in the eCRF.

All treatments received within 14 days prior to Cycle 1 Day 1 will be recorded in the eCRF, including the name of the drug, route, indication, start date, and stop date (if applicable). In addition, any and all prior therapies for the treatment of pancreatic cancer (or cancer-relatedevents such as bone pain) will also be recorded in the eCRF.

Patients may receive supportive care for disease-related symptoms and for toxicity associated with study treatment. The date of supportive medication administration as well as the name anddosage regimen of each medication must be recorded in the eCRF.

The following treatments are permitted during the study:

Transfusion of blood products. Transfusion of fresh frozen plasma should be minimized, as it provides an exogenous source of asparagine. It is preferable to administer antithrombin III (AT III) concentrates in case of a decrease in AT III following eryaspase administration. Hemoglobin must be 9 g/dL at Cycle 1 Day 1 and must be 8 g/dL before all subsequent doses. If hemoglobin is <8 g/dL, then appropriate measures must be taken according to standard clinical practice prior to further administration of chemotherapy.

Systemic and inhaled steroid treatment. A standard 3- to 5-day course of dexamethasone following the institutional standard of care is permitted for the prevention of treatment-induced nausea and vomiting. In addition, oral glucocorticoids at a daily dose of 1.5 mg dexamethasone (or equivalent) are permitted.

Palliative radiotherapy (e.g., for painful bone metastases). Prophylactic granulocyte colonystimulating factor (GCSF) in case of neutropenia. Details and indications for use of GCSF should follow American Society for Cooperative Oncology (ASCO) guidelines [42],

During the treatment phase, the following treatments are prohibited: 1) concomitant vaccination with live vaccines, due to increased risk of serious infection; 2) immunization with live or live-attenuated vaccines should take place no earlier than 3 months after completion of eryaspase treatment; 3) other L- ASNase products; 4) highly hemolytic agents that may lead to RBC hemolysis: e.g. acetanilide, antipyrine (phenazone), and chloroquine and derivatives; and 5) prophylactic phenytoin.

Discontinuation from eryaspase and/or chemotherapy treatment does not imply discontinuation from the study, and remaining study procedures should be completed as indicated by the study protocol. If a clinically significant finding is identified after enrollment, the investigator or qualified designee will determine whether any change in patient management is needed. Any new clinically relevant finding will be reported as an adverse event (AE).

A patient may be withdrawn from treatment for any of the following reasons: 1) unacceptable toxicity; 2) objective disease progression following radiological assessment as determined by the investigator (per modified RECIST 1.1 criteria); 3) patient withdrawal of consent for treatment; 4) pregnancy; 5) investigator decision for other reasons, with documentation of reason(s) if not included in any of the above categories. NOTE: Clinical progression must be confirmed radiologically before treatment discontinuation and patient withdrawal. Sponsor decision, including discontinuation of the study, with documentation of reason(s) in the eCRF. The reason for withdrawal will be recorded in the eCRF. The EOT visit must be performed and documented within 30 days after the last dose of eryaspase or chemotherapy. AEs/SAEs and concomitant medications must be collected up to 90 days after the last dose of study treatment or until start of new anti-cancer treatment, whichever is first. For details of the assessments to be performed, refer to the SOE, Table 2. The Sponsor reserves the right to close the investigational site or terminate the study at any time for any reason. If the study is terminated, the Sponsor will notify the Investigators in writing. If the study is prematurely terminated or suspended, the Investigator will promptly inform study participants and the relevant IEC/IRB, and the Sponsor will provide the reason(s) for the termination or suspension to competent regulatory authorities. Study participants will be contacted as appropriate and will be informed of any changes to the study visit schedule. The Investigator may initiate site closure at any time, provided there is reasonable cause and sufficient notice is given in advance of the intended termination. Reasons for the early closure of an investigational site by the sponsor may include but are not limited to:

1) Failure of the Investigator to comply with the protocol, the sponsor procedures, or Good Clinical Practice (GCP) guidelines; 2) Inadequate recruitment of patients by the Investigator. Discontinuation of further drug development; 3) The Investigator will be responsible for completing all study close-out procedures as required by the Sponsor and/or local regulatory authorities if a site is closed or the study is terminated early.

Screening and study procedures and their timings are summarized in the SOE in Table 2. Adherence to the study requirements, including those specified in the SOE, is essential and required for study conduct. Protocol waivers or exemptions are not allowed (with the exception of those in response to immediate safety concerns). All screening evaluations must be completed within 3 weeks prior to randomization, with confirmation that the patient continues to meet eligibility criteria prior to dosing on Cycle 1 Day 1.

All patients must sign and date an Institutional Review Board (IRB)/lndependent Ethics Committee (lEC)-approved Informed Consent Form (ICF) prior to any screening assessments. However, an evaluation conducted as part of the patient routine clinical management (e.g., blood counts, disease evaluation) that is performed before signature of the ICF may be utilized for screening or baseline purposes provided that the procedure met the protocol-specified criteria and was performed within the time frame defined in the SOE (Table 2). Separate informed consent for the optional PGx assessment will be requested at the same time.

Eligibility criteria are to be evaluated within 3 weeks prior to randomization, with confirmation that the patient continues to meet eligibility pre-dose on Cycle 1 Day 1. NOTE: screening period longer than 3 weeks may be allowed due to COVID-19 related restrictions. Screening Assessments to Be Completed within 3 Weeks prior to Randomization: 1) Demography, including gender and date of birth;

2) Medical history, including cancer history and prior anticancer therapies; 3) Complete physical exam, including height and weight (height to be collected at screening only).

Vital signs: temperature, heart rate, and blood pressure. ECOG PS, and KPS if ECOG PS is 1. Laboratory assessments (to be completed within 14 days of randomization): hematology, clinical chemistry, and coagulation panels will be completed at the local laboratory for the parameters defined in Table 8 below. Serum pregnancy test (for patients of childbearing potential): a negative human chorionic gonadotropin test is required before the patient may receive any chemotherapy agent or eryaspase. Moreover, a woman is considered of childbearing potential unless post-menopausal or permanently sterile by one of following methods: hysterectomy, bilateral salpingectomy, and bilateral oophorectomy. A woman is considered postmenopausal in she has not had menses for 12 months without an alternative medical cause.

Collection of a paraffin-embedded tumor tissue block, or a minimum of 10 unstained slides, obtained from an archived or newly obtained tumor tissue sample for biomarker analysis. Whole blood sample for PGx assessment (optional) 12-lead ECG. Baseline disease assessments: Radiological imaging studies: Chest CT scans; Abdomen CT scans; Brain MRI is indicated in patients with known/suspected brain metastasis at study entry. Brain scans will be repeated during the study at 12-week intervals if clinically indicated; Bone scan is indicated in patients with known/suspected bone metastasis at study entry. Bone scans will be repeated during the study at 12-week intervals if clinically indicated. Additional imaging may be performed as clinically indicated. In general, CT scans are the preferred method of radiological assessment of the chest and abdomen; however, other techniques may be used (i.e., MRI). The same imaging technique must be used for a given patient throughout the study. Radiological images must be collected in a de-identified manner, quality controlled, stored, and available for future review and reading as necessary.

Prior and concomitant medications: record all medications the patient has received from 14 days prior to randomization, as well as any and all prior therapies for the treatment of pancreatic cancer (or cancer-related events such as bone pain), and any currently ongoing medications. Collection of AEs and SAEs from the date of informed consent.

Table 8. List of Laboratory Assessments

Patients who are found to be eligible for the study will be randomized to study treatment within 3 days prior to Cycle 1 Day 1. Randomization will be done through the IWRS as described in Section 5.4.2. The following assessment will be performed at least 5 days prior to Cycle 1 Day 1: 1) RBC phenotype (including D, C, E, c, e, K and other antigens as per site practice), ABO blood group status, and Rhesus factor, all assessed on 2 separate samples, whichcan be collected on the same day. This information is required at least 5 working daysbefore the first eryaspase infusion; 2) A prescription form indicating patient identifiers and body weight, the recipient of the product (Investigator or designee), and the place and time of the delivery must be sent as soon as possibleonce the first infusion is scheduled (at the latest 5 working days before planned administration).

The following assessments will be performed on Cycle 1 Day 1 prior to initiation of study treatment. Screening laboratory tests and procedures may be substituted for those on Cycle 1 Day 1 if performed within 5 days prior to Cycle 1 Day 1: 1) Symptom-directed physical examination, including body weight; 2) Vital signs: temperature, heart rate, and blood pressure; 3) ECOG PS, and KPS if ECOG PS is 1 (see APPENDIX 2 and APPENDIX 3). EORTC QLQ-C30; 4) Hematology, clinical chemistry, and coagulation panels and CA19-9, Table 8. Urine pregnancy test for patients of childbearing potential. A negative test is required before administration of any chemotherapy agent or eryaspase; 5) Baseline blood and plasma samples for pharmacokinetic and pharmacodynamic analysis (for patients in the eryaspase arm); 6) Baseline blood samples for immunogenicity (for patients in the eryaspase arm). Baseline blood and plasma samples for biomarker analysis (both arms), such as ctDNA, proteomics, and transcriptomics.

Prior to administration of the first infusion of eryaspase, (and each subsequent infusion during the treatment period), an IAST and complete compatibility (cross-match) test between the patient's blood and eryaspase should be performed using the removable segment tubes to confirm compatibility. In case a patient receives multiple bags of eryaspase to achieve the intended prescribed dose, a separate crossmatch test must be performed with each eryaspase bag to confirm compatibility. In case of incompatibility, an additional blood sample may be required for further investigations. Details of collection and shipment are provided in the IMP Manual.

Radiological disease assessments. CT/MRI of the chest and abdomen will be performed every 8 weeks (±3 days) calculated from the date of randomization until disease progression, patient's withdrawal from study, or death. Every effort should be made to adhere to the assessment schedule, irrespective of any treatment delays or modifications. The same imaging technique must be utilized for a given patient throughout the study. Bone and/or brain scans are to be repeated every 12 weeks if clinically indicated.

Patient-reported outcomes will be measured using a standardized instrument, the EORTC QLQ- C30. The self-administered questionnaires will be completed every 4 weeks during the treatment phase. For patients in the eryaspase arm, blood/plasma samples for pharmacokinetic and pharmacodynamic assessment will be collected, where possible, at the following time points of Cycles 1 and 3: 1) Day 1 prior to eryaspase administration; 2) Day 1 at 5-10 minutes post- eryaspase infusion; and 3) At 5-8 days postinfusion (at the Investigator's discretion) and At Day 15 pre-dose. Samples will be analyzed for whole blood and plasma concentrations of ASNase and amino acids. The sparse PK data will be combined with previous data as part of a Population PK (POP PK) analysis. To this effect, sample collection for PK and pharmacodynamics may be stopped based on the outcome of the evaluation of sample results during the course of the trial.

For patients in the eryaspase arm, samples for assessment of anti-ASNase antibodies and neutralizing antibodies will be collected pre-dose at Cycle 1 Day 1, and Day 15 and at Day 1 of every second cycle thereafter (i.e., at Cycle 3 Day 1, Cycle 5 Day 1, and so on), and upon determination of disease progression, and EOT, whichever is sooner. Plasma samples for biomarker analysis will be collected predose on Cycle 1 Day 1 and Day 15 and at Day 1 of every second cycle thereafter (i.e., at Cycle 3 Day 1, Cycle 5 Day 1, and so on), upon determination of disease progression, and EOT, whichever is sooner. An optional blood sample for pharmacogenetic (PGx) analysis will be obtained once during the study, preferably during the screening phase. A separate informed consent must be signed before the sample is obtained.

The following safety assessments will be performed pre-dose for all patients on Day 1, and Day 15, unless otherwise specified: 1) Hematology, clinical chemistry, and coagulation panels and CA19-9; 2) For patients under eryaspase arm, in addition to regular evaluation of liver function tests before treatment administration, patients will be monitored on weekly basis in the event of occurrence of >Grade 2 elevation of liver enzymes and bilirubin levels; 3) For women of childbearing potential, additional pregnancy testing should be performed in case of delayed menstrual period and is recommended to be performed monthly and until the end of treatment exposure extended by 30 days, in case of sexual activity; 4) Vital signs: temperature, heart rate, and blood pressure. Symptom-directed physical examination, including body weight. ECOG PS; 5) 12-lead ECG: as clinically indicated; 6) Collection of AEs: from time of informed consent until 90 days after the last study treatment or start of new anti-cancer treatment, whichever is sooner; 7) Review/recording of all concomitant medications added and/or changed from 14 days prior to randomization until 90 days after the last study treatment or start of new anti-cancer treatment, whichever is sooner.

The End of Treatment (EOT) visit should be conducted as soon as possible (but no later than 30 days) after the patient discontinuation of the study drug, and will include the following assessments: 1) Hematology, clinical chemistry, and coagulation panels and CA19-9. Serum pregnancy test for patients of childbearing potential; 2) Vital signs: temperature, heart rate, and BP; 3) Symptom-directed physical exam, including body weight; 4) ECOG PS (see APPENDIX 2). EORTC QLQ-C30; 5) Review of AEs and concomitant medications; and 6) Samples for immunogenicity (for patients in the eryaspase arm) and biomarker analysis (both arms).

During the follow-up phase, the extent of efficacy evaluation will depend on the disease status. In the absence of disease progression or starting new anti-cancer therapy: 1) Radiological imaging of Tumor lesions to be carried out every 8 weeks until disease progression; 2) And then OS follow up every 8 weeks until death, lost for follow up or withdrawal from study; 3) In the presence of objective disease progression or start of anticancer therapy; 4) OS follow up every 8 weeks until death, lost for follow up or withdrawal from study.

Collection and follow-up of AEs and SAEs will occur until 90 days after discontinuation of the study drug, beginning of a new cancer treatment, or death, whichever occurs first. SAEs will be followed until resolution. For all patients who discontinue study treatment, the EORTC QLQ-C30 questionnaire will be completed every 8 weeks during the follow-up phase until withdrawal from the study or death. Information will be collected about any subsequent cancer therapy given after discontinuation of study drug (s), including both medications and procedures.

Survival information will be collected by phone, follow-up visit, or from medical records approximately every 8 weeks from the date of the EOT visit until the patient death, until the patient is lost to follow-up, or until study closure. Survival follow-up will include collection of information about any subsequent anticancer therapy received after discontinuation of study drug.

If a patient fails to return for scheduled visits or is unreachable for survival follow-up, the following efforts should be made to contact him/her: 3 phone attempts, including the date and time, should be documented in the patient chart. If there is no response to the phone calls, a certified letter or local equivalent should be sent. After these efforts have been exhausted with no response, a patient should be considered as lost to follow-up.

CT/MRI scans of the chest and abdomen will be performed every 8 weeks, calculated from the date of randomization, until disease progression, or until withdrawal from the study, or death. Every effort should be made to adhere to the assessment schedule. The same imaging technique must be utilized for a given patient throughout the study. Tumor response and disease progression will be evaluated in this study using a modified version of the criteria proposed by the RECIST Committee, version 1.1 (RECIST 1.1). The modifications to RECIST 1.1 will be used to assess responses more accurately and consistently in specific cases that are not addressed in RECIST 1.1.

Definitions. "Measurable non-nodal lesions" means those that can be accurately measured in at least 1 dimension, longest diameter (LD) to be recorded as >10 mm by CT scan (CT slice thickness no greater than 5 mm). "Malignant lymph nodes" are to be considered pathologically enlarged and measurable, a lymph node must measure >15 mm in the short axis (SA) when assessed by CT scan (CT slice thickness recommended to be no greater than 5 mm). It is recommended that slice gaps not be utilized for the image acquisition procedures. All radiographic measurements should be taken and recorded in millimeters utilizing an electronic measurement method based on lesion boundary definition. Lesions on chest X-ray will not be considered measurable. All other lesions (or sites of disease), including small lesions (LD <10 mm or pathological lymph nodes >10 mm to <15 mm in the SA) as well as truly non- measurable lesions, are considered non-measurable disease. Lesions considered truly non-measurable include leptomeningeal disease, ascites, pleural or pericardial effusions, inflammatory breast disease, lymphangitic involvement of skin or lung, and masses or abdominal organomegaly identified by physical exam that are not measurable by reproducible imaging techniques. Target lesions must be measurable lesions. All lesions up to a maximum of 5 lesions per organ and 10 lesions in total, representative of all involved organs, will be identified as target lesions, recorded and measured at baseline, and followed consistently throughout the study. Target lesions should be selected on the basis of their size (based on LD for non-nodal lesions or SA for lymph nodes), their suitability for accurate repeated measurements, and on their being most representative of the patient's tumor burden. A sum of the diameters for all target lesions will be calculated and reported for each time point. The baseline sum of diameters will be used as a reference according to which the objective tumor response will be characterized.

All other lesions (or sites of disease, including any measurable lesions that were not selected as target lesions) will be identified as non-target lesions and indicated as present at baseline. It is possible to record multiple non-target lesions involving the same organ as a single item (e.g., multiple enlarged pelvic lymph nodes or multiple liver metastases). Measurements of these non-target lesions will not be performed, but the presence, absence, or unequivocal progression of these lesions should be noted for subsequent assessments.

A separate assessment category of "new lesions" is defined as part of the independent reading. The finding of a new lesion should be unequivocal (i.e., not attributable to differences in scanning technique, change in imaging modality, etc.). If a new lesion is equivocal (i.e., because of its small size), and follow-up imaging confirms that it is definitely a new lesion, then the lesion will be considered to have appeared on the date of the initial scan, indicating disease progression as of that date. A lesion identified on a follow-up scan in an anatomical location that was not scanned at baseline is considered a new lesion and will indicate disease progression.

A lesion that reappears at follow-up after a response of PR or stable disease is not considered new. The lesion's LD (or SA for lymph nodes) is added back into the sum of diameters. A lesion that reappears at follow-up after a CR is automatically considered PD.

At baseline, lymph nodes with tumor burden will be considered "target lesions" if the SA is > 15 mm and "non-target lesions" if the SA is >10 mm and <15 mm, and "normal" if the SA is <10 mm. At subsequent assessments, a lymph node must measure >10 mm along the SA to be considered a new lesion. A lymph node that was identified as a target lesion at baseline that falls below the measurable threshold at subsequent assessment and then gets larger is not considered new if it follows a PR or SD. The lymph node SA measurement is added back into the sum of diameters. A lymph node that was identified as a target lesion at baseline that falls below the measurable threshold at subsequent assessment and then gets larger following a CR is automatically considered PD.

Previously irradiated lesions cannot be selected as target lesions. These lesions can be monitored to assess progression as non-target lesions. When non-nodal and nodal lesions split or fragment, the individual diameters of the fragmented portions should be added together to calculate the target lesion sum. As lesions merge, a boundary between the lesions should be drawn so the LD or SA of each individual lesion can continue to be measured. If the lesions have merged in such a way that they can no longer be separated by this boundary, the LD or SA of the merged lesion should be measured. A non-nodal target lesion that is present but too small to measure accurately at evaluations after baseline (<5 mm but greater than 0 mm in unilateral dimension) will be classified as Too small to measure and will be assigned a value of 5 mm for the purposes of determining the sum of diameters. All other lesions (i.e. >5 mm) will have actual size recorded.

A target lymph node should always have the actual SA measurement recorded, even if the lymph node regresses to <10 mm on study. This means that when lymph nodes are included as target lesions, the sum of diameters may not be zero even if CR criteria are met, since a normal lymph node is defined as having an SA of <10 mm.

Target lesion response criteria. 1) Complete response (CR): Disappearance (or normalization) of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in SA to <10 mm; 2) Partial response (PR): At least 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum of diameters; 3) Stable disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study; 4) Progressive disease (PD): At least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum of diameters on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm; and 5) Non-evaluable (NE): Patient time points that have inadequate or missing images, including the inability to visualize >25% of target disease.

Non-target lesion response criteria. 1) CR: Disappearance (or normalization) of all non-target lesions. All lymph nodes must be non-pathological in size (<10 mm SA); 2) Non-complete response, nonprogressive disease (Non-CR/Non-PD): Persistence of 1 or more non-target lesion(s); 3) PD: Unequivocal progression of existing non-target lesions; 4) NE: Patient time points that have inadequate or missing images, including the inability to visualize >50% of non-target disease; 5) No disease (ND): No non-target disease noted; and 6) The absence of non-target lesions at follow-up time points is designated as ND and not SD when there is no non-target disease noted at baseline.

A separate assessment of the appearance of one or more new lesions will be provided in the read. If at least one new lesion is present, the patient is considered to have progressive disease overall. If no lesions were identified at baseline in a specific body region (e.g., chest or abdomen) and the scan of that body region is unavailable at follow-up, then the response assessment will be based on the scans of the other regions. Best overall response (BOR) is the best response recorded from the start of treatment until the end of treatment (taking as reference for PD the smallest measurement recorded since the start of treatment). Patient-reported outcomes will be measured using a standardized instrument, the EORTC QLQ-C30, at the following time points: 1) Cycle 1 Day 1 before treatment; 2) Every 4 weeks during the treatment phase, Day 1 of each cycle before treatment. At the End of Treatment visit; and 3) Every 8 weeks during the follow-up phase until end of study. The following scores will be calculated: 5 functional scales, 3 symptom scales, a global health status/QoL scale, and 6 single items (Table 9).

Table 9. EORTC QLQ-30 Scales and Items [43]

*ltem range is the difference between the possible maximum and the minimum response to individual items;most items take values from 1 to 4, giving range = 3. t(revised) scales are those that have been changed since version 1.0, and their short names are indicated by a suffix "2" - for example, PF2.

Table 10. Performance Status (ECOG Grading Scale)

Table 11. Karnofsky Performance Status

The Karnofsky Performance Scale Index allows patients to be classified as to their functional impairment. This can be used to compare effectiveness of different therapies and to assess the prognosis in individual patients. The lower the Karnofsky score, the worse the survival for mostserious illnesses.

Eryaspase PK will be evaluated using sparse sampling, where possible. Whole blood and plasma concentrations of ASNase from these samples will be combined with previous data as part of a POP-PK analysis. The PD effect of eryaspase on amino acid levels will also be evaluated. For all patients who provide samples, PK and PD of eryaspase will be assessed at each measurement in terms of: Total ASNase activity (U/L), Plasma ASNase activity (U/L); Plasma concentrations of asparagine (pmol/L), and; Plasma concentrations of glutamine (pmol/L).

The development of anti-ASNase antibodies and neutralizing antibodies will be determined for patients in the eryaspase arm. This study will collect samples for biomarker assessments in all patients. Sample types collected include tumor samples, whole blood samples and plasma samples. Biomarkers will be tested prospectively and summarized at baseline and, where applicable, at subsequent assessments. These assays will be performed at central laboratories. In addition, biomarkers identified in other clinical studies may also be assessed in the biomarker samples collected from patients enrolled in this study. Detailed instructions for processing, storage, and shipping of samples will be provided in the Laboratory Manual.

A paraffin-embedded tissue block (FFPE), or a minimum of 10 unstained slides, obtained from an archived or newly obtained tumor tissue sample (primary tumor or metastatic lesion) will be required. All tumor samples may be used to assess the effects of eryaspase and tumor-relevant pathway dysregulation (such as mutations, amplifications) that may be important in the development ofprogression of cancer as well as for the potential use in diagnostic development. Assays to evaluate biomarkers that may correlate with clinical outcome may be performed at central laboratories, such as: 1) Mutational analyses and detection of gene amplification of relevant oncogenes (e.g., KRAS, MYC, PI3KCA, and FGFR) and genes involved in activated pathways of survivaland proliferation (e.g., Erkl/2, STAT3, AKT, and MARK); 2) Expression profiling may be done by analysis of ribonucleic acid (RNA) extracted from tissue; 3) Depending on tissue availability, additional assays including immunohistochemistry (IHC), gene expression profiling, or protein activation may be performed; 4) If applicable, mechanisms of resistance to eryaspase treatment will be evaluated.

Plasma samples will be collected for the following biomarker analyses: 1) Circulating tumor DNA (ctDNA) may be analyzed in plasma samples to identify plasma-borne somatic mutations as predictors of clinical outcome with eryaspase. Proteomic and transcriptomic analyses of biomarker samples may be conducted toidentify profiles that can be related to treatment response. Examination of pre- and post-treatment protein and transcript profiles may uncover novel blood-borne candidate biomarkers/profiles that could be used to predict response to eryaspase. Markers of immunological response may be evaluated for correlation with response.

Pharmacogenetic analysis is the study of the variability in drug response due to hereditary factors. Individual genetic composition (genotype) may have an impact on the PK and the pharmacodynamic effects of the drug. Examples of pharmacogenetic analyses include genotyping of metabolizing enzymes such as uridine 5'-diphosphate glucuronosyltransferases (UGTs) and transporters, evaluation of a whole-genome single nucleotide polymorphism (SNP), or other genetic marker sets. The information obtained is solely used to further characterize drug effects and does nof’_s have clinical, diagnostic, or therapeutic implications for the individual patients. This PGx research is not designed to determine whether other members of the patient family are at risk of developing pancreatic cancer. The Sponsor will be blinded as to the subject's identity and since the analysis is done for research purposes only, individual results will not be shared with the Investigator and/or subject of the subject's relatives. Any information obtained is not intended for inclusion in the medical record. This research will not change the care the subject receives in this study.

A whole blood sample (5 mL) will be drawn using a tube containing ethylenediaminetetraacetic acid (EDTA) from patients who provide separate written informed consent for this optional procedure. Patient participation in the PGx analysis is voluntary, and refusal of consent will not disqualify the patient from participating in the study. Furthermore, a patient can withdraw consent for the PGx analysis at any time during the study.

An "adverse event" (AE) is any untoward medical occurrence in a patient or clinical trial subject administered a medicinal product and which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. A serious adverse event (SAE) is any untoward medical occurrence that at any dose: 1) Results in death; 2) Is lifethreatening; 3) Requires inpatient hospitalization or prolongation of existing hospitalization; 4) Results in persistent or significant disability/incapacity; 5) Is a congenital anomaly/birth defect, or 6) Is otherwise medically significant (any event not meeting the above criteria that, based upon medical judgment, jeopardizes the patient, and may require medical or surgical intervention to prevent one of the other outcomes listed in the definition of an SAE). If the SOC or institutional practice requires hospitalization of patients for the sole purpose of conducting study procedures or delivering study therapy, then such hospital admission will not be considered or reported as meeting SAE criteria. However, should an SAE develop during such a hospitalization, it will be considered serious and reported as such. That said, disease progression should not be reported as an SAE and where an AE changes to a SAE, this date should be used as the onset date of the SAE.

The severity of AEs is to be graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0. For AEs not listed in the CTCAE scale, the following definitions should be used: Mild (Grade 1): The AE is noticeable to the patient but does not disrupt normal daily activities; it does not require discontinuation of the study drug, but may require additional therapy; Moderate (Grade 2): The AE is moderately uncomfortable and interferes with the patient daily activities; it does not require discontinuation of the study drug but may require additional therapy; Severe (Grade 3): The AE is intolerable and disrupts normal daily activities, and may require additional therapy or hospitalization and/or discontinuation of the study drug; Life-threatening (Grade 4): The AE exposes the patient to risk of death at the time of the event; this does not refer to an event that might have caused death if it had been more severe. Any event which is considered lifethreatening is considered to be serious; Death related to AE (Grade 5): An event that results in death is considered a Grade 5 AE, and by definition, is an SAE.

An Investigator who is qualified in medicine must assess the causal relationship between eryaspase, chemotherapy, or both and each occurrence of each AE/SAE. The Investigator should decide whether, in his/her medical judgment, there is a "reasonable possibility" that event may have been caused by the study drug (IMP and non-IMP background treatment). If no valid reason exists for suggesting a causal relationship between the study drug (IMP or non-IMP background treatment) and the occurrence of the AE, then the AE should be classified as "unrelated". If there is any valid reason for suspecting a possible causal relationship between the study drug (IMP and non-IMP background treatment) and the occurrence of the AE, then the AE should be considered "related".

Alternative causes, such as underlying disease(s), concomitant therapy, and other risk factors, as well as the temporal relationship of the event to study treatment administration, should be considered and investigated. The Investigator should also consult the IB in making his/her assessment. There may be situations in which an SAE has occurred and the Investigator has minimal information to include in the initial report to the Sponsor. However, it is critical that the Investigator provide an assessment of causal relationship for every event before the initial transmission of the SAE report to the Sponsor, as it is one of the criteria used when determining regulatory reporting requirements. The Investigator may change his/her opinion of causality in light of follow-up information; in this case an SAE follow-up report should be sent with the updated causal relationship assessment.

Definitions of the causal relationship of an AE to the use of the study medication are as follows: 1) "Related" means there is a reasonable possibility of a causal relationship between the administration of the study drug (IMP and non-IMP background treatment) and the adverse event emergence. Reasonable possibility means there is evidence to suggest a causal relationship, e.g. temporal relationship, biological plausibility, between study intervention and the AE; 2) "Not Related" means there is no reasonable possibility that the administration of the study intervention caused the event, there is no temporal relationship between the study intervention and the event onset, or an alternate etiology has been established.

The drug safety group will be responsible for determining whether an AE is expected or unexpected, based on available reference safety information in the IB. An AE will be considered unexpected if its nature, severity, or frequency is not consistent with the reference safety information described for eryaspase in the IB.

All AEs that are reported by a patient or observed by study personnel from the date of patient informed consent until 90 days after the last study treatment or start of new anti-cancer treatment, whichever is sooner, will be reported. Any medical condition that is present at the time that the patient is screened will be considered as a baseline condition and not reported as an AE. All AEs must be followed until they resolve, return to baseline, or are determined to be permanent at the EOT visit.

All AEs, whether serious or non-serious, must be recorded in the source documents and in the eCRF using standard medical terminology. For each event, the Investigator will evaluate and report the onset date, resolution date, severity, causality, action taken, seriousness, outcome (ifappl icable), and whether or not it caused any change in study drug administration (dose delay, dose reduction, or study treatment discontinuation). Changes in the severity of an AE must be documented to allow the duration of the event at each level of severity to be assessed. AEs characterized as intermittent require documentation of the onset and duration of each episode.

Whenever possible, diagnoses should be provided when signs and symptoms represent a common etiology (e.g., cough, runny nose, sneezing, sore throat, and head congestion should be reported as "upper respiratory infection"). A specific disease or syndrome, rather than individual associated signs and symptoms, should be identified whenever possible. Where there is no link between different clinical symptoms occurring at the same time, each sign should be recorded as a separate AE. The following AEs are of special interest: venous thrombotic events, hepatic toxicity, pancreatitis, and transfusion-related events.

Table 12. Minimum Safety Assessment

Statistical considerations. The primary efficacy endpoint is Overall Survival (OS) and the study is designed to test the superiority of eryaspase plus chemotherapy over chemotherapy alone. The primary analysis will test the following hypotheses: Null Hypothesis: The hazard ratio for OS between eryaspase plus chemotherapy and chemotherapy alone is equal to one; Alternative Hypothesis: The hazard ratio for OS between eryaspase plus chemotherapy and chemotherapy alone is less than one. The key secondary efficacy endpoints are Progression-free Survival (PFS), Objective Response Rate (ORR), Duration of Response (DoR) and Disease Control Rate (DCR). The null and alternative hypotheses for PFS will be as for OS. The primary analyses of the endpoints ORR and DCR will test the following hypotheses: Null

Hypothesis: The ORR/DCR in the eryaspase plus chemotherapy group is equal to the ORR/DCR in the chemotherapy alone group; Alternative Hypothesis: The ORR/DCR is greater in the eryaspase plus chemotherapy group versus the chemotherapy alone group. There will be no formal statistical testing for DoR as this is not a randomized comparison and these data will be evaluated through the presentation of descriptive statistics. All statistical testing will be undertaken based on a one-sided 2.5% significance level subject to adjustments for interim analysis.

With a power of 88.4% and an overall one-sided type I error of 2.5%, and including one interim analysis for efficacy with an O'brien-Fleming type stopping rule, a total of 390 deaths are required to detect a treatment effect hazard ratio of 0.725 for eryaspase plus chemotherapy versus chemotherapy alone. The hazard ratio in the Phase 2 study for eryaspase was 0.63 (95% Cl: 0.39 to 1.10), and 0.725 represents a conservative estimate based on these data that is viewed as being highly clinically relevant. The interim analysis for efficacy will take place once 261 (67%) events have been observed. Assuming a recruitment period of 26 months, a median overall survival in the control group of 6.0 months, a 10% probability of dropping out during the course of the study, and a minimum follow-up of 9 months, the study size will be based on the recruitment of 482 patients.

The primary endpoint is OS which is measured from the date of randomization to the date of death from any cause. Patients who are not known to have died will be censored at the date of last contact, and this will apply as of the date of data cut-off for any particular analysis. The primary analysis will be the comparison of OS between the two treatment arms in the ITT population using the one-sided stratified log-rank test, with stratification factors as used in the randomization. Data will be summarized in Kaplan-Meier curves together with medians and 95% confidence intervals for those medians. The Cox Proportional Hazards model, stratified for the randomization factors, will be used to obtain a hazard ratio together with its 95% confidence interval.

The secondary endpoint, PFS, will be compared between the two treatment arms using the same methods of analysis as for OS. The following efficacy analysis will also be performed in the ITT population: 1) ORR, defined as the proportion of patients who achieve objective tumor response (CR or PR) per RECIST 1.1. Each patient BOR will be summarized (CR, PR, SD, PD, or unknown). This comparison will be based on the Cochran-Mantel-Haenszel test, with stratification factors as for the analysis of OS and PFS. Results will be reported in terms of an odds ratio and associated 95% confidence interval; 2) DCR, defined as the proportion of patients who achieve CR, PR and SD; and 3) DoR will be evaluated in patients who achieve CR/PR. It will be measured from the time CR/PR (whichever is first recorded) is first met until the first date that recurrence or PD is objectively documented. Kaplan-Meier curves will be provided in association with this analysis; however, there will be no p-value calculations, as this is not a randomized comparison.

Extensive evaluation of the consistency of treatment effect for OS and PFS across the population as a whole will be undertaken by providing analyses in subgroups, with displays in forest plots and p- values for treatment by interactions.

Additional disease control endpoints will be evaluated as exploratory analyses, namely DCR8, DCR12, and DCR16 defined as the disease control rates when SD is required to last for at least 8, 12, and 16 weeks, respectively. These analyses will follow the methodology for DCR. All primary and secondary efficacy analyses will be repeated in the PP population. Confirmatory testing for the primary and secondary efficacy endpoints will be performed hierarchically (OS->PFS->ORR->DCR) in order to account for multiplicity. Any statistically significant findings occurring below a non-significant result in the hierarchy will be considered as exploratory findings.

Interim Analyses. This study will have an event-driven interim analysis for OS, based on O'Brien- Fleming boundaries. The interim analysis for OS will take place following 261 deaths. The number of deaths required to trigger the final analysis will be 390.

Hazard ratios and 95% confidence intervals will be calculated for OS within subgroups using the unadjusted Cox proportional hazards model in order to evaluate the consistency of treatment effect. These subgroups will include those defined by the stratification factors. Other subgroups may also be considered, with a full list being set down in the SAP. Subgroup analyses of PFS will be performed in a similar way. ORR and DCR will also be evaluated within these same subgroups, based on the calculation of unadjusted odds ratios and corresponding 95% confidence intervals.

The study will be considered completed with completion of the last visit of the last patient participating in the study.

Example 2. Eryaspase in First Line Pancreatic Cancer

The rESPECT 1ST (NCT04292743) is a single arm, dose escalating Phase 1 clinical trial to evaluate the safety of eryaspase in combination with modified FOLFIRINOX. The trial includes pancreatic cancer patients who have received no prior chemotherapy for the treatment of locally advanced or metastatic pancreatic cancer. FOLFIRINOX is one of the most commonly utilized first-line chemotherapy regimens for the treatment of pancreatic cancer, despite its toxicity.

At the time of this filing, a total of nine patients have been enrolled with a mean age of 70. Four patients had locally advanced disease and five had metastatic disease. Patients were treated across two dose cohorts of 75 U/kg and 100 U/kg eryaspase, with three and six patients included, respectively.

After review of the safety data, the dose escalation committee concluded that the novel combination of mFOLFIRINOX plus eryaspase was well tolerated with no DLT. Consequently, the MTD has been declared at a therapeutic dose of 100 U/kg eryaspase.

Interestingly, of the six patients that were evaluated for response, there were four partial responses (PRs) and two stable disease (SD) outcomes, or an objective response rate (ORR) of 67% and 100% disease control rate (DCR) . It is now planned for the study to enroll additional patients at the 100 U/Kg dose level to further assess clinical activity and safety.

The declared MTD of 100 U/Kg eryaspase corresponds with the dose currently being used in clinical trial in second-line patients and it can now be taken forward into future late-stage clinical studies in first-line pancreatic patients.

Example 3. Top Line Results

The Phase 3 Trial described in Example 1 was conducted and the following recounts the initial findings.

Briefly, ~500 people >18 years of age were enrolled in this trial. Each patient had Stage III or IV PAC and had received one prior PAC. Each patient also had measurable disease and an ECOG PS of 0 or 1. The primary endpoint was Overall Survival and the key secondary endpoints were Progression- free survival, Objective response rate, Disease control rate, Safety and tolerability, and Quality of life. In total, ~90 clinical sites were activated in 11 countries in Europe and the United States.

Notably, we observed a trend for an improved survival in the irinotecan-based back chemotherapy and the effect of treatment was very stable across various subgroups. Further, treatment was exceptionally well-tolerated and eryaspase did not enhance toxicity of either backbone chemotherapy. Overall, the study was robust, with clean well balanced baseline characteristics. The study also addressed several critical questions for the oncology community, including the role of asparaginase in treating pancreatic cancer and the feasibility of gemcitabine/abraxane in 2L setting.

As discussed in Example 1, OS was a primary endpoint. With a power of 88.4% and an overall one-sided type I error of 2.5% and including one interim analysis for efficacy with an O'Brien-Fleming type stopping rule, a total of 390 deaths are required to detect a treatment effect hazard ratio of 0.725 for eryaspase plus chemotherapy versus chemotherapy alone. This translates to an approximate 2.28 months increase from an assumed median overall survival of 6 months in the control arm. The choice of 6 months in the control arm is based on reported OS in Napoli-1 study.

And while the data indicated that the pre-selected OS primary endpoint was not met, a surprising increased benefit was observed for patients receiving asparaginase plus the irinotecanbased chemotherapy vs. patients receiving eryaspase plus gemcitabine/abraxane.

Table 13. Patient disposition

Table 14. Baseline characteristics - ITT

Table 15. Efficacy outcome - ITT

Table 16. Efficacy outcome - PP

FIG. 1 is a Kaplan-Meier plot showing Overall survival - ITT.

FIG. 2 is a Kaplan-Meier plot showing Overall survival - Per Protocol.

FIG. 3 is a summary of Overall Survival by Treatment Group backbone chemotherapy - ITT. FIG. 4 is a Kaplan-Meier plot showing Overall survival - gemcitabine-based chemotherapy - PP.

FIG. 6 is a Forest plot of OS hazard ratios in subgroups - PP, part 1.

FIG. 7 is a Forest plot of OS hazard ratios in subgroups - PP, part 2.

FIG. 8 is a Forest plot of PFS hazard ratios in subgroups - ITT, part 1. FIG. 9 is a Forest plot of PFS hazard ratios in subgroups - ITT, part 2.

Table 16. Subsequent anti-cancer therapy

Table 17. Treatment emergent adverse events - safety population

As indicated in FIGs. 4 and 5, eryaspase provided a superior technical effect when combined with the irinotecan-based chemotherapy, but not when added to the gemcitabine-based chemotherapy. Based on the findings disclosed herein, Applicant envisions that any asparaginase having a suitable half-life and safety profile would provide a comparable enhancement of the activity of the irinotecan-based chemotherapy. Moreover, Applicant envisions that asparaginase should produce comparable results when combined with topoisomerases other than irinotecan. And while the full primary endpoint was not achieved, all efficacy indicators showed a trend of increased efficacy compared to chemotherapy alone arm. In particular, there was a trend towards improving OS in patients who received eryaspase and irinotecan-based therapy compared to irinotecan alone: mOS: 8 vs 5.7 months, HR 0.77, P value = 0.99. Moreover, the treatment effect (or lack of) was consistent and stable across all subgroups. Treatment also well-tolerated, and the addition of eryaspase did not enhance the cytotoxicity of chemotherapy.

Accordingly, in some embodiments, the invention comprises a method of enhancing the solid tumor anti-cancer efficacy of an irinotecan-based chemotherapy— but not a gemcitabine-based chemotherapy— comprising administering both the irinotecan-based chemotherapy and asparaginase.

In view of this full and enabling disclosure, the skilled artisan may routinely determine other combinations of asparaginase and Topoisomerase I inhibitor-based chemotherapeutic regimens.

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Claims

78 CLAIMS

1. A method of treating second line (2L) pancreatic cancer comprising the steps of administering to a patient in need thereof an effective amount of a combination of an effective amount of chemotherapeutic(s) and an effective amount of asparaginase, irrespective of the nature of prior 1^st line treatments, wherein the asparaginase is optionally selected from red blood cells encapsulating asparaginase (eryaspase), RYLAZE, or any modified asparaginase or asparaginase formulations (e.g. a nanoparticle) having an in-vivo half-life of at least about 4 to about 14 days.

2. A method of treating second line (2L) pancreatic cancer in an patient in need thereof, comprising administering to the individual an effective amount of a composition comprising the asparaginase of claim 1 and a Topoisomerase I inhibitor (e.g. irinotecan, in particular Onivyde®)/5- fluorouracil/leucovorin, wherein the asparaginase is administered at about 75 to about 150 U/Kg, about 80 to about 140 U/Kg, about 85 to about 130 U/Kg, about 90 to about 120 U/Kg, or about 100 U/Kg, given on Days 1 and 15 of each 4-week cycle, and wherein each chemotherapeutic agent is administered according to its label and standard of practice.

3. The method of claim 1 or 2 wherein the patient experiences significant improvements in Overall Survival (OS), Progression-free Survival (PFS), Objective Response Rate (ORR), Duration of Response (DoR), Disease Control Rate (DCR), Pharmacokinetics (PK) and Pharmacodynamics (PD) induction of Anti-ASNase antibodies (Abs), Adverse events (AEs), and/or Quality of life (QoL), when compared with any improvements elicited by the respective chemotherapeutic regimen without eryaspase; optionally wherein: a) the patient's cancer has progressed on at least one line of therapy for metastatic disease; and/or b) the patient's cancer has recurred with metastatic disease less than or equal to 12 weeks of completion of systemic chemotherapy; and/or c) the patient's cancer progressed to metastatic disease less than or equal to 12 weeks after completion of systemic chemotherapy; and/or d) the patient's cancer has recurred with metastatic disease less than or equal to 12 weeks of completion of systemic chemotherapy; and/or e) the patient's cancer exhibits elevated levels of CA 19-9; and/or 79 f) the patient's cancer cells exhibit an energy production profile characterized by a high OXPHOS to glycolysis ratio, which is associated with a poor prognosis.

4. A method for increasing overall survival (OS) in a patient afflicted with pancreatic cancer, said method comprising administering to said patient a therapeutically effective amount of about 100 U/kg eryaspase and therapeutically effective amounts of either gemcitabine/Abraxane or Onivyde®/5- fluorouracil/leucovorin.

5. A method for increasing progression free survival (PFS) in a patient afflicted with pancreatic cancer, said method comprising administering to said patient a therapeutically effective amount of about 100 U/kg eryaspase and therapeutically effective amounts of either gemcitabine/Abraxane or Onivyde®/5-fluorouracil/leucovorin.

6. A method for increasing overall response rate (ORR) in a patient afflicted with pancreatic cancer, said method comprising administering to said patient a therapeutically effective amount of about 100 U/kg eryaspase and therapeutically effective amounts of either gemcitabine/Abraxane or Onivyde®/5-fluorouracil/leucovorin.

7. The method of any one of the preceding claims, wherein the chemotherapeutics are administered as follows: Onivyde (irinotecan nanoliposomal) + 5-FU/leucovorin are administered on Days 1 and 15 of each 4-week cycle as follows: Onivyde 70 mg/m2 IV over 90 minutes (recommended starting dose of Onivyde in patients homozygous for UGT1A1*28 is 50 mg/m2). Leucovorin 400 mg/m2 IV over 30 minutes, though modifications to the leucovorin dose per sites' standard administration protocol may be allowed in the study following approval from the medical monitor. 5-FU 2400 mg/m2 over 46 hours. FOLFIRI (irinotecan, 5-FU, and leucovorin) is to be administered every 2 weeks on Day 1 and Day 15 of each 4-week cycle as follows: Irinotecan 180 mg/m2 IV infusion over 90 minutes (recommended starting dose of irinotecan in patients homozygous for UGT1A1*28 is 150 mg/m2); Leucovorin 400 mg/m² IV infusion over 2 hours, though the leucovorin dose may be modified as discussed immediately supra. 5-FU 400 mg/m² IV bolus injection over 2-4 minutes, immediately following leucovorin infusion, and 5-FU 2400 mg/m² IV continuous infusion over 46 hours, immediately following bolus 5-FU.

8. A method of decreasing a risk of hospitalization in a patient suffering from pancreatic cancer, said method comprising administering to said patient an effective amount of asparaginase and irinotecan/5-fluorouracil/leucovorin.

9. A method of preventing the progression of locally advanced pancreatic cancer to 80 metastatic cancer comprising administering to said patient an effective amount of asparaginase and irinotecan/5-fluorouracil/leucovorin.

10. A method of increasing Overall Survival (OS), Progression-free Survival (PFS), Objective Response Rate (ORR), Duration of Response (DoR) and/or Disease Control Rate (DCR), in a patient afflicted with pancreatic cancer, wherein the method comprises administration of an effective amount of asparaginase and irinotecan/5-fluorouracil/leucovorin.

11. A method of additively or synergistically enhancing the solid tumor anti-cancer efficacy of a Topoisomerase I inhibitor-based chemotherapy without significantly increasing toxicity comprising administering an enhancing-effective amount of asparaginase and an effective amount of the Topoisomerase I inhibitor-based chemotherapy to a patient in need thereof.

12. The method of claim 11, wherein the patient is suffering from 2L PAC, irrespective of prior IL chemotherapy.

13. The method of claim 12, wherein the patient previously received a gemcitabine-based treatment.

14. The method of claim 12, wherein the patient previously received FOLFIRINOX.

15. The method of any one of claims 11 to 13, wherein the asparaginase is eryaspase.

16. The method of any one of claims 11 to 13, wherein the asparaginase is a modified asparaginase or is a nanoparticle-encapsulated asparaginase, wherein the asparaginase has a comparable PK/PD profile when compared to eryaspase.

17. The method of any one of claims 11-13, wherein the Topoisomerase I inhibitor is irinotecan, optionally Onivyde®, and the other chemotherapeutic agents are 5-fluorouracil and leucovorin, and wherein the asparaginase is administered at about 75 to about 150 U/Kg, about 80 to about 140 U/Kg, about 85 to about 130 U/Kg, about 90 to about 120 U/Kg, or about 100 U/Kg, given on Days 1 and 15 of each 4-week cycle, and wherein each chemotherapeutic agent is administered according to its label and standard of practice.

18. The method of any one of claims 11-13, wherein the asparaginase is PEGylated or PASylated.

19. The method of any one of claims 11-13, wherein the asparaginase synergistically enhances the efficacy of the Topoisomerase I inhibitor-based chemotherapy or the Topoisomerase I 81 inhibitor-based chemotherapy synergistically enhances the efficacy of the asparaginase.

20. The method of claim 17, wherein the asparaginase synergistically enhances the efficacy Topoisomerase I inhibitor-based chemotherapy or the Topoisomerase I inhibitor-based chemotherapy synergistically enhances the efficacy asparaginase.

21. A combination of chemotherapeutic(s) and asparaginase for use in treating second line (2L) pancreatic cancer, irrespective of the nature of prior 1^st line treatments, wherein the asparaginase is optionally selected from red blood cells encapsulating asparaginase (eryaspase), RYLAZE, or any modified asparaginase or asparaginase formulations (e.g. a nanoparticle) having an in-vivo half-life of at least about 4 to about 14 days.

22. A combination of asparaginase of claim 21 and Topoisomerase I inhibitor (e.g. irinotecan, in particular Onivyde®)/5-fluorouracil/leucovorin for use in treating second line (2L) pancreatic cancer in an patient in need thereof, wherein the asparaginase is administered at about 75 to about 150 U/Kg, about 80 to about 140 U/Kg, about 85 to about 130 U/Kg, about 90 to about 120 U/Kg, or about 100 U/Kg, given on Days 1 and 15 of each 4-week cycle, and preferably wherein each chemotherapeutic agent is administered according to its label and standard of practice.

23. The combination for the use of claim 21 or 22, wherein the patient experiences significant improvements in Overall Survival (OS), Progression-free Survival (PFS), Objective Response Rate (ORR), Duration of Response (DoR), Disease Control Rate (DCR), Pharmacokinetics (PK) and Pharmacodynamics (PD) induction of Anti-ASNase antibodies (Abs), Adverse events (AEs), and/or Quality of life (QoL), when compared with any improvements elicited by the respective chemotherapeutic regimen without eryaspase; optionally wherein: a) the patient's cancer has progressed on at least one line of therapy for metastatic disease; and/or b) the patient's cancer has recurred with metastatic disease less than or equal to 12 weeks of completion of systemic chemotherapy; and/or c) the patient's cancer progressed to metastatic disease less than or equal to 12 weeks after completion of systemic chemotherapy; and/or d) the patient's cancer has recurred with metastatic disease less than or equal to 12 weeks of completion of systemic chemotherapy; and/or 82 e) the patient's cancer exhibits elevated levels of CA 19-9; and/or f) the patient's cancer cells exhibit an energy production profile characterized by a high OXPHOS to glycolysis ratio, which is associated with a poor prognosis.

24. A combination of about 100 U/kg eryaspase and therapeutically effective amounts of either gemcitabine/Abraxane or Onivyde®/5-fluorouracil/leucovorin, for use in increasing overall survival (OS), or increasing progression free survival (PFS), or for increasing overall response rate (ORR), in a patient afflicted with pancreatic cancer.

25. A combination for the use of any one of claims 21 to 24, wherein the chemotherapeutics are administered as follows: Onivyde (irinotecan nanoliposomal) + 5-FU/leucovorin are administered on Days 1 and 15 of each 4-week cycle as follows: Onivyde 70 mg/m2 IV over 90 minutes (recommended starting dose of Onivyde in patients homozygous for UGT1A1*28 is 50 mg/m2). Leucovorin 400 mg/m2 IV over 30 minutes, though modifications to the leucovorin dose per sites' standard administration protocol may be allowed in the study following approval from the medical monitor. 5-FU 2400 mg/m2 over 46 hours. FOLFIRI (irinotecan, 5-FU, and leucovorin) is to be administered every 2 weeks on Day 1 and Day 15 of each 4-week cycle as follows: Irinotecan 180 mg/m2 IV infusion over 90 minutes (recommended starting dose of irinotecan in patients homozygous for UGT1A1*28 is 150 mg/m2); Leucovorin 400 mg/m² IV infusion over 2 hours, though the leucovorin dose may be modified as discussed immediately supra. 5- FU 400 mg/m² IV bolus injection over 2-4 minutes, immediately following leucovorin infusion, and 5-FU 2400 mg/m² IV continuous infusion over 46 hours, immediately following bolus 5-FU.

26. A combination of asparaginase and irinotecan/5-fluorouracil/leucovorin, for use in decreasing a risk of hospitalization in a patient suffering from pancreatic cancer, or preventing the progression of locally advanced pancreatic cancer to metastatic cancer, or increasing Overall Survival (OS), Progression-free Survival (PFS), Objective Response Rate (ORR), Duration of Response (DoR) and/or Disease Control Rate (DCR), in a patient afflicted with pancreatic cancer..

27. A combination of asparaginase, preferably an enhancing-effective amount of asparaginase, and an effective amount of the Topoisomerase I inhibitor-based chemotherapy, for use in additively or synergistically enhancing the solid tumor anti-cancer efficacy of a Topoisomerase I inhibitorbased chemotherapy without significantly increasing toxicity in a patient in need thereof.

28. The combination for the use of claim 27, wherein the patient is suffering from 2L PAC, irrespective of prior IL chemotherapy, and optionally the patient previously received a gemcitabine-based 83 treatment.

29. The combination for the use of claim 27 , wherein the patient previously received FOLFIRINOX.

30. The combination for the use of claim 27 or 28, wherein the asparaginase is eryaspase; or a modified asparaginase or a nanoparticle-encapsulated asparaginase, wherein the asparaginase has a comparable PK/PD profile when compared to eryaspase.

31. The combination for the use of claim 27, 28 or 30, wherein the Topoisomerase I inhibitor is irinotecan, optionally Onivyde®, and the other chemotherapeutic agents are 5-fluorouracil and leucovorin, and wherein the asparaginase is administered at about 75 to about 150 U/Kg, about 80 to about 140 U/Kg, about 85 to about 130 U/Kg, about 90 to about 120 U/Kg, or about 100 U/Kg, given on Days 1 and 15 of each 4-week cycle, and preferably wherein each chemotherapeutic agent is administered according to its label and standard of practice.

32. The combination for the use of claim 27, 28, 30 or 31, wherein the asparaginase is PEGylated or PASylated.

33. The combination for the use of claim 27 to 32, wherein the asparaginase synergistically enhances the efficacy of the Topoisomerase I inhibitor-based chemotherapy or the Topoisomerase I inhibitor-based chemotherapy synergistically enhances the efficacy of the asparaginase.