Method of treating a tumor

Abstract

The present invention provides a method for treating a tumor in a patient comprising the steps of: (i) administering an oncolytic virus and one or more anticancer drugs to the patient: and (ii) irradiating the tumor thereafter.

Description

[0001] Attorney Docket No. 4456-0340PWO1

[0002] METHOD OF TREATING A TUMOR CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U. S. Application No. 63 / 747,502, filed January 21, 2025, the entirety of which is incorporated by reference herein.

[0004] [0 0 0 1 ]

[0005] This invention was made with government support under Grant Nos. U10CA180868 and U10CA180822 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] [0 0 0 2]

[0008] The present invention relates to a method of treating a tumor by combining an oncolytic virus, an antitumor agent, and radiation therapy.

[0009] BACKGROUND ART

[0010] [0 0 0 3 ]

[0011] An oncolytic virus growing selectively in tumor cells has an antitumor effect by itself because the viral growth induces cell death in tumor cells. However, combined use of such a virus with a substance having an antitumor effect makes it possible to inhibit cancer cell growth and thereby to secure sufficient time for the virus to grow in cancer cells. Besides, the virus that has grown in cancer cells is destroyed by anticancer agents, which promotes viral release. As a result, rapid viral diffusion into surrounding cancer cells and remarkable antitumor effect can be expected. Further, since the mode of action of cell death caused by viruses and the mode of action of cell death caused by conventional anticancer agents are different, the possibility that respective antitumor effects when they are combined will be inhibited would be presumably low. Actually, it is reported that the antitumor effects of herpes viruses growing selectively in tumor cells have been enhanced pre-clinically and clinically when they were used in combination with conventional anticancer therapies such as with anticancer agents or radiation (Post DE, et al., Curr Gene Ther. 2004 Mar; 4(1):41-51; and Bennett JJ, et al., FASEB J. 2004 Jun; 18(9): 1001-3).

[0012] [0 0 0 4]

[0013] Definitive chemoradiation (CRT) is a standard-of-care for patients (Pts) with medically inoperable esophageal cancer (EC). The NRG / RTOG 0436 study of cisplatin / paclitaxel and RT (+ / - cetuximab) as definitive therapy showed that 58% of control arm Pts have locallyAttorney Docket No. 4456-0340PWO1

[0014] persistent disease. OBP-301 is a conditionally-restricted, replication-competent adenovirus derived from human adenovirus type 5 that adds a human Telomerase Reverse Transcriptase gene promoter, replicating only in tumor cells to cause lysis. It may cause immunogenic cell death, enhance RT increasing radiosensitization by blocking DNA repair and improve local control.

[0015] DISCLOSURE OF INVENTION

[0016] [0 0 0 5 ]

[0017] It is an object of the present invention to provide a method of treating a tumor by using an oncolytic virus in combination with anticancer drugs and radiation.

[0018] The present inventors have found that combined use of an oncolytic virus comprising a polynucleotide containing a promoter for human telomerase, an El A gene, an IRES sequence and an E1B gene in this order with a substance having an antitumor effect enhances anticancer activity.

[0019] The present invention relates to the following.

[0020] [0 0 0 6 ]

[0021] [1] A method for treating a tumor in a patient comprising the steps of:

[0022] (i) administering an oncolytic virus and one or more anticancer drugs to the patient: and

[0023] (ii) irradiating the tumor thereafter.

[0024] [2] A method for treating a tumor, comprising administering to a patient an oncolytic virus and two types of anticancer drugs, and irradiating the patient with radiation.

[0025] [3] The method according to [1], wherein the oncolytic virus is an oncolytic adenovirus.

[0026] [4] The method according to [1], wherein the tumor is at least one selected from the group consisting of stomach cancer, gastroesophageal junction cancer, esophageal cancer, duodenal cancer, pancreatic cancer, colon cancer, head and neck cancer, anal cancer, rectal cancer, small intestine cancer, lung cancer, liver cancer, brain tumors, cervical cancer, jaw cancer, maxillary cancer, submandibular gland cancer, oral cancer, salivary gland cancer, sublingual gland cancer, parotid gland cancer, nasal cavity cancer, paranasal sinus cancer, laryngeal cancer, breast cancer, biliary tract cancer, bladder cancer, kidney cancer, prostateAttorney Docket No. 4456-0340PWO1

[0027] cancer, uterine cancer, ovarian cancer, thyroid cancer, pharyngeal cancer, sarcoma, malignant lymphoma, leukemia, skin cancer, and melanoma.

[0028] [5] The method according to [1], wherein the oncolytic adenovirus is a virus having incorporated into its genome a polynucleotide comprising a human telomerase promoter (hTERT promoter), an E1A gene, an IRES sequence, and an E1B gene in this order (OBP-301), or a virus having incorporated into the E3 region of OBP-301 a polynucleotide comprising an Egr-1 promoter and a p53 gene in this order (OBP-702).

[0029] [6] The method according to [1], wherein the anticancer drug is selected from the group consisting of alkylating agents, platinum agents, topoisomerase inhibitors, metabolic antagonists, microtubule inhibitors, antibiotic anticancer agents, and molecular targeted drugs.

[0030] [7] The method according to [6], wherein the alkylating agent is selected from the group consisting of cyclophosphamide, melphalan, chlorambucil, ifosfamide, mechlorethamine, uracil mustard, thiotepa, busulfan, dacarbacin, nimustine, ranimustine, and temozolomide.

[0031] [8] The method according to [6], wherein the platinum agent is selected from the group consisting of cisplatin, carboplatin, nedaplatin, and oxaliplatin.

[0032] [9] The method according to [6], wherein the topoisomerase inhibitor is selected from the group consisting of irinotecan, etoposide, and nogitecan.

[0033]

[0010] The method according to [6], wherein the antimetabolite is selected from the group consisting of gemcitabine, methotrexate, fluorouracil, tegafur, doxifluridine, pentostatin, and cladribine.

[0034]

[0011] The method according to [6], wherein the microtubule inhibitor is selected from the group consisting of eribulin, paclitaxel, docetaxel, vinblastine, vinorelbine, and vincristine.

[0035]

[0012] The method according to [6], wherein the antibiotic anticancer agent is selected from the group consisting of doxorubicin, daunorubicin, mitomycin C, and actinomycin.Attorney Docket No. 4456-0340PWO1

[0036]

[0013] The method according to [1], wherein the anticancer drug is a combination of the platinum agent and microtubule inhibitor.

[0037]

[0014] The method according to [1], wherein the anticancer drug is a combination of carboplatin and paclitaxel.

[0038]

[0015] The method according to [1], wherein the oncolytic virus is administered at least three times at intervals of 6 to 10 days.

[0039]

[0016] The method according to [1], wherein the anticancer drug is administered at least five times, with each type of agent being administered once a week.

[0040]

[0017] The method according to [1], wherein the oncolytic virus is administered at a concentration of 1x1010vp / mL to IxlO13vp / ml in a dose of 1-2 ml.

[0041]

[0018] The method according to

[0014] , wherein the carboplatin is administered at a dose of AUC 1-6.

[0042]

[0019] The method according to

[0014] , wherein the carboplatin is administered at a dose of AUC 1-3.

[0043]

[0020] The method according to

[0014] , wherein the carboplatin is administered at a dose of AUC 2.

[0044]

[0021] The method according to

[0014] , wherein the paclitaxel is administered at a dose of 25-100 mg / m2.

[0045]

[0022] The method according to

[0014] , wherein the paclitaxel is administered at a dose of 25-70 mg / m2.

[0046]

[0023] The method according to

[0014] , wherein the paclitaxel is administered at a dose of 50Attorney Docket No. 4456-0340PWO1

[0047]

[0024] The method according to

[0014] , wherein the radiation is administered at a dose of 1.5-2 Gy in at least 28 doses.

[0048]

[0025] The method according to [1], wherein the oncolytic virus is administered at a concentration of 1 x 1012vp / mL and in a dose of 1 to 2 ml on days 3, 12, and 26 after the start of treatment, carboplatin is administered at a dose of AUC1-3 once a week for 5 doses, paclitaxel is administered at a dose of 25-70 mg / m2once a week for 5 doses, and radiation is administered at a dose of 1.8 Gy for 28 doses.

[0049]

[0026] The method according to [1], wherein the oncolytic virus is administered at a concentration of 1 x 1012vp / mL and in a dose of 1 to 2 ml on days 3, 12, and 26 after the start of treatment, carboplatin is administered at a dose of AUC2 once a week for 5 doses, paclitaxel is administered at a dose of 50 mg / m² once a week for 5 doses, and radiation is administered at a dose of 1.8 Gy for 28 doses.

[0050]

[0027] A composition comprising an oncolytic virus and one or more anticancer drugs for use in a method of treating a tumor in a patient.

[0051]

[0028] A pharmaceutical composition for treatment of tumor to be used in combination with one or more anticancer drugs and radiation therapy, comprising an oncolytic virus as an active ingredient.

[0052]

[0029] A pharmaceutical composition for treatment of tumor to be used in combination with one or more anticancer drugs, oncolytic virus, and radiation therapy, comprising a platinum agent an active ingredient.

[0053]

[0030] A pharmaceutical composition for treatment of tumor to be used in combination with one or more anticancer drugs, oncolytic virus, and radiation therapy, comprising a microtubule inhibitor an active ingredient.

[0054] BRIEF DESCRIPTIONOF DRAWINGS

[0055] [0 0 0 7 ]

[0056] Figure 1 shows the construct of OBP-301.Attorney Docket No. 4456-0340PWO1

[0057] ΔE1 = delta E1 adenovirus vector, E1A, E1B = transcriptional units, hTERT = human telomerase reversetranscriptase, IRES = internal ribosomal entry site, ITR = inverted terminal repeat.

[0058] Figure 2 shows a study design of clinical trial.

[0059] *Surgery is permitted post-chemoradiation if medically indicated.

[0060] Figure 3 shows injection syringe.

[0061] Figure 4 shows example of mapping into up to 5 tracks.

[0062] DESCRIPTION OF EMBODIMENTS

[0063] [0 0 0 8 ]

[0064] The present invention relates to a method for treating a tumor, comprising administering to a patient an oncolytic virus and two types of anticancer drugs, and irradiating the patient with radiation.

[0065] [0 0 0 9 ]

[0066] The term “tumor” is understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. Preferably, the inventive methods disclosed herein are used to prevent and treat malignant tumors. The term “cancer” is also understood in the art, for example, an uncontrolled growth of tissue that has the potential to spread to distant sites of the body (i.e., metastasize).

[0067] [0 0 1 0 ]

[0068] The recombinant oncolytic virus of the present invention (e.g. OBP-301) means a virus in which a polynucleotide comprising a promoter for human telomerase, an El A gene, an IRES sequence and an E1B gene in this order has been integrated into its genome. The type of virus used in the present invention is not particularly limited, but adenoviruses are preferable from the viewpoint of safety. Among adenoviruses, type 5 adenovirus is especially preferable because of its easiness in handling, etc. The recombinant oncolytic adenovirus can be obtained by the method described in WO 2004 / 5511. Alternatively, the recombinant oncolytic adenovirus, OBP-301 can be obtained from Oncolys BioPharma Inc. as “Telomelysin” ®. In addition, OBP-702, in which a polynucleotide containing an Egr-1 promoter and a p53 gene in the above order is incorporated in the E3 region of OBP-301, can also be preferably used. OBP-702 is available from Oncolys BioPharma Inc.

[0069] [ 0 0 1 1 ]

[0070] Antitumor AgentsAttorney Docket No. 4456-0340PWO1

[0071] Antitumor agents used in combination with oncolytic virus in the present invention are not particularly limited. For example, the following antitumor agents can be used:

[0072] ■ Alkylating agents: cyclophosphamide, melphalan, chlorambucil, ifosfamide, mechlorethamine, uracil mustard, thiotepa, busulfan, dacarbacin, nimustine, ranimustine, temozolomide, etc.

[0073] ■ Platinum agents: cisplatin, carboplatin, nedaplatin, oxaliplatin, etc.

[0074] ■ Topoisomerase inhibitors: irinotecan, etoposide, nogitecan, etc.

[0075] ■ Antimetabolites (metabolic antagonists): gemcitabine, methotrexate, fluorouracil, tegafur, doxifluridine, pentostatin, cladribine, etc.

[0076] ■ Microtubule inhibitors: eribulin, paclitaxel, docetaxel, vinblastine, vinorelbine, vincristine, etc.

[0077] ■ Antibiotic anticancer agents: doxorubicin, daunorubicin, mitomycin C, actinomycin, etc.

[0078] ■ Molecularly targeted drugs (including small molecule compounds and antibody drugs): trastuzumab, imatinib, gefitinib, bevacizumab, crizotinib, cetuximab, lapatinib, bortezomib, tofacitinib, olaparib, venetoclax, etc.

[0079] • Immunotherapeutic agents: anti-PD-1 antibody, anti-PD-Ll antibody, anti-CTLA-4 antibody, etc.

[0080] [0 0 1 2 ]

[0081] In the present invention, two of the above antitumor drugs can be selected and used.

[0082] In a preferred embodiment of the invention, a combination of the platinum agent and microtubule inhibitor can be used. In one embodiment of the invention, a combination of carboplatin and paclitaxel can be used.

[0083] [0 0 1 3 ]

[0084] In the present invention, the target tumor to be treated is not particularly limited.

[0085] For example, stomach cancer, gastroesophageal junction cancer, esophageal cancer, duodenal cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), colon cancer, head and neck cancer, anal cancer, rectal cancer, small intestine cancer, lung cancer, liver cancer, brain tumors (including pituitary adenoma and glioma), cervical cancer, jaw cancer, maxillary cancer, submandibular gland cancer, oral cancer, salivary gland cancer, sublingual gland cancer, parotid gland cancer, nasal cavity cancer, paranasal sinus cancer, laryngeal cancer, breast cancer, biliary tract cancer (including bile duct cancer and gallbladder cancer), bladder cancer, kidney cancer, prostate cancer, uterine cancer (including cervical cancer and uterine body cancer), ovarian cancer, thyroid cancer, pharyngeal cancer, sarcoma (e.g., bone sarcoma, chondrosarcoma, Kaposi's sarcoma,Attorney Docket No. 4456-0340PWO1

[0086] myosarcoma, angiosarcoma, fibrosarcoma, etc.), malignant lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemia (including, for example, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL), lymphoma, multiple myeloma (MM), myelodysplastic syndrome, etc.), skin cancer, melanoma, etc. are exemplified.

[0087] [0 0 1 4 ]

[0088] In the present invention, various pharmaceutical (medicinal) acceptable carriers can be included in the anticancer drug depending on the form of use, as long as the active ingredient is contained in an effective amount without losing its activity, and can be used as a pharmaceutical composition. For example, carriers commonly used as diluents, excipients, etc. can be applied. Further, water, physiological buffer solutions (PBS, etc.), and various organic solvents can be used. Examples of organic solvents include aqueous alcohol (ethanol, etc.), glycerol, olive oil, etc. Furthermore, various fillers, extenders, binders, surfactants, dyes, fragrances, etc. can be added.

[0089] [0 0 1 5 ]

[0090] The pharmaceutical composition can be in the form of a liquid, suspension, emulsion, aerosol, foam, granules, powder, tablet, capsule, ointment, aqueous gel, etc. In addition, for use in injections, etc., it can be made into a lyophilized product or granulated product that is dissolved in physiological saline or an appropriate buffer solution (e.g. PBS) immediately before use to prepare a medicinal solution.

[0091] [0 0 1 6 ]

[0092] The pharmaceutical composition can be used in the form, method, and dosage according to the purpose of treatment and the patient. In the present invention, the administration route of the anticancer drugs or pharmaceutical composition is not limited, but may be either oral or parenteral, and may be administered to the living body (target cells or organs) by, for example, intravenous, intramuscular, intraperitoneal, intratumoral or subcutaneous injection; inhalation from the nasal cavity, oral cavity or lungs; or as a suppository, topical agent, etc.

[0093] [0 0 1 7 ]

[0094] The dose of the oncolytic virus and anticancer drugs can be appropriately changed according to the drug used, the type of cancer, the age and weight of the patient, and the like.

[0095] The oncolytic virus can be administered at least three times at intervals of 6 to 10 days. For example, the first dose of oncolytic virus can be administered during the Day 1 to Day 3 period, Day 12 to Day 18 period, and Day 26 to Day 32 period.Attorney Docket No. 4456-0340PWO1

[0096] The oncolytic virus can be administered at a concentration of 1x1010vp / mL to 1x1013vp / ml in a dose of 1-2 ml.

[0097] [0 0 1 8 ]

[0098] Anticancer agent can be administered at least five times, with each type of agent being administered once a week.

[0099] Radiation can be administered at a dose of 1.5-2 Gy in at least 28 doses.

[0100] [0 0 1 9 ]

[0101] In one embodiment of the invention, the dosage and administration of the oncolytic virus and anticancer drug, as well as the dosage and administration of radiation are as follows: Oncolytic virus (e.g. OBP-301): Total of 3 times (Days 1-3, 12-18, 26-32, respectively), 1-2ml of 1x1012vp / ml.

[0102] Anticancer drug: 1x weekly x 5 times,

[0103] Carboplatin AUC1-6 + Paclitaxel 25-100mg / m2

[0104] Radiation: 1.5-2Gy x 28-30 times

[0105] [0 0 2 0]

[0106] In one preferred embodiment of the invention, the dosage and administration of the oncolytic virus and anticancer drug, as well as the dosage and administration of radiation are as follows:

[0107] Oncolytic virus (e.g. OBP-301): Days 3, 12, 26, l-2ml of 1x1012vp / ml

[0108] Anticancer drug: Carboplatin AUC2 + Paclitaxel 50mg / m2, lx weekly x 5 times Radiation: 1.8Gy, 28 times (Monday -Friday, 5 weeks + 3 days)

[0109] [0 0 2 1 ]

[0110] Hereinbelow, the present invention will be described more specifically with reference to the following Examples. However, the present invention is not limited to these Examples.

[0111] [0 0 2 2]

[0112] [EXAMPLE 1]

[0113] Overview

[0114] Methods: In NRG-GI007, a phase 1 study (NCT04391049), OBP-301 was added to weekly carboplatin / paclitaxel and RT (50.4 Gy / 28 fxs) for medically inoperable EC Pts with pathologically proven adenocarcinoma or squamous cell carcinoma (SCC) of the esophagus or Siewert Type I / II gastroesophageal junction. Pts receive l-2mL of intratumoral OBP-301 1×1012virus particles / ml via endoscopy 3 days prior to and then at days 12 and 26 of CRT. The primary endpoint was protocol-defined dose-limiting toxicity, already reported.Attorney Docket No. 4456-0340PWO1

[0115] Secondary endpoints reported here are treatment-related (definitely or probably related to any protocol treatment) AEs - all and grade 4+ non-hematologic, and cCR in the primary tumor, defined as negative EGD / biopsy 6-8 weeks post-CRT. Per the protocol design, no statistical testing is done for these endpoints.

[0116] [0 0 2 3 ]

[0117] Results: Initially, 6 evaluable (eligible and started protocol treatment) Pts were enrolled from June 2020 to April 2023. As initial dose level was deemed safe, an additional 9 Pts were enrolled from August 2023 to July 2024, totaling 15 evaluable Pts for secondary endpoints. Median age was 74 years, 9 Pts (60%) with ECOG PS 1. 11 Pts (73%) had adenocarcinoma and 4 had SCC; 11 Pts had N+ disease. 14 Pts (93%) received all planned OBP-301 injections and received 50.4 Gy RT. The most common treatment-related grade 3 / 4 AEs were decreased neutrophil (6 Pts; 40%) and lymphocyte counts (5 Pts; 33%), expected with CRT. No grade ≥3 OBP-301-related non-hematologic AEs were reported. 2 Pts died prior to restaging neither definitely or probably related to OBP-301: 1 Pt developed grade 5 respiratory failure 6 weeks after CRT (suspected RT pneumonitis), while a 2ndPt had an unrecognized tumor invasion of the bronchus at baseline, which worsened during week 3 of CRT, resulting in a grade 5 tracheo- esophageal fistula. 2 Pts have not yet reached time for restaging. All 11 Pts who have been restaged had a cCR, for a preliminary cCR rate of 100% (95% CI: 74.1%, 100%).

[0118] [0 0 2 4 ]

[0119] Conclusions: OBP-301 + CRT is safe and the preliminary cCR rate compares favorably to the historical control from NRG / RTOG 0436. Updated safety and cCR data will be presented. A randomized study is being planned.

[0120] [0 0 2 5 ]

[0121] DETAILS

[0122] Background: Definitive chemoradiation (CRT) is a standard-of-care for patients (Pts) with medically inoperable esophageal cancer. The NRG / RTOG 0436 study of cisplatin / paclitaxel and RT (+ / - cetuximab) as definitive therapy showed that 58% of control arm Pts had a cCR at 6-8 weeks after CRT completion. OBP-301 is a conditionally-restricted, replication-competent adenovirus derived from human adenovirus type 5 that adds a human Telomerase Reverse Transcriptase (hTERT) gene promoter, replicating only in tumor cells to cause lysis. It may cause immunogenic cell death, enhance RT and improve local control.

[0123] [0 0 2 6 ]

[0124] Methods: In NRG-GI007, a phase 1 study (NCT04391049), OBP-301 was added to weeklyAttorney Docket No. 4456-0340PWO1

[0125] carboplatin / paclitaxel and RT (50.4 Gy / 28 fractions) for Pts with medically inoperable EC. Pts receive 1-2mL of intratumoral OBP-301 1×1012virus particles / ml via endoscopy (EGD) 3 days prior to and then at Days 12 and 26 of CRT. The primary endpoint was protocol-defined doselimiting toxicity (DLT). Initial dose safe - additional Pts enrolled. Secondary endpoints for all Pts reported here are AEs and cCR in the primary tumor. Pts have a cCR if:

[0126] EGD grossly free of disease (biopsy not required) or EGD residual / suspicion of disease with negative biopsy

[0127] [0 0 2 7]

[0128] OBP-301 (1×1012vp / mL) was administered as an intratumoral injection into the primary esophageal tumor mass via endoscopy. A total volume of 2.0 mL of the virus solution was injected into 5 sites, with 0.4 mL administered at each site. OBP-301 was administered on Day 3 (± 1 day), Day 12 (± 1 day), and Day 26 (± 1 day).

[0129] Chemotherapy Protocol Patients received premedication consisting of dexamethasone 12 mg IV, diphenhydramine 25 mg IV, famotidine 20 mg IV, and palonosetron 250 mcg.

[0130] Following the premedication, paclitaxel was administered at a fixed dose of 50 mg / m² over 1 hour, followed by the administration of carboplatin at a fixed dose of AUC 2 over 30 minutes. Paclitaxel and carboplatin were administered on Day 1 (± 1 day), Day 8 (± 2 days), Day 15 (± 2 days), Day 22 (± 2 days), and Day 29 (± 2 days).

[0131] [0 0 2 8 ]

[0132] Results: From June 2020 to July 2024, 15 evaluable Pts were enrolled (6 for DLT, 9 additional). Median age was 74 years, 9 Pts (60%) with ECOG PS 1. 11 Pts (73%) had adenocarcinoma and 4 had squamous cell carcinoma; 11 Pts had N+ disease. 14 Pts (93%) received all planned OBP-301 injections and received 50.4 Gy RT. No DLT was seen. The most common treatment-related (definitely or probably related to CRT and / or OBP-301) grade 3 & 4 AEs were decreased neutrophil (6 Pts; 40%) and lymphocyte counts (5 Pts; 33%). 2 Pts died prior to restaging: 1 Pt developed gd 5 respiratory failure 6 weeks after CRT (suspected RT pneumonitis), while a 2nd Pt had an unrecognized tracheoesophageal fistula at baseline, which worsened during Week 3 of CRT. The cCR rate for patients undergoing restaging (n=13) is 100%, and 87% for all 15 patients including the 2 patients that died prior to restaging.

[0133] Conclusions: OBP-301 + CRT can be safely given with chemoradiation. The cCR rate compares favorably to the historical control from NRG / RTOG 0436. A randomized study is being planned.

[0134] [0 0 2 9 ]Attorney Docket No. 4456-0340PWO1

[0135] Definitive chemoradiation

[0136] Definitive chemoradiation is a standard-of-care for Pts with locally advanced esophageal / GE junction cancer who are medically inoperable [RTOG 85-01 ]2

[0137] RTOG 0436 randomized 344 Pts to cisplatin / paclitaxel and RT to 50.4 Gy ± cetuximab, an anti-EGFR Ab3:

[0138] - 62% of Pts had adenoCA

[0139] - No benefit for cetuximab

[0140] - The cCR rate - defined as -ve EGD - was 58% (control arm)

[0141] - The 24-month local failure rate was 48%

[0142] [0 0 3 0]

[0143] Telomerase

[0144] Telomerase is responsible for maintaining the length of telomeres at the 3’ end of chromosomes

[0145] Expressed only in cancer cells

[0146] [0 0 3 1 ]

[0147] OBP-301 (Telomelysin™)

[0148] OBP-301 is an oncolytic virus that incorporates a telomerase (TETR) promoter and is activated only in tumor cells

[0149] [0 0 3 2 ]

[0150] OBP-301 + RT

[0151] A Japanese phase I study treated esophageal CA Pts with OBP-301 + RT (NCT03213054)1:

[0152] RT to 60 Gy (2 Gy / fx *30 fx) starting on Day 4

[0153] - OBP-301 Days 1, 18 and 32

[0154] 13 Pts were treated at increasing doses of OBP-301 + RT (3 at highest dose of 1×1012vp / ml). Only grade 3 / 4 toxicities were leukopenia (8%) and lymphopenia (77%).

[0155] 8 of 12 evaluable Pts (67%) achieved a local complete response (compared to historical rate of 40% with RT alone).

[0156] Subsequently, 37 Japanese esophageal cancer Pts were treated in a phase II study with OBP-Attorney Docket No. 4456-0340PWO1

[0157] 301 and RT4:

[0158] “Local complete response” rate was 41.7%, which exceeded predefined threshold of 30.2%

[0159] 1-year OS rate was 71.4%, which exceeded historical rate of 57.4%

[0160] NDA submission to Japanese regulatory authority is planned.

[0161] [0 0 3 3 ]

[0162] STUDY DESIGN

[0163] Study design is shown in Figure 2.

[0164] [0 0 3 4]

[0165] KEY ELIGIBILITY CRITERIA

[0166] Pathologically proven esophageal or gastroesophageal junction (GEJ) adenocarcinoma or SCC / Siewert type l-ll tumors.

[0167] Patients must, in the opinion of a thoracic surgeon or multidisciplinary team, be a candidate for initial non-operative management.

[0168] ECOG PS 0-2

[0169] Adequate hematologic, renal, and hepatic function.

[0170] [0 0 3 5 ]

[0171] OBJECTIVES

[0172] Primary Objective:

[0173] To determine if the addition of OBP-301 to chemoradiation with carboplatin / paclitaxel is safe. Secondary Objectives:

[0174] Assess toxicities.

[0175] Assess the cCR rate,

[0176] Assess the number of patients alive without progression (PFS) and number alive (OS) at 1 and 2 years.

[0177] [0 0 3 6 ]

[0178] STATISTICAL PLAN

[0179] Dose-limiting toxicity (DLT) defined as following AEs definitely or probably attributed to OBP-301, occurring up to 30 days after CRT completion:Attorney Docket No. 4456-0340PWO1

[0180] Any grade > 3 toxicity EXCEPT:

[0181] - Grade 3 nausea / vomiting

[0182] - Grade 3 esophagitis or dehydration

[0183] The first occurrence of grade 3 / 4 neutropenia

[0184] - Grade 3 / 4 lymphopenia

[0185] Any toxicity that leads to >14-day cumulative delay in CRT.

[0186] As OBP-301 1×1012dose was safe, 9 more patients will be accrued and secondary endpoints will be reported for the cohort of 15 evaluable patients treated at that dose, including the 6 used for the DLT assessment.

[0187] [0 0 3 7]

[0188] cCR Assessment:

[0189] The number of cCRs will be reported, along with percent estimate and 95% Cl. No formal statistical testing will be done for this endpoint

[0190] [0 0 3 8 ]

[0191] BASELINE CHARACTERISTICS (n=15)Attorney Docket No. 4456-0340PWO1

[0192] Age (years) Median (min-max) 74 (62-87) Sex Male 12 (80%) Female 3 (20%) Race White 15 (100%) Zubrod Performance Status i|||||||[|^ 6 (40%) iiiiijiii 9 (60%) Histology Adenocarcinoma 11 (73%) SCC 4 (27%) Location Esophagus 12 (80%) GEJ 3 (20%) Clinical T category T1b 2 (13%)

[0193] 4 (27%) 9 (60%) Clinical N category NO 3 (20%) N1-3 11 (73%)

[0194]

[0195] NX 1 (7%) [0 0 3 9 ]

[0196] TOXICITIES (n=15)

[0197] Notable toxicities seen in >20% of Pts REGARDLESS of attributionAttorney Docket No. 4456-0340PWO1

[0198] Grade 2 3 4 Anemia ® (47%) 3 (20%) 1 (7%) Anorexia3(20%) 3 {20%) 1 (7%) Constipation 7 (47%) 2 (13%) Dehydration 2 (13%) 1 (7%) Diarrhea4(27%) 4 {27%) Esophageal pain 1 (7%) 3 (20%) 1 (7%) Fatigue7(47%) 4 (27%) 2 (13%) Lymphocyte count 3 {20%) 3 (20%) 6 (40%) decreased Nausea6(40%) 3 (20%) Neutrophil decreased 2 (13%) 7 (47%) 1 (7%) Platelet decreased7(47%) 1 (7%)

[0199]

[0200] Attorney Docket No. 4456-0340PWO1

[0201] [0 0 4 0 ]

[0202] Notable toxicities definitely or probably related to OBP-301

[0203] Grade Toxicity Chills 2 (20%) Esophagitis 1 (7%) Fever 3 (20%)

[0204]

[0205] Headache 1 (7%) [0 0 4 1 ]

[0206] 2 Pts died prior to disease reassessment:

[0207] o 81M with T3Nx GEJ adenoCA and baseline history of interstitial lung disease completed protocol therapy. 6 weeks later, he developed hypoxic respiratory failure, died week later. The grade 5 respiratory failure was assessed to be due possibly to radiation pneumonitis and to underlying ILD; it was unlikely to be due to OBP-301 o 77F with T3N3 esophageal SCC developed respiratory failure during Week 3 of CRT following 2ndOBP-301 injection then died from recurrent aspiration. In retrospect, it was recognized that tracheal invasion by the primary tumor was present at baseline and a fistula developed due to brisk tumor shrinkage. The grade 5 fistula was probably related to CRT and possibly related to OBP-301

[0208] [0 0 4 2 ]

[0209] COMPLETE RESPONSE ASSESSMENT

[0210] 13 Pts underwent a restaging EGD

[0211] o 100% (95% CI: 79.6, 100) of Pts had a cCR

[0212] o 11 Pts had normal EGDs (2 no and 9 negative biopsy)

[0213] o 2 Pts had EGDs which showed abnormal mucosa but had negative biopsies All 15* Pts: 13 had cCR, 87% (95% CI: 62.1, 96.3)

[0214] * including 2 Pts who died prior to restaging

[0215] oAttorney Docket No. 4456-0340PWO1

[0216] [0 0 4 3]

[0217] CONCLUSION OBP-301 can be safely given in combination with CRT.

[0218] cCR compares favorably to the cCR rate in RTOG 0436 and 2 Japanese studies with RT alone. Number of pts alive without progression (PFS) and number alive (OS) at 1 and 2 years are pending. A randomized study is being designed.Attorney Docket No. 4456-0340PWO1

[0219] [EXAMPLE 2]

[0220] The following is the study protocol for Example 1.

[0221] 1. OBJECTIVES

[0222] 1.1 Primary Objective

[0223] To determine if the addition of OBP-301 to chemoradiation with carboplatin / paclitaxel is safe.

[0224] 1.2 Secondary Objectives

[0225] 1.2.1 To assess toxicities associated with the addition of OBP-301 to chemoradiation.

[0226] 1.2.2 To assess the number of clinical complete responses (cCR).

[0227] 1.2.3 To assess the number of patients alive / without progression (PFS) and the number of

[0228] patients alive (OS) at 1 and 2 years.

[0229] 1.3 Exploratory Objectives

[0230] To report correlate outcomes - cCR, PFS and OS - with immune and virus-based

[0231] correlative assays

[0232] 2. BACKGROUND

[0233] 2.1 Definitive Chemoradiation (11 -JAN-2022)

[0234] In the U. S., esophageal cancer is a rare but deadly malignancy. In 2020, it is estimated

[0235] that 18,440 patients will be diagnosed, with 16,170 deaths (Siegel 2020). It ranks as the

[0236] seventh cause of cancer deaths, despite its rarity, underlining the challenge of treating

[0237] such patients. These poor outcomes are in part because approximately half of patients in

[0238] the U. S. are diagnosed with metastatic disease.

[0239] For patients with localized disease, surgery remains the cornerstone of curative therapy.

[0240] However, esophageal cancer is a disease of older patients, many of whom cannot tolerate the morbidity of an esophagectomy because of underlying cardiopulmonary

[0241] comorbidities or because of a borderline performance status. Complications include

[0242] respiratory problems in 11% to 20%, anastomotic leak in 3% to 7%, and wound infection in 5%. Operative mortality ranges from zero to 4%, even in high-volume institutions

[0243] (Mathisen 1988, Griffin 2002, Nichols 2005).

[0244] As such, a standard-of-care that has emerged for patients with locally advanced

[0245] esophageal and gastroesophageal junction (GEJ) cancer who are medically inoperable is definitive chemoradiation. This approach is based on the seminal Radiation Therapy

[0246] Oncology Group (RTOG) 85-01 study, which revealed superior outcomes for 5- fluorouracil (FU) / cisplatin chemotherapy and radiation vs. radiation alone (Herskovic

[0247] 1992); long-term survival was reported in both squamous cell cancer (SCC) patients and

[0248] the small group of adenocarcinoma patients treated on this study (Cooper 1999).Attorney Docket No. 4456-0340PWO1

[0249] Definitive chemoradiation is a particularly strong consideration for patients with

[0250] esophageal SCC. Many of these patients have co-morbidities associated with alcohol and tobacco use, and surgery carries with it a higher rate of post-operative mortality

[0251] following neoadjuvant chemoradiation secondary to increased risks of cardiopulmonary complications. In addition, observation for patients with esophageal SCC who achieve a clinical complete response to chemoradiation is supported by the results of two

[0252] randomized European trials, which did not show a clear improvement in overall survival for surgery following chemoradiation (Stahl 2005, Bedenne 2007).

[0253] Platinum / Taxane with Radiation (11 -JAN-2022)

[0254] While the RTOG 85-01 study treated patients with 5-FU / cisplatin and radiation, the

[0255] Dutch CROSS study established carboplatin / paclitaxel as a contemporary reference chemotherapy regimen in combination with radiation prior to surgery for operable tumors

[0256] (van Hagen 2012). This regimen resulted in a 23% and 49% pathologic complete

[0257] response rate (pCR) respectively in patients with esophageal adenocarcinomas and SCC.

[0258] Compared to surgery alone, it also reduced the risk of distant metastases by 6%

[0259] (Oppedijk 2014).

[0260] Of note, the widespread adoption of this regimen is in large part because it was very well-tolerated. For example, the rate of grade 3 / 4 esophagitis was only 1% and there was only

[0261] 1 esophageal perforation in 171 patients. The grade 3 / 4 nausea rate was also only 1%.

[0262] Unfortunately, the relatively low pCR rate with chemoradiation means that the majority

[0263] of patients who do not undergo surgery will have locally persistent / recurrent disease,

[0264] which is a source of significant morbidity, including dysphagia and bleeding. In addition, these patients are at significant risk for developing metastatic disease. As such, this

[0265] population represents a high unmet medical need in what is already a highly malignant disease.

[0266] Relatively few studies have focused on this population as the majority of studies have mandated pre-operative therapy prior to surgery. A recent exception is the RTOG 0436

[0267] study, which randomized 344 patients (62% of whom had adenocarcinomas) to cisplatin / paclitaxel and radiation to 50.4 Gy (1.8 Gy / fraction ×28 fractions) with or

[0268] without cetuximab, an antibody against epidermal growth factor receptor

[0269] (Suntharalingam 2017). The study demonstrated there was no improvement in overall

[0270] survival (OS), which was the primary endpoint; the 24-month OS rate was about 44%.

[0271] Similarly, the clinical CR (cCR) rate - defined as endoscopic clearance of the primary

[0272] tumor - was about 56% in both arms and the 24-month local failure rate for all patients

[0273] was about 48%.

[0274] Oncolytic viruses

[0275] Oncolytic immunotherapy employs viruses that are designed to preferentially replicate in and lyse cancer cells and through this process trigger anti-tumor immunity. Following the first description of a virus engineered to replicate selectively in cancer cells over 20 yearsAttorney Docket No. 4456-0340PWO1

[0276] ago, the field of oncolytic immunotherapy has expanded dramatically. Over 10 different viral species have now been assessed or are under assessment in human studies, including the Hl 01 adenovirus product, which was approved for use in combination with chemotherapy as a treatment for head and neck cancer by the Chinese State Food and Drug Administration in November 2005.

[0277] More recently, a phase III pivotal trial of talimogene laherparepvec, an oncolytic herpes

[0278] virus expressing granulocyte-macrophage colony stimulating factor (GM-CSF) in

[0279] patients with advanced melanoma met its primary endpoint, demonstrating a significant improvement in durable response rate versus GM-CSF alone (16% vs 2%, p<0.0001) in patients with advanced melanoma (Andtbacka 2015). Based on these results, in October

[0280] 2015, it was granted approval as the first oncolytic viral therapy in the U. S. as local

[0281] therapy for recurrent melanoma. The European Medicines Agency also approved

[0282] talimogene laherparepvec for the treatment of adults with unresectable melanoma.

[0283] Conditionally replicable oncolytic viruses are engineered for selective replication in

[0284] cancer cells that express certain oncogenic phenotypes (Hawkins 2001, Nemunaitis

[0285] 2010). To this end, multiple viral backbones have been employed, although the most commonly utilized is derived from the adenovirus serotype 5 (Ad5).

[0286] OBP-301: Background

[0287] OBP-301 (Telomelysin™) is a novel, condition-restricted, replication-competent

[0288] adenovirus serotype 5-based adenoviral construct that incorporates a human telomerase

[0289] reverse transcriptase gene (hTERT) promoter. hTERT encodes the catalytic protein

[0290] subunit of telomerase, a polymerase that acts to stabilize telomere lengths and is highly expressed in tumors but not in normal, differentiated adult cells. Thus infection with

[0291] OBP-301 leads to viral cytolytic activity preferentially in cancer cells but not normal

[0292] cells.

[0293] The adenoviral reproductive cycle is a highly orchestrated process. At the molecular

[0294] level, the adenoviral genome contains 8 transcriptional units that are activated at different phases of infection. They are referred to as early (El A), immediate early (E1B, E2, E3,

[0295] E4), intermediate (IX and IVa2), and late genes. It has been reported previously that the transcriptional control of the El A expression via the hTERT promoter could restrict adenoviral replication to telomeres-positive cells and efficiently lyse tumor cells

[0296] (Fujiwara 2007). In the construct of OBP-301 (Figure 1), the normal transcriptional

[0297] regulatory element of the E1A gene is replaced by the hTERT promoter and additional modifications to enhance specificity include the replacement of the normal transcriptional element of viral E1B gene by an internal ribosomal entry site (IRES) sequence to

[0298] minimize “leakiness”). Furthermore, OBP-301 is the first replication-competent

[0299] adenovirus that retains a fully functional viral E3 region, which codes for proteins that

[0300] regulate the immune response to the virally infected cell (Hawkins 2001).

[0301] Telomerase activation is considered to be a critical step in carcinogenesis and its activityAttorney Docket No. 4456-0340PWO1 is closely correlated with hTERT expression. Thus, expression of hTERT is generally

[0302] low in differentiated adult cells and active in cells with proliferative capacity such as

[0303] tumors, gastrointestinal endothelium, or stem cells;(Shay 1997, Shay 2001) the majority

[0304] of malignant tumors shown to demonstrate high telomerase activity. Previous studies

[0305] have demonstrated control over the expression of exogenous genes to telomerase-positive cancer cells by the hTERT promoter (Kamradt 2003) and the hTERT promoter is an

[0306] excellent candidate for generating cancer-specific oncolytic adenovirus.

[0307] There are several mechanisms by which adenoviruses in general, and OBP-301 in

[0308] particular, can destroy tumor cells in vivo. Direct cell lysis occurs following viral

[0309] replication and data from animal models have shown that non-replicating adenovirus in

[0310] rodent tissue will induce both an acute (2-4 days) and chronic (14 days) inflammatory infiltration. During the acute phase, tumor necrosis factor (TNF-a) and interleukin (IL)-l and IL-6 are produced in high concentrations locally within the tissue. These cytokines

[0311] have both direct cytopathic effects and indirect effects through immune effector cell recruitment into the local tissue. Finally, adenovirus infection of a tumor cell may

[0312] augment tumor antigen presentation and enhance tumor antigenicity, leading in turn to immunogenic cell death, which leads to release of danger-associated molecular patterns, thereby attracting innate immune cells, particularly dendritic cells to the tumor and

[0313] leading to the recruitment and maturation of tumor specific T-cells in the tumor microenvironment, improved recognition and destruction of tumors by cytotoxic T cells

[0314] (van Vloten 2018).

[0315] In vitro studies have validated the selective infectivity and direct cytolysis of OBP 301 in cancer cells and in vivo, intratumoral injection has demonstrated anti-tumor activity

[0316] without significant toxicity. Additionally, distant viral uptake was observed followingAttorney Docket No. 4456-0340PWO1

[0317] intratumoral injection, evidenced by the presence of adenoviral protein in non-injected

[0318] tumors following treatment of the contralateral tumor. These encouraging pre-clinical

[0319] findings of safety and directed anti-tumor activity were supportive of the continuing

[0320] clinical development of OBP 301 as an oncolytic therapeutic agent (Fujiwara 2007).

[0321] OBP-301: Clinical Experience

[0322] In a phase I study, OBP-301-001, (OBP-301), 16 patients were treated with single intratumoral injections of OBP-301(Nemunaitis 2010). No grade 3 / 4 toxicities were observed and common grade 1 / 2 toxicities included injection site reactions and systemic symptoms (fevers / chills in 6 patients). Viral DNA was transiently detected in 13 of the 16 patients.

[0323] Of 11 patients with RECIST-evaluable lesions, 1 patient had a partial response, while 7

[0324] had stable disease.

[0325] In OBP-301 -003 -CV (OBP-301 + Radiation phase I / II study), OBP-301 was combined

[0326] with radiation to 60 Gy in Japanese esophageal cancer patients who were assessed not to

[0327] be candidates for surgery or chemoradiation. OBP-301 were administered at three dose

[0328] levels; 1 mL of 1x1010viral particles (vp) / mL, 1 mL of 1x1011vp / mL, and ImL of 1x1012vp / mL, administered 3 times via endoscopic injection (Tanabe 2019). In total, 13 patients were treated (3 at the highest dose level of 1×1012vp / ml). Toxicities were manageable.

[0329] The only observed grade 3 / 4 toxicities were leukopenia (8%) and lymphopenia (77%).

[0330] Grade 1 / 2 toxicities include transient fever without evidence of infection (62%), chills

[0331] (15%), nausea (25%), esophagitis (31%) anorexia (39%) and pneumonitis (23%). Of the

[0332] 13 patients treated, 8 achieved a local cCR to the combination of OBP-301 and radiation

[0333] and 3 patients have achieved a partial response. The overall response rate was 91.7%.

[0334] The clinical CR was 83.3 % in stage I and 60% in stages II and III. These results compare favorably with a historical cCR rate of about 37.2% for stages I-IV with radiation alone

[0335] (Toh 2020, Fujiwara 2019).

[0336] Beyond augmenting the local effects of radiation, OBP-301 has been shown in vitro to

[0337] inhibit DNA repair mechanisms, which may sensitize the infected tumor cells to radiation (Kuroda 2010), leading to synergistic effect. In addition to a local effect, OBP-301 may

[0338] also potentiate immune responses. Viral replication is highly immunogenic and oncolysis induced by such viruses releases tumor epitopes and provides costimulatory danger

[0339] signals (Endo 2008).

[0340] Combining OBP-301 with Chemoradiation (11 -JAN-2022)

[0341] Given the excellent safety profile of both carboplatin / paclitaxel and radiation and OBP-301 with radiation (in an elderly population that was assessed not to be a eligible for even chemoradiation alone), there is a compelling rationale to combine OBP-301 with carboplatin / paclitaxel and radiation as definitive therapy for patients with

[0342] esophageal / GEJ cancer who are not surgical candidates. In addition to improvement in locoregional control, there is also the potential to augment anti-tumor immunity, which

[0343] may also impact positively on distant micrometastatses through an abscopal effect.Attorney Docket No. 4456-0340PWO1

[0344] 3. ELIGIBILITY AND INELIGIBILITY CRITERIA

[0345] Note: Per NCI guidelines, exceptions to inclusion and exclusion criteria are not

[0346] permitted. For questions concerning eligibility, please contact the Biostatistical / Data Management Center (see protocol cover page). For radiation therapy-related eligibility

[0347] questions, please contact RTQA (see protocol cover page).

[0348] 3.1 Eligibility Criteria ( 19-MAY-2023 )

[0349] A patient cannot be considered eligible for this study unless ALL of the following

[0350] conditions are met.

[0351] 3.1.1 Pathologically (histologically or cytologically) proven diagnosis of adenocarcinoma or squamous cell carcinoma (SCC) of the esophagus or gastroesophageal junction (GEJ) within 90

[0352] days prior to registration;

[0353] • Gastroesophageal junction tumors must be Siewert Type I / II;

[0354] 3.1.2 Required diagnostic workup for study entry:

[0355] • History / physical examination prior to registration;

[0356] • CT of the chest / abdomen with intravenous contrast within 28 days prior to registration;

[0357] If CT contrast is contraindicated MRI of the chest / abdomen without contrast is permitted;

[0358] • Bronchoscopy for squamous cell carcinoma (SCC) tumors that are adjacent to the

[0359] airway to exclude a tracheoesophageal fistula within 42 days prior to registration;

[0360] • Endoscopic ultrasound (if technically feasible) within 90 days prior to registration;

[0361] • Whole body PET / CT scan within 42 days prior to registration: Note: scan will be

[0362] used for radiation treatment planning, in addition to ruling out metastatic disease;

[0363] 3.1.3 Age > 18;

[0364] 3.1.4 ECOG Performance Status of 0-2 within 14 days prior to registration;

[0365] 3.1.5 Adequate hematologic function within 14 days prior to registration defined as follows:

[0366] • Absolute Neutrophil Count >l,500 / mcL

[0367] • Hemoglobin > 9 gm / dL

[0368] • Platelets > 100, 000 / mcL

[0369] 3.1.6 Adequate renal function within 14 days prior to registration defined as follows:

[0370] • Creatinine clearance of >50 ml / min (as calculated by Cockcroft-Gault equation)

[0371] 3.1.7 Adequate hepatic function within 14 days prior to registration defined as follows:

[0372] • Total Bilirubin < 1.5 x ULN (patients with known Gilbert Syndrome can have a Total Bilirubin <2.5 x ULN)

[0373] • AST / ALT < 2.5 x ULNAttorney Docket No. 4456-0340PWO1

[0374] atients for whom non-operative management is a viable option in the opinion of a thoracic surgeon and / or multidisciplinary team and are candidates for chemoradiation; this does not preclude patients from receiving surgery after chemoradiation if felt to me medically indicated;

[0375] Patients must, in the opinion of a treating gastroenterologist, have a tumor that is amenable to intratumoral injection with at least 1 mL (1 x1012vp / mL) of OBP-301 and be a candidate for 3 endoscopy procedures.

[0376] Female patients of child bearing potential must have a negative serum / urine pregnancy test within 14 days prior to study entry. A female not of childbearing potential is one who has undergone a hysterectomy, bilateral oophorectomy, tubal ligation, or who has had no menses for 12 consecutive months.

[0377] Patients of reproductive potential must agree to use effective contraception for the duration of study treatment as well as 6 months (for women) or 12 months (for men) after the last administered injection of OBP-301. Effective contraception includes oral contraceptives, implantable hormonal contraception, double-barrier method or intrauterine device.

[0378] he patient must provide study-specific informed consent prior to study entry.

[0379] Patients with a prior or concurrent malignancy whose natural history or treatment does not have the potential to interfere with the safety or efficacy assessment of the investigational regimen are eligible for this trial.

[0380] Known acute or chronic hepatitis B or C infection (testing not required prior to study entry in patients with no known history of hepatitis B or C);

[0381] • For patients with evidence of chronic hepatitis B virus (HBV) infection, the HBV viral load must be undetectable on suppressive therapy, if indicated.

[0382] • For patients with a history of hepatitis C virus (HCV) infection, they must (i) have been treated and cured, (ii) for patients with HCV infection who are currently on treatment, they are eligible if they have an undetectable HCV viral load

[0383] HIV-infected patients on effective anti-retroviral therapy with undetectable viral load within 6 months prior to study entry are eligible for this trial.

[0384] Ineligibility Criteria (11 -JAN-2022)

[0385] Patients with any of the following conditions are NOT eligible for this study.

[0386] Definitive clinical or radiologic evidence of metastatic disease including;

[0387] • Positive malignant cytology of the pleura, pericardium or peritoneum;

[0388] • Radiographic evidence of involvement of any adjacent mediastinal structure, e.g. aorta, trachea, which would increase the risk of repeated endoscopic interventions;

[0389] • Tracheoesophageal fistula

[0390] • Radiographic evidence of distant organ involvement;Attorney Docket No. 4456-0340PWO1

[0391] • Non-regional lymph nodes that cannot be contained within a radiation field;

[0392] More than 1 esophageal lesion;

[0393] Prior systemic chemotherapy for the study cancer;

[0394] Prior radiotherapy to the region of the study cancer that would result in overlap of radiation therapy fields;

[0395] Biopsy-proven tumor invasion of the tracheobronchial tree or presence of

[0396] tracheoesophageal fistula or recurrent laryngeal or phrenic nerve paralysis.

[0397] For patients with known history or current symptoms of cardiac disease, or history of treatment with cardiotoxic agents, a New York Heart Association Functional Classification

[0398] 2C or worse.

[0399] ncontrolled diabetes;

[0400] Infection requiring IV antibiotics at the time of registration;

[0401] Patients requiring immunosuppressive medications including chronic suppressive steroid therapy (greater than the equivalent of 20mg / day of prednisone), methotrexate,

[0402] azathioprine and TNF-a blockers within 7 days prior to study entry; See section 5.4.2

[0403] for further details;

[0404] Received live vaccine within 30 days prior to registration;

[0405] Received a blood transfusion, hematopoietic agent; granulocyte-colony stimu / ating factor

[0406] (G-CSF), and / or oxygen supplementation within 7 days before the screening lab.

[0407] Breast feeding females.Attorney Docket No. 4456-0340PWO1

[0408] 4. REQUIREMENTS FOR STUDY ENTRY, TREATMENT, AND FOLLOW-UP

[0409] (19-MAY-2023)

[0410] PRE-TREATMENT ASSESSMENTS

[0411] Assessments Prior to Registration Prior to Initial OBP-301

[0412] (calendar days) Injection

[0413] (calendar days)

[0414] Optional Patient History X

[0415] Form (Patient Reported)*

[0416] Endoscopy with biopsy 90

[0417] Endoscopic Ultrasound (If 90

[0418] technically feasible)

[0419] Bronchoscopy (for SCC 42

[0420] tumors that are adjacent to the

[0421] airway)

[0422] Whole body PET / CT scan 42

[0423] CT / MRI chest / abdomen** 28

[0424] CBC (to include absolute 14

[0425] neutrophil count, Hgb and

[0426] platelets)

[0427] CMP (to include creatinine, 14

[0428] AST / ALT, and total bilirubin)

[0429] History and physical exam X

[0430] ECOG Performance status 14

[0431] Concomitant Medications* 14

[0432] Urine or Serum Pregnancy 14 (for women of child X

[0433] test bearing potential)

[0434] Optional collection of (see Section 10)

[0435] archival tumor tissue

[0436] Optional Blood Collection <2

[0437]

[0438] (see Section 10)

[0439] *In person preferred; may be conducted by telehealth visit at the discretion of the site-identified qualified healthcare professional.

[0440] **CT of the chest / abdomen with intravenous contrast. If CT contrast is contraindicated, MRI of

[0441] the chest / abdomen without contrast is permitted.Attorney Docket No. 4456-0340PWO1

[0442] ASSESSMENTS DURING TREATMENT

[0443] Assessments Prior to OBP-301 injection Weekly during concurrent chemotherapy and radiation Physical exam per X

[0444] Institutional Standard

[0445] ECOG PS assessment X

[0446] Adverse Event Assessment X

[0447] Concomitant Medications X

[0448] CBC (to include WBC, X (see section 5.2) X

[0449] absolute neutrophil count,

[0450] Hgb and platelets)

[0451] CMP (to include creatinine, X

[0452] AST / ALT, and total

[0453] bilirubin)

[0454] Optional Collection of tumor X(see Section 10)

[0455] and blood for correlative

[0456] assays

[0457] Urine or Serum Pregnancy Only prior to first OBP-301

[0458] test treatment only (for women of

[0459]

[0460] child bearing potential)Attorney Docket No. 4456-0340PWO1

[0461] ASSESSMENTS IN FOLLOW UP

[0462] Assessment 1 week 6-8 weeks Q 3 mo Q 3-6 mo Q 6 mo

[0463] post post RT from end from end from end chemoRT completion RTX2 RT X 2 YRS RTX2

[0464] YRS YRS

[0465] Physical Exam X

[0466] CBC and CMP X X

[0467] Whole Body X

[0468] PET / CT (if not

[0469] covered by insurance

[0470] CT / MRI is allowed

[0471] and must include

[0472] chest, abdomen, and

[0473] pelvis)

[0474] CT or MRI X

[0475] chest / Abdomen

[0476] Endoscopy with X X (only for

[0477] / without Biopsy (see clinical

[0478] definition of disease complete

[0479] assessments below) responders at

[0480] 6-8 weeks post

[0481] RT

[0482] completion)

[0483] Physical Exam X X

[0484] Adverse Event X X

[0485] Evaluation

[0486] ECOG PS X X

[0487] Optional Collection X

[0488] of Tumor and Blood

[0489] for Correlative

[0490] Assays (See Section

[0491]

[0492] 10)

[0493] Definition of Disease Assessments

[0494] The same method of imaging assessment and the same technique should be used to characterize baseline and post-treatment extent of disease. Imaging-based evaluation is preferred to

[0495] evaluation by clinical exam when both methods have been used to assess the anti-tumor effect of therapy.

[0496] While it is recognized that it is not always possible to obtain pathologic proof of progressive

[0497] disease, biopsy or autopsy material confirming recurrent cancer is preferred and every reasonable attempt to obtain such is encouraged.

[0498] Local DiseaseAttorney Docket No. 4456-0340PWO1

[0499] 6-8 weeks post RT completion for clinical complete response (cCR)

[0500] At the time of the 6-8 weeks post RT completion endoscopy, a visual inspection of the

[0501] site of the original primary disease must be documented. Patients will be scored as

[0502] follows:

[0503] • Those patients who are found to be grossly free of disease are NOT required to

[0504] undergo biopsy and will be scored as achieving a clinical complete response

[0505] • Patients deemed to have residual disease or suspicion of residual disease must

[0506] undergo a biopsy in order to pathologically confirm findings. Any patient with pathologically confirmed residual disease will be scored as a local failure. Patients

[0507] who are pathologically proven to have no evidence of disease will be scored as

[0508] achieving a clinical complete response

[0509] Follow-up after initial cCR assessment

[0510] • Clinical complete responders: If there is evidence or suspicion of disease on followup CT / MRI scans, biopsy is required to confirm recurrence of disease. In the absence of clinical symptoms or radiographic findings, patients should undergo a surveillance EGD every 6 months for up to 2 years from the start of protocol therapy.

[0511] • Patients with residual disease: These patients should be followed for local disease per institution’s standard of care.

[0512] Distant Disease

[0513] In the absence of histologic or cytologic proof of recurrence, clinical evidence (including e.g.

[0514] new masses on CT scan, new lesions on bone scan, ascites not explained by other causes or

[0515] enlarging mass by endoscopic ultrasound) should be considered highly suspicious of recurrent disease. It is strongly suggested that these findings lead to a biopsy to confirm progression.

[0516] However, if the patient’s clinical condition does not allow biopsy to be performed and these

[0517] findings are unequivocal, they may be acceptable as progression of disease.

[0518] 5. TREATMENT PLAN / REGIMEN DESCRIPTION

[0519] Protocol treatment must begin within 2 weeks after protocol registration.

[0520] 5.1 Treatment Summary (19-MAY-2023)

[0521] Patients undergo an intra-tumoral injection via endoscopy (EGD) with OBP-301 (1×1012vp / mL).

[0522] Patients will then receive chemoradiation with carboplatin and paclitaxel administered on a Monday or Tuesday and radiation administered Monday through Friday.Attorney Docket No. 4456-0340PWO1

[0523] Treatment Regimen with Initial OBP-301 Dose Level: OBP-301 injection must start Thursday or Friday. Chemoradiation must start Monday or Tuesday.

[0524] Week Week 2 Week Week 4 Week

[0525] 1 3 5

[0526] Day -3 Day 1 Day 8 Day 12 Day 15 Day 22 Day 26 Dav 29

[0527] (+ / - 1 (+ / - 1 (+ / - 2 (+ / - 1 (+Z- 2 (+1- 2 (+ / - 1 (+ / - 2

[0528] day) day) day) day) day) day) day) day)

[0529] OBP-301 by EGD 1 xlO12X X X

[0530] vp / mL (ql4d ±1 day)

[0531] Carboplatin AUC 2 X X X X X

[0532] Paclitaxel 50mg / m2X X X X X

[0533]

[0534] Radiation 1.8 Gy / fraction Daily Monday-Friday for 5 ½ weeks (28 fractions)Attorney Docket No. 4456-0340PWO1

[0535] IF NEEDED: Treatment Regimen with DE-ESCALATED OBP-301 Dose Level:

[0536] Week Week 2 Week Week 4 Week

[0537] 1 3 5

[0538] Day *3 Day 1 Day Day Dav Dtn Dav 26 Day 19

[0539] (+ / - I (+ / ♦ 1 8 12 15 22 {47- 1 (+7*2

[0540] da>) day ) (+A (+A 1 {47-2 (+7*2 day) day )

[0541] 2day) day) day) day)

[0542] □BP-301 by EGD I x l<i" X X X

[0543] vp / mL{qI4d±l day)

[0544] Carbapkitffi AUC2 X X X

[0545] Paclitaxel 5(hng / in- X iOiOiOi X

[0546]

[0547] Radiation 1.8 Gy / fraction Daily Monday-Friday for 5 ½ weeks (28 fractions)

[0548] 5.2 Systemic Therapy (26-JUL-2021)

[0549] 5.2.1 OBP-301 Treatment

[0550] OBP-301 (l><1012vp / mL) is administered as intratumoral injection into a primary esophageal

[0551] tumor mass that is suitable for injection, and in a location amenable for endoscopic injection.

[0552] Irrespective of the size of the tumor, every effort should be made to inject 1.0 to 2 ml of OBP-301; the maximal volume of 2 ml is preferred and should be injected as long as this is technically possible.

[0553] Pre-inj ection planning is the key to success, therefore, a sketch of the tumor and the planned injection sites should be made. The sketch will be helpful in determining the total quantity of study drug to inject. Please refer to Appendix I.

[0554] The optimum technique for injection of a primary tumor is to inject the viral particles into the

[0555] lesion. This will ensure that the viral particle will maximally penetrate into the lesion.

[0556] Endoscopists who perform a polypectomy will be eligible to perform the procedure. A training

[0557] video (prepared by Oncolys Biopharma) is available as well.

[0558] The procedure will be performed in the Endoscopy Unit with standard sterilization procedures. A standard gastroscope and needle (e g. GIF-HQ190 Olympus gastroscope with 23 or 25 gauge

[0559] injection needle made by Boston Scientific / or Olympus (Needl eMaster) is used for the

[0560] procedure.

[0561] Guidelines for each injection procedure are provided below:

[0562] Pre-Injection

[0563] • Review of medications

[0564] • Abbreviated physical exam and performance statusAttorney Docket No. 4456-0340PWO1

[0565] • Vital signs

[0566] Post-Injection

[0567] • Monitor patient for 60-90 minutes for any symptoms of injection site pain, bleeding, nausea.

[0568] • Vital signs (except weight)

[0569] Standard sedation procedures used for endoscopy will be used, and consists of monitored

[0570] anesthesia care with an anesthesiologist and the patient monitored at all times.

[0571] For patients who consent to the optional collection of tumor (see Section 10), biopsy of the

[0572] primary tumor should be performed prior to OBP-301 injection. Up to 3 biopsies should be

[0573] performed and processed by Pathology per standard procedure. If <3 core biopsies are

[0574] performed, the reason should be documented in the procedure note.

[0575] The tumor area to be injected will be >1 cm and can be mapped into (preferably at least) 5

[0576] sections. The dose (volume) delivered to the lesion should be within the range of 1.0 to 2.0 mL.

[0577] Refer to the OBP-301 Pharmacy Manual (Appendix I) for details to complete the endoscopic

[0578] injection of OBP-301.

[0579] Parameters for Treatment

[0580] ■ A CBC is required within 3 days prior to OBP-301 administration.

[0581] Parameters for treatment on Day -3 are as follows:

[0582] • WBC>3,000 / pL

[0583] • ANC>l,500 / pL

[0584] • Platelets >100, 000 / pL

[0585] • Hgb >9 g / dL

[0586] Parameters for treatment on any other subsequent day are as follows and based on a CBC

[0587] obtained at any time during that week prior to OBP-301 administration.

[0588] • ANC>l,000 / pL

[0589] • Platelets >75, 000 / pL

[0590] FOR THE DE-ESCALATED COHORT:

[0591] Preparation Step: Use injection syringe (Figure 3) to suck the 1 ml from 1x1012vp / mL vial and

[0592] use diluted solution syringe to suck the 9 mL D5W.

[0593] Dilution Step: Slowly push 1 mL OBP-301 to Diluted solution syringe, and mix (The

[0594]

[0595] concentration of diluted solution syringe would be 1 x 1011vp / mL.)Attorney Docket No. 4456-0340PWO1

[0596] Dose Levels

[0597] Dose Level Number Details

[0598] forDLT of OBP- Assessment 301 Doses

[0599] Initial 3 ■ OBP-301 1 x 1012viral particles (vp) / mL (up to 2mL of OBP- COHORT 301 should be injected based on the tumor volume.) will be

[0600] given by an intra-tumoral injection via EGD every 14 days on

[0601] Days -3, 12 and 26 (±1 day).

[0602] IF NEEDED; • OBP-301 1×1011viral particles (vp) / mL (up to 2mL of OBP-301 should be injected based on the tumor volume)) will be given by an intra-tumoral injection via EGD every 14 days on Days -3, 12 and 26 days (±1 day).

[0603]

[0604] 5.2.2 Paclitaxel / Carboplatin Chemotherapy

[0605] Paclitaxel

[0606] Paclitaxel will be administered at a fixed dose of 50 mg / m² over 60 minutes weekly;

[0607] rounding dose down to the nearest 10 mg is permitted as long as within 10% of the

[0608] calculated dose. This is the recommended dose administration guidance but sites may

[0609] follow institutional standards.

[0610] The paclitaxel infusion is administered prior to the carboplatin infusion.

[0611] The first dose will be given on Monday or Tuesday during the first week of radiation.

[0612] Subsequent doses will be given every 7 days + / -1 day for a total of 5 doses.

[0613] Recommended pre-medications or per institutional standards:Attorney Docket No. 4456-0340PWO1

[0614] • Dexamethasone lOmg IVP, completed 30 minutes prior

[0615] • Diphenhydramine 50mg IVP or equivalent

[0616] • H-2 blocker of choice (Cimetidine 300mg IV or ranitidine 50mg IV or famotidine

[0617] 20mg IV) 30 minutes prior

[0618] Carboplatin

[0619] See Carboplatin Dose Calculation Instruction in Appendix V

[0620] Carboplatin will be administered at a fixed dose AUC 2 over 30 minutes weekly. The

[0621] first dose will be given on either Monday or Tuesday during the first week of radiation.

[0622] Subsequent doses will be given every 7 days + / -1 day for a total of 5 doses.

[0623] The maximum dose of carboplatin will be capped at 300 mg.

[0624] Recommended pre-medications or per institutional standards:

[0625] • Antiemetic regimen including a 5HT3-antagonist and steroid

[0626] Radiation Therapy (10-JUL-2023)

[0627] Radiation must begin on a Monday or Tuesday following the first intratumoral

[0628] injection with OBP-301.

[0629] Radiation Therapy Schema

[0630] Radiation will be given in 1.8 Gy / fraction *28 fractions (50.4 Gy total)- 5

[0631] fractions / week Monday -Friday starting on Day 1. Radiation can either precede or follow chemotherapy on days when they are given concurrently.

[0632] Treatment Technology

[0633] Proton treatments are not allowed, IMRT only.

[0634] Photon beam intensity modulated radiation therapy (IMRT) / VMAT with effective

[0635] energies between 6 and 10 MV is required. IMRT may be delivered using multiple fixed

[0636] fields employing dynamic multi-leaf collimator, helical arc therapy or volumetric

[0637] modulated arc therapy using any of the commercially available delivery systems. Daily

[0638] image-guidance (IGRT) is also required for all patients.

[0639] Immobilization and Simulation

[0640] Proper immobilization is important for this protocol. Patient setup reproducibility must

[0641] be achieved using appropriate clinical devices.

[0642] Assessment and Management of Internal Organ Motion

[0643] Special considerations must be made to account for the effect of internal organ motion

[0644] (e.g., primarily breathing associated motion) on target positioning and reproducibility. As a first step, it is required that the treatment team quantify the specific motion of a target

[0645] so as to determine if management strategies are needed. The patient should be in normal

[0646] free breathing at the time of initial tumor motion assessment. Deep inspiration or

[0647] expiration breath hold is not allowed for initial tumor motion assessment as such

[0648] assessment generally overestimates free breathing tumor motion. Options for motionAttorney Docket No. 4456-0340PWO1

[0649] assessment include real time fluoroscopy and 4D CT scanning. Any strategy, including

[0650] 4D CT should incorporate appropriate image review and quality assurance to ensure

[0651] suitability for treatment planning and target delineation.

[0652] In some tumor locations, tumor motion measurement may demonstrate motion

[0653] exceeding the required small tumor expansions per this protocol (resulting in marginal

[0654] miss or excessive volume of irradiation) unless a motion management strategy is

[0655] employed.

[0656] Acceptable maneuvers for motion management include reliable abdominal compression, accelerator beam gating with the respiratory cycle, tumor tracking, and active breathholding techniques or other methods approved by the study committee. These measures

[0657] are recommended in cases where the extent of motion quantified on motion assessment exceeds 1 cm. Internal organ management maneuvers must be reliable enough to insure

[0658] that the GTV does not deviate beyond the confines of the PTV with any significant

[0659] probability (i.e., < 5%).

[0660] Table 5.3.2 highlights the recommended and minimum requirements for motion

[0661] assessment and treatment planning imaging.

[0662] Table 5.3.2 Motion Assessment / Management Guidelines for Simulation

[0663] Treatment Recommended Minimum Method Scan(s)

[0664] Technique Method for Motion for Motion Recommended for Assessment During Assessment During Treatment Planning Simulation Simulation

[0665] Free breathing 4D CT or fluoroscopy Repeated slow Average intensity

[0666] treatment as long as tumor can be acquisition CT projection (AveIP) scan

[0667] using an directly visualized scanning through from a full field of view

[0668] internal gross the target (to sample 4D CT for dose

[0669] target volume motion) fused to the calculations; the

[0670] (IGTV) planning CT dataset maximum intensity

[0671] approach, projection (MIP) scan may including be desirable to aid iGTV abdominal definition; Free-breathing compression scans are not

[0672] recommended for

[0673]

[0674] treatment planning.

[0675] Gating with a 4D CT Exhale CT plus Reconstructed average of

[0676] gating window fluoroscopy (free- gating window scans from breathing + 4D CT.

[0677] fluoroscopy strongly

[0678] discouraged due to

[0679] baseline shift)

[0680] Breath hold Reproducibility of N / A Scan in breath hold

[0681] (Le, ABC) breath hold confirmed position (inhale

[0682]

[0683] (examples: multiple low recommended since itAttorney Docket No. 4456-0340PWO1 dose scans Over tumor,

[0684]

[0685] maximizes lung volume);Attorney Docket No. 4456-0340PWO1

[0686] repeat fluoroscopy or

[0687]

[0688] scout images)

[0689] Tracking 4D CT or breath hold N / A 4D CT or breath hold CT

[0690]

[0691] CT Simulation Imaging

[0692] Motion assessment during simulation should be performed on all patients to account for

[0693] the tumor and nodal excursion with respiration and the appropriate type of treatment

[0694] planning CT required. Respiratory motion may be significant for esophageal (particularly distal) and GE junction lesions. When 4D-CT planning or other motion management techniques are used, margins may be modified to account for observed motion and may

[0695] also be reduced if justified. The 4D-CT data may also be used to create an internal target volume (IGTV) from which subsequent clinical target volume (CTV) and planning target volume (PTV) expansions can be made.

[0696] A motion management technique-specific treatment planning CT (e.g., 4D-CT, breathhold, gated CT, etc.) should be used during simulation to define gross tumor volume

[0697] (GTV), internal gross tumor volume (IGTV), clinical target volume (CTV), and planning

[0698] target volume (PTV) (see definitions in Section 5.3.4). Contiguous CT slices, having no

[0699] more than 3 mm thickness are to be obtained starting from the level of the cricoid

[0700] cartilage and extending inferiorly through the entire liver and kidney(s) volume. The field of view must be large enough so that none of the patient’s anatomy along the path of the treatment beams is cut off.

[0701] A treatment planning FDG PET / CT scan (or FDG-PET alone) (if available for

[0702] simulation) with the patient in the treatment position is encouraged for treatment

[0703] planning. In the case where the PET / CT is obtained in the treatment position as part of radiation simulation, the CT from this study may be used as the planning CT scan, but a motion management technique-specific treatment planning CT should still be done.

[0704] Intravenous contrast during the planning CT is optional provided a diagnostic chest CT

[0705] was done with contrast to delineate the major blood vessels. If not, intravenous contrast

[0706] should be given during the planning CT. If contrast is used, the densities or RLSPs

[0707] should be over-ridden or the contrast scan must be registered to a non-contrast scan for planning purposes.

[0708] .3.3 Imaging for Structure Definition, Image Registration / Fusion

[0709] Limited extent FDG-PET / CT + / - contrast (unless contraindicated) imaging will be

[0710] preferred as part of staging and to assist in volume delineation in all eligible patients. CT

[0711] + contrast (unless contraindicated) will be allowed as a substitute if for some reason

[0712] FDG-PET is not able to be acquired.

[0713] .3.4 Definition of Target Volumes and Margins

[0714] Note: All structures must be named for digital RT data submission as listed in the tableAttorney Docket No. 4456-0340PWO1 below. The structures marked as “Required” in the table must be contoured andAttorney Docket No. 4456-0340PWO1

[0715] submitted with the treatment plan. Structures marked as “Required when applicable”

[0716] must be contoured and submitted when applicable.

[0717] Resubmission of data may be required if labeling of structures does not conform to the

[0718] standard DICOM name listed. Capital letters, spacing and use of underscores must be

[0719] applied exactly as indicated.

[0720] Standard Validation Required / Required

[0721] Description

[0722] Name When applicable / Optional

[0723] Primary disease GTV to

[0724] GTVp_5040 receive 5040 cGy Required

[0725] Nodal disease GTV (if

[0726] applicable) as confirmed

[0727] by PET / CT or EUS

[0728] GTVn_5040 FNA to receive 5040 Required

[0729] cGy. Peri turn oral nodal

[0730] disease are included

[0731] with the GTVp.

[0732] Required if 4D-CT used to

[0733] assess and account for GTV

[0734] Primary disease GTV

[0735] motion. Not required if 4D- that accounts for motion

[0736] CT is not obtained or motion

[0737] over the respiratory

[0738] IGTVp_5040 is minimal on 4D-CT

[0739] cycle to receive 5040

[0740] (<10mm). If treatment is to

[0741] cGy.

[0742] be delivered with breath hold

[0743] or gated techniques, this is not

[0744] required.

[0745] Nodal disease GTV (if Required if 4D-CT used to

[0746] applicable) as confirmed assess and account for GTV

[0747] by PET / CT or EUS motion. Not required if 4D- FNA that accounts for CT is not obtained or motion

[0748] IGTVn_5040 over the respiratory is minimal on 4D-CT

[0749] cycle to receive 5040 (<10mm). If treatment is to cGy. Peri turn oral nodal be delivered with breath hold disease are included or gated techniques, this is not with the IGTVp 5040. required.

[0750] Sum of

[0751] GTVp / IGTVp_5040 and

[0752] GTV_5040 Required for GTV 5040.

[0753] GTVn / IGTVn_5040,

[0754] and Required for IGTV 5040,

[0755] which is the volume

[0756] IGTV_5040 unless IGTVs not made.

[0757] enveloping all GTV(s)

[0758] or IGTV(s).

[0759] CTV to receive 5040

[0760] CTV_5040 Required

[0761]

[0762] cGyAttorney Docket No. 4456-0340PWO1

[0763] PTV to receive 5040

[0764] PTV_5040 Required

[0765]

[0766] cGy

[0767] Detailed Specifications

[0768] Target volumes: The definitions of volumes will be in accordance with the 1999 ICRUReport#62.

[0769] GTVp_5040 and IGTVp_5040: The GTVp includes the GTV defined as the primary

[0770] tumor in the esophagus as delineated on planning scan and other pre-treatment diagnostic studies (e.g. PET / CT, EUS). GTVp should be contoured on the primary CT dataset (see Section 5.3.8 for definition). When 4DCT simulation is performed for non-gated free-breathing delivery, the phases of the breath cycle could be used to construct the

[0771] composite primary GTV volume (IGTVp). Ideally, targets should be contoured on all

[0772] phase images in order to construct IGTVp. Some commercial software may be used to populate the physician’s GTVp contour on one phase images to the other phase images. If such software is unavailable, at least 4 phase images (end of inspiration, mid-inspiration, end of expiration and mid-expiration) should be used to assure IGTVp accuracy.

[0773] Maximum Intensity Projected (MIP) images should not be used to generate IGTV (see

[0774] Chen X, Lu H, Tai A, et al. Determination of internal target volume for radiation

[0775] treatment planning of esophageal cancer by using 4-dimensional computed tomography (4DCT). Int J Radiat Oncol Biol Phys. 2014;90(1):102-9).

[0776] GTVn_5040 and IGTVn_5040: The GTVn defined as any grossly involved regional

[0777] lymph nodes, either considered suspicious on PET / CT or proven by EUS biopsy. GTVn

[0778] should be contoured on the primary CT dataset (see 5.3.8 for definition). When 4DCT simulation is performed for non-gated free-breathing delivery, the phases of the breath

[0779] cycle could be used to construct the composite nodal GTV volume (IGTVn). Ideally,

[0780] targets should be contoured on all phase images in order to construct IGTVn. Some commercial software may be used to populate the physician’s GTVn contour on one

[0781] phase images to the other phase images. If such software is unavailable, at least 4 phase

[0782] images (end of inspiration, mid-inspiration, end of expiration and mid-expiration) should be used to assure IGTVn accuracy. Maximum Intensity Projected (MIP) images should

[0783] not be used to generate IGTVn (see Chen X, Lu H, Tai A, et al. Determination of internal target volume for radiation treatment planning of esophageal cancer by using 4-dimensional computed tomography (4DCT). Int J Radiat Oncol Biol Phys.

[0784] 2014;90(1):102-9).

[0785] CTV_5040: The CTV is defined as the GTVp with a 3.5-4.0 cm expansion superiorly

[0786] and inferiorly along the length of the esophagus and cardia and a 1.0-1.5 cm radial

[0787] expansion. It should also include GTVn with a 1.0-1.5 cm expansion in all dimensions.

[0788] This CTV expansion needs to be trimmed away from the edges of anatomic boundaries of microscopic disease spread, including major blood vessels, vertebral body, pericardial lining / heart, pleura, liver, pancreas, kidneys and spleen. This volume should be

[0789] expanded if needed to cover the paraesophageal, celiac and supraclavicular lymph node regions. If not involved, the celiac nodes (for distal and gastroesophageal junctionAttorney Docket No. 4456-0340PWO1

[0790] tumors) or the bilateral supraclavicular nodes (for upper thoracic tumors) are

[0791] recommended to be included for NO status, but required to be covered for N+ status. Such rules also apply to any large mid esophageal tumors with extension to either upper

[0792] thoracic or distal esophagus. The 3.5-4 cm superior and inferior expansion should follow

[0793] the contour of the esophagus and proximal stomach. The intent is to extend the margin

[0794] along the length of the esophagus and proximal stomach to provide a margin for coverage of submucosal extension of tumor and lymphatics. For cervical esophageal cancer, nodal coverage of the cervical lymph node regions in the head and neck (e g. neck levels 3, 4,

[0795] etc.) is at the discretion of the treating physician, but recommended for N+ status.

[0796] PTV_5040: Additional margin shall be added to the CTV for set up error and movement.

[0797] This expansion should be 0.5 to 1.0 cm and does not need to be uniform in all

[0798] dimensions. 4DCT data is allowed to customize PTV expansion.

[0799] Definition of Critical Structures and Margins

[0800] Note: All structures must be named for digital RT data submission as listed in the table

[0801] below. The structures marked as “Required” in the table must be contoured and

[0802] submitted with the treatment plan. Structures marked as “Required when applicable”

[0803] must be contoured and submitted when applicable.

[0804] Resubmission of data may be required if labeling of structures does not conform to the

[0805] standard DICOM name listed. Capital letters, spacing and use of underscores must be

[0806] applied exactly as indicated.

[0807] Validation

[0808] Standard Name Description Required / Required when applicable / Optional

[0809] SpineCanal Spinal Canal Required

[0810] Lungs Right + Left Lung Required

[0811] Lung R Right Lung Required

[0812] Lung L Left Lung Required

[0813] Esophagus minus

[0814] Esophagus-GTV Required

[0815] GTV

[0816] Esophagus minus

[0817] Esophagus-IGTV Required if IGTV made

[0818] IGTV

[0819] Heart Heart Required

[0820] Required for all mid / distal

[0821] Liver Liver

[0822] tumors

[0823] Required for all mid / distal

[0824] Kidney R Right Kidney

[0825]

[0826] tumorsAttorney Docket No. 4456-0340PWO1

[0827] Required for all mid / distal

[0828] Kidney L Left Kidney

[0829] tumors

[0830] Required for all mid / distal

[0831] Spleen Spleen

[0832] tumors

[0833] Stomach-GTV required for

[0834] Stomach- Stomach minus all mid / distal tumors.

[0835] GTV / IGTV GTV / IGTV Stomach-IGTV required if

[0836] IGTV made.

[0837] Required for all mid / distal

[0838] Bowel Small Small bowel

[0839] tumors

[0840] Required for all mid / distal

[0841] Bowel Large Large bowel

[0842]

[0843] tumors

[0844] Detailed Specifications

[0845] SpineCanal: Boundaries: Cranial: 1stslice of CT; Caudal: last slice of CT;

[0846] Lungs: Boundaries: Cranial: From apex bilaterally; Caudal: to bottom of L2

[0847] Esophagus-GTV / IGTV: Boundaries: Cranial: Bottom of cricoid; Caudal: GE junction; subtract

[0848] IGTV from structure

[0849] Heart: Boundaries: Base: Bottom of the aortic arch; Inferior: Apical most of the ventricle

[0850] Liver: Boundaries: Cranial: From dome; Caudal: Inferior tip

[0851] Kidneys: Entire kidneys contoured separately as Kidney _R and Kidney L

[0852] Spleen: Entire spleen

[0853] Stomach-GTV / IGTV: Boundaries: whole stomach minus GTV / IGTV

[0854] Bowel_Small: Boundaries: Level of PTV

[0855] Bowel_Large: Boundaries: Level of PTV

[0856] 5.3.6 Dose Prescription

[0857] Note: The information provided in this section can be used for adjusting the dose constraints for treatment planning purposes. This table together with the planning priority table should be used

[0858] during dose optimization. It is important to remember that ideal plans might not be achievable in all cases. Thus, the Compliance Criteria table could be different than the information given here.

[0859] Cases will be scored using the Compliance Criteria table.

[0860] Target Dose (Gy) Fraction Size #of Frequency Dose specification Standard Name (Gy) fractions technique Daily Covering 95% of PTV_5040 50.4 1.8 28

[0861] PTV* Daily Covering 95-99% CTV_5040 50.4 1.8 28

[0862] ofCTV IGTVp_5040 Daily

[0863] Covering >=99% IGTVn_5040 50.4 1.8 28

[0864] of IGTV

[0865] IGTV_5040

[0866]

[0867] Attorney Docket No. 4456-0340PWO1

[0868] GTVp_5040 Daily

[0869] Covering >=99% GTVn_5040 50.4 1.8 28

[0870] of GTV

[0871] GTV_5040

[0872]

[0873] ^Prescribe to the isodose line that covers 95% of the PTV, or inverse plan to cover 95%> of the PTVwith the

[0874] prescription dose

[0875] 5.3.7 Compliance Criteria

[0876] The compliance criteria listed here will be used to score each case. Given the limitations

[0877] inherent in the treatment planning process, the numbers given in this section can be different than the prescription table. The Per Protocol and Variation Acceptable categories are both considered to be acceptable. The Per Protocol cases can be viewed as ideal plans, and the Variation

[0878] Acceptable category can include more challenging plans that do not fall at or near the ideal

[0879] results. A final category, called Deviation Unacceptable, results when cases do not meet the requirements for either Per Protocol or Variation Acceptable. Plans falling in this category are considered to be suboptimal and additional treatment planning optimization is recommended.

[0880] Normalization of Dose: The plan is normalized such that 95% of the PTV 5040 volume

[0881] receives prescription dose of 50.4 Gy.

[0882] Note: Deviation Unacceptable occurs when dose limits for Variation Acceptable are not

[0883] met

[0884] Target Volume Constraints and Compliance Criteria

[0885] Variation

[0886] Name of Structure Dosimetric parameter Per Protocol

[0887] Acceptable*

[0888] PTV_5040 D0.03cc[%] <= 110% Rx Dose <= 113% Rx Dose

[0889]

[0890] V50.4Gy[%] >=95% >90%

[0891] *Per Protocol range is excluded from Variation Acceptable range.

[0892] Normal Structure Constraints and Compliance Criteria

[0893] Dosimetric Variation

[0894] Name of Structure Per Protocol

[0895] parameter Acceptable*

[0896] Lungs D0.03cc[%] <= 110%Rx <= 113% Rx Dose

[0897] Dose

[0898] MeanfGy] <= 20 Gy <= 21 Gy

[0899] V30Gy[%] <= 20% <= 25%

[0900] V20Gy[%] <= 30% <= 35%

[0901] V10Gy[%] <= 50% <= 55%

[0902] V5Gy[%] <= 65% <= 75%

[0903] Heart D0.03cc[Gy] <= 52Gy <= 54 Gy

[0904] MeanfGy] <= 38 Gy <= 40 Gy

[0905] V40Gy[%] <= 50% <= 55%

[0906]

[0907] Kidney_L D0.03cc[Gy] <= 45 Gy <= 50 GyAttorney Docket No. 4456-0340PWO1

[0908] MeanfGy] <= 18 Gy <= 20 Gy

[0909] V20Gy[%] <= 30% <= 40%

[0910] Kidney _R D0.03cc[Gy] <= 45 Gy <= 50 Gy

[0911] Mean[Gy] <= 18 Gy <= 20 Gy

[0912] V20Gy[%] <= 30% <= 40%

[0913] SpineCanal D0.03cc[Gy] <= 45 Gy <= 50 Gy

[0914] Liver MeanfGy] <= 21 Gy <= 25 Gy

[0915] V30Gy[%] <= 30% <= 40%

[0916] Bowel Small D0.03cc[Gy] <= 52Gy <= 54 Gy

[0917] Bowel Large D0.03cc[Gy] <= 52Gy <= 54 Gy

[0918] Stomach-GTV / IGTV MeanfGy] <= 40 Gy <= 45 Gy

[0919] (parameters apply to

[0920] Stomach-IGTV if made,

[0921] otherwise they apply to

[0922] Stomach-GTV)

[0923]

[0924] Spleen MeanfGy] <= 45 Gy <= 50 Gy

[0925] *Per Protocol range is excluded from Variation Acceptable range.

[0926] Recommended dose acceptance criteria for other normal tissue, but not to be used for plan score.

[0927] Heart Mean [Gy] <=30 Gy

[0928]

[0929] Lungs Mean [Gy] <=18 Gy

[0930] Delivery Compliance criteria

[0931] Per Protocol Variation Deviation

[0932] Acceptable Unacceptable

[0933] Radiation Start date Day 1 2-3 days >3 days

[0934] Overall Treatment time <40 days 41-52 days >52 days

[0935]

[0936] Interruptions None <1 week >1 week

[0937] 5.3.8 Treatment Planning Priorities and Instructions

[0938] Critical Structure and Target priorities are listed in order of decreasing importance. Use these priorities when not all planning constraints can be met:

[0939] 1. Spinal Cord

[0940] 2. PTV

[0941] 3. Lungs

[0942] 4. Heart

[0943] - Required algorithms

[0944] The dose calculation algorithm in treatment planning system (TPS) should be one of thoseAttorney Docket No. 4456-0340PWO1

[0945] approved by IROC, which includes Convolution Superposition, Collapsed Cone Convolution,

[0946] AAA and Monte Carlo (see http: / / rpc.mdanderson.org / RPC / home.htm for details). These

[0947] algorithms have been tested using the IROC lung phantom to provide acceptable dose calculation accuracy within a heterogeneous medium. The dose calculation algorithms like Clarkson or

[0948] pencil beam should not be used for photon treatment planning.

[0949] For Convolution / Superposition type algorithms, dose should be reported as computed inherently by the given algorithm. For Monte Carlo or Grid Based Boltzmann Solver algorithms,

[0950] conversion of Dm (dose-to-medium) to Dw (dose-to-water) should be avoided. Dm, computed inherently by these algorithms, should be reported.

[0951] - Primary dataset for dose calculation

[0952] The primary dataset for dose calculation must be a free-breathing CT that is generated from

[0953] 4DCT, such as an average intensity pixel CT (AvelP), mid-ventilation CT or, the breath-hold / gated CT, or the free-breathing CT acquired with no other motion management. Maximum Intensity Pixel (MIP) generated images from 4DCTs may not be used as the primary dose

[0954] calculation dataset. In the case in which contrast is present during the treatment planning CT, the density of the contrast should be overridden to a representative background electron density or RSLP.

[0955] -Dose matrix resolution

[0956] Dose grid size should be < 3 mm in all directions.

[0957] Planning Procedures

[0958] • Using a motion management technique-specific treatment planning CT from the

[0959] simulation, primary tumor motion should be evaluated.

[0960] • For tumor motion > 1.0 cm, motion management techniques such as breathhold or

[0961] respiratory gating techniques should be considered for planning and treatment but not required, as long as the primary tumor motion is accounted for in the planning for both modalities and that daily image guidance and weekly anatomic imaging verification are

[0962] done to ensure proper set up and targeting.

[0963] Planning Procedures

[0964] • For IMRT (multiple fixed fields or arc therapy) the PTV will be treated with any

[0965] combination of coplanar, non-coplanar, or dynamically arcing fields. Please refer to

[0966] Section 8.4 for credentialing requirements. Margins to be used are stated in Section

[0967] 5.3.4.

[0968] 5.3.9 Patient-Specific OA

[0969] Any patient-specific QA that needs to be acquired should follow institutional guidelines

[0970] and AAPM task group report recommendations. Patient specific QA is highly

[0971] recommended. The recommended patient specific QA criteria is for >90% of the

[0972] comparison points to pass a ±3% / 3mm Gamma Index analysis.Attorney Docket No. 4456-0340PWO1

[0973] 5.3.10 Daily Treatment Localization / IGRT

[0974] Image-guided radiation therapy (IGRT) is radiation therapy using imaging to facilitate accuracy and precision throughout its entire process. In this section we use the

[0975] terminology IGRT to focus on image-guidance at the time of radiation delivery to ensure its adherence to the planned treatment, with computer assisted process, i.e. image

[0976] handling together with calculation of shift and rotations (if available) must be determined with computer assistance.

[0977] ■ Daily IGRT is required for all treatments on this protocol and may be achieved using any

[0978] one or more of the following techniques:

[0979] o Orthogonal kilovoltage (KV) images, e.g. ExacTrac; on-board imagers (OBI) or

[0980] similar systems;

[0981] o Linear-accelerator mounted kV and MV conebeam CT images;

[0982] o Linear-accelerator mounted MV CT images (e.g., Tomotherapy);

[0983] o MRI-on-rails, ViewRay and MR-Linac.

[0984] ■ The institution’s procedure to register treatment day image dataset with the reference

[0985] dataset should comply with the following recommendations:

[0986] o Region-of-Interest (ROI) or “clip box” for fusion should be set to encompass the high

[0987] dose PTV and adjacent vertebral bodies. (Note: The same strategy should be used for repeat CT scans required for verification, QA or replanning.)

[0988] o If the fusion software allows the user to create an irregular ROI (e.g., ExacTrac), treatment room objects seen on in-room X-rays should be excluded from the registration.

[0989] o Automatic (e.g., based on mutual information bone or soft tissue fusion) types of registration should be used; the result of the fusion must be visually checked for alignment of the target or bony structures, such as vertebral bodies when appropriate.

[0990] Manual adjustments (using drag-and-drop capabilities) should be made when necessary.

[0991] ■ Following the registration, the translational and (if the appropriate technology is available) rotational corrections should be applied to the treatment couch. If all the variances are less than 3 mm, the treatment can proceed without correction. If one or more corrections are 3- 5 mm, adjustment is necessary prior to treatment; however, re-imaging is not recommended. If one or more of the corrections are larger than 5 mm, the imaging can be repeated in addition to performing table / positioning adjustments.

[0992] ■ If orthogonal projection imaging is used for setup, this fact should be communicated to the therapists (i.e. bony anatomy or fiducial). The relationship between the image surrogate ofAttorney Docket No. 4456-0340PWO1

[0993] internal anatomy and the soft tissue targets shall be verified, at a minimum, as part of the repeat CTs done for QA, verification or replanning.

[0994] ■ If in-room CT is available but orthogonal projection X-ray imaging is used for daily setup, weekly verification of soft tissue with setup surrogate is recommended.

[0995] ■ If in-room CT is used for daily setup, the setup surrogate needs to be communicated to the therapists (i.e. bony anatomy, IGTV, or other). If, due to changing anatomy, a compromise must be made between multiple target structures, the therapists shall be guided by the treating physician as to the best compromise.

[0996] General Concomitant Medication and Supportive Care Guidelines (19-MAY-2023)

[0997] 1 Permitted Supportive / Ancillary Care, Concomitant Medications, and PostChemoradiation Resection

[0998] All supportive therapy for optimal medical care will be given during the study period at the discretion of the attending physician(s) within the parameters of the protocol and documented on each site’s source documents as concomitant medication.

[0999] Concurrent use of hormones for non-cancer-related conditions (e.g., insulin for diabetes and hormone replacement therapy) is acceptable.

[1000] Surgery is permitted post-chemoradiation if medically indicated.

[1001] Prohibited Therapies

[1002] ■ Any other investigational anti-cancer therapy

[1003] ■ Any other concurrent chemotherapy, radiotherapy, immunotherapy, biologic or

[1004] hormonal therapy for cancer treatment other than the treatments stated in this

[1005] protocol.

[1006] ■ Immunosuppressive medications including, but not limited to systemic corticosteroids

[1007] at doses exceeding 20 mg / day of prednisone or equivalent, methotrexate,

[1008] azathioprine, and TNF-a blockers. Use of immunosuppressive medications in subjects with contrast allergies is acceptable. In addition, use of inhaled and intranasal corticosteroids is permitted. The shortest possible duration of steroids will be allowed for different indications, at the discretion of the principal investigator (e.g., chronic obstructive pulmonary disease, radiation, nausea, etc.)

[1009] ■ Live attenuated vaccines within 30 days of OBP-301 dosing, i.e., 30 days prior to the

[1010] first dose, during treatment with OBP-301 and for 30 days post discontinuation of

[1011] OBP-301. Inactivated vaccines, such as the injectable influenza vaccine, are

[1012] permitted

[1013] Nutritional and Herbal Supplements

[1014] The concomitant use of herbal therapies is not recommended, as their pharmacokinetics, safety profiles, and potential drug-drug interactions are generally unknown. However the use of general nutritional foundation supplements will be allowed including: calcium withAttorney Docket No. 4456-0340PWO1

[1015] vitamin D and / or minerals, 0mega3s (fish oil), Vitamin B6, Vitamin B12, a basic multivitamin, L-glutamine, or probiotics oral supplements will be permitted as long as at or below recommended dosing by a healthcare provider. Herbal-based multivitamins are not allowed.

[1016] Participation in Other Trials

[1017] Patients are not to participate in other therapeutic trials during this study. However, trials that do not add experimental agents are allowed (e.g. imaging trials, quality of life, etc).

[1018] Duration of Therapy

[1019] In the absence of treatment delays due to adverse event(s), treatment may continue as

[1020] specified in the above treatment modality sections or until one of the following criteria applies:

[1021] • Disease progression,

[1022] • Intercurrent illness that prevents further administration of treatment;

[1023] • Unacceptable adverse event(s), as described in Section 6;

[1024] • Patient decides to withdraw consent for participation in the study, or

[1025] • General or specific changes in the patient’s condition render the patient unacceptable

[1026] for further treatment in the judgment of the investigator;

[1027] TREATMENT MODIFICATIONS / MANAGEMENT

[1028] General Guidelines (26-JUL-2021)

[1029] The following side effects are anticipated: nausea, vomiting, myelosuppression (including lymphopenia), esophagitis, dysphagia, mucositis and fatigue.

[1030] • If doses of chemotherapy are missed, they should not be made up.

[1031] • Any delay of > 3 weeks requires discontinuing paclitaxel and carboplatin chemotherapy. If a dose is missed it will not be made up once RT is completed;

[1032] • If chemotherapy needs to be permanently discontinued, patients may continue with radiation and OBP-301 alone if it is considered to be in the patient’s best interests.

[1033] Use of filgrastim is recommended, at the discretion of the treating investigator if chemoradiation is held for neutropenia. All dose reductions are permanent, i.e. drug doses will not be re-escalated after resolution of toxicity.

[1034] Dose levels for carboplatin and paclitaxel are as follows:

[1035] Dose level Weekly dose

[1036] Carboplatin Paclitaxel

[1037] 0 AUC 2 50 mg / m²

[1038]

[1039] -1 AUC 1.5 40 mg / m2

[1040] There will not be any additional dose reductions.

[1041] Hematologic toxicity _ _

[1042] [ Week 2-5 blood counts during chemoradiation

[1043]

[1044] Dosage

[1045]

[1046] Attorney Docket No. 4456-0340PWO1

[1047] ANC Platelet count

[1048] ≥1,000 / µL AND ≥75,000 / µL Chemotherapy at full dose

[1049] <l,000 / pL OR <75,000 / pL First occurrence

[1050] Hold all treatments (chemo, OBP-301

[1051] and RT). Re-check CBC. Consider filgrastim. When ANC ≥1,000 / µL

[1052] and platelets ≥75,000 / µL, resume chemotherapy at dose level -1

[1053] Second occurrence

[1054] Hold all treatments and resume when ANC>l,000 and platelets >75,000.

[1055] Continue chemo at the -1 dose level,

[1056] i.e. no further dose reductions are

[1057]

[1058] permitted.

[1059] Non-hematologic toxicity

[1060] Toxicity CTCAE version Modality Modification

[1061] 5 grade

[1062] Nausea / vomiting, >3 Carboplatin First occurrence

[1063] dehydration Paclitaxel Hold all treatment until

[1064] (despite maximum OBP-301 toxicities improve to grade <2. medical support) Radiation Resume chemo at dose level - Therapy 1. OBP-301 injections will

[1065] resume at the next previously scheduled time-point.

[1066] Second and further occurrence

[1067] Hold all treatment until

[1068] toxicities improve to grade <2.

[1069] Continue chemo at the -1 dose

[1070] level, i.e. no further dose

[1071] reductions are permitted.

[1072] OBP-301 injections will

[1073] resume at the next previously scheduled time-point.

[1074] Esophagitis >3 Carboplatin First occurrence

[1075] Paclitaxel Hold all treatment until

[1076] OBP-301 toxicities improve to grade <2.

[1077] Radiation Resume chemo at dose level - Therapy 1. OBP-301 injections will

[1078] resume at the next previously scheduled time-point.

[1079] Second and further occurrence

[1080] Hold all treatment until

[1081]

[1082] toxicities improve to grade <2.Attorney Docket No. 4456-0340PWO1

[1083] Continue chemo at the -1 dose

[1084] level, i.e. no further dose

[1085] reductions are permitted.

[1086] OBP-301 injections will

[1087] resume at the next previously scheduled time-point.

[1088] If grade >3 in last week of

[1089] treatment-Hold chemo but

[1090] continue the RT at the

[1091] discretion of the treating

[1092] physician.

[1093] Mucositis (despite > 3 Carboplatin First occurrence

[1094] maximum Paclitaxel Hold all treatment until

[1095] supportive care OBP-301 toxicities improve to grade <2.

[1096] Radiation Resume Carboplatin and

[1097] Therapy Paclitaxel at dose level -1.

[1098] OBP-301 injections will

[1099] resume at the next previously scheduled time-point.

[1100] Second and further occurrence

[1101] Hold all treatment until

[1102] toxicities improve to grade <2.

[1103] Continue Carboplatin and

[1104] Paclitaxel at the -1 dose level,

[1105] i.e. no further dose reductions

[1106] are permitted.

[1107] OBP-301 injections will

[1108] resume at the next previously scheduled time-point.

[1109] Fatigue >3 Carboplatin First occurrence

[1110] Paclitaxel Hold all treatment until

[1111] OBP-301 toxicities improve to grade <2.

[1112] Radiation Resume Carboplatin and

[1113] Therapy Paclitaxel chemo at dose level

[1114] -1. OBP-301 injections will

[1115] resume at the next previously scheduled time-point.

[1116] Second and further occurrence

[1117] Hold all treatment until

[1118] toxicities improve to grade <2.

[1119] Continue Carboplatin and

[1120] Paclitaxel at the -1 dose level,

[1121] i.e. no further dose reductions

[1122] are permitted.

[1123]

[1124] OBP-301 injections willAttorney Docket No. 4456-0340PWO1

[1125] resume at the next previously scheduled time-point.

[1126] Any other toxicity >3 Carboplatin First occurrence

[1127] Paclitaxel Hold all treatment until

[1128] OBP-301 toxicities improve to grade <2.

[1129] Radiation Resume Carboplatin and

[1130] Therapy Paclitaxel at dose level -1.

[1131] OBP-301 injections will

[1132] resume at the next previously scheduled time-point.

[1133] Second and further occurrence

[1134] Hold all treatment until

[1135] toxicities improve to grade <2.

[1136] Continue Carboplatin and

[1137] Paclitaxel at the -1 dose level,

[1138] i.e. no further dose reductions

[1139] are permitted.

[1140] OBP-301 injections will

[1141] resume at the next previously scheduled time-point.

[1142] Diarrhea (despite >3 Paclitaxel First occurrence

[1143] maximum medical Only Hold Paclitaxel until diarrhea support) improves to grade <2. Dose

[1144] reduce Paclitaxel by 1 dose

[1145] level.

[1146] Second occurrence

[1147] Hold all Paclitaxel until

[1148] toxicities improve to grade <2.

[1149] Continue Paclitaxel at the -1

[1150] dose level, i.e. no further dose reductions are permitted Hepatotoxicity >1 Paclitaxel Decrease paclitaxel by 1 dose

[1151] (ALT, AST, blood Only level. Resume paclitaxel if

[1152] bilirubin) Total Bili ≤1.5× ULN OR

[1153] AST / ALT ≤2.5× UL N.

[1154] Carboplatin dose will remain

[1155] at current dose level.

[1156] Sensory 2 Paclitaxel Hold Paclitaxel. When toxicity neuropathy Only is grade <1, resume Paclitaxel

[1157] at -1 dose level for all

[1158] subsequent cycles. Carboplatin

[1159] dose will remain at current

[1160] dose level.

[1161] >3 Patients will not receive any

[1162]

[1163] more paclitaxel.Attorney Docket No. 4456-0340PWO1

[1164] Infusion related >3 Carboplatin Permanently discontinue

[1165]

[1166] reaction Paclitaxel Paclitaxel and Carboplatin.

[1167] Hypersensitivity Reaction Management per Institutional Standard

[1168] Hypersensitivity reactions include anaphylactic-like reactions. Tachycardia, bronchoconstriction, hypotension, facial edema and erythema may occur and should be

[1169] treated with antihistamine, corticosteroids, and epinephrine as per institutional

[1170] hypersensitivity management protocol. If the treating Physician feels that it is appropriate

[1171] to continue with protocol therapy, options include pre-medication with steroids and diphenhydramine the night before and at the time of carboplatin or paclitaxel infusion. For patients who develop a grade 1 / 2 carboplatin reaction, a desensitization protocol can be considered, after consultation with the Principal Investigator

[1172] OBP-301

[1173] OBP-301 injection will be delayed for any ongoing non-hematologic Grade 3 toxicity or hematologic Grade 4 toxicity (except for Grade 4 Lymphopenia) at the time of the next scheduled injection. Patients who require a delay of OBP-301 injection of > 4 weeks (i.e., approximately 6 weeks from the previous injection) due to lack of resolution to Grade <

[1174] toxicity will not receive subsequent OBP-301 treatments.

[1175] Any patient who experiences any of the following grade >3 toxicities directly attributable

[1176] to OBP-301 and / or endoscopic injections will not receive further OBP-301 treatments:

[1177] • Perforation

[1178] • Bleeding

[1179] • Infection

[1180] • Pain lasting >3 days despite maximal medical management

[1181] If a dose of OBP-301 is missed within the 2 day window, unless there is a specific reason

[1182] that OBP-301 administration via EGD is contraindicated, administration during the same calendar week is permitted but must not be on the same day as chemotherapy administration.

[1183] Injection site pain is the one of the more common adverse events in connection with intratumoral injections. Therefore, the following pain control measures are suggested to improve the comfort of the patient.

[1184] 1. Review the analgesics already prescribed for the patient and determine the level of pain control.

[1185] 2. Additional pain medication will with all likelihood be needed during the day of the intratumoral injections and for several days afterwards. Local anesthesia may be required just before or during the intratumoral injection at the discretion of the treating gastroenterologist.

[1186] Radiation Therapy (26- JUL-2021)

[1187] Radiation therapy will typically continue as prescribed without modifications while these

[1188] drug-related adverse events are being managed.Attorney Docket No. 4456-0340PWO1

[1189] Fatigue, esophagitis, cough, nausea and vomiting can be common acute side effects of

[1190] radiation to the esophagus.

[1191] For nausea and vomiting, anti-emetics should be initiated at the onset of symptoms and continued as directed by the treating physician until resolution of symptoms to grade 0-1.

[1192] Additional supportive care measures, e.g. oral or intravenous rehydration, etc., should be instituted as required by the patient’s clinical condition. Additional medical evaluation is recommended for those patients with continued > grade 2 nausea / vomiting, lasting > 48

[1193] hours despite institution of optimal supportive care measures. Primary prophylaxis should be initiated once nausea or vomiting has occurred with the prior treatment. Admit to

[1194] hospital and administer IV fluids and all supportive measures for symptoms unresponsive to medication and signs of dehydration.

[1195] For cough, dextromethorphan with or without guaifenesin, benzonatate, or mild narcotic medications (e.g. codeine syrup) can be used.

[1196] Esophagitis can be managed by the preference of the treating physician and may

[1197] commonly include medications such as sucralfate, Magic Mouthwash (e.g. custom mix of Benadryl, Maalox, lidocaine / xylocaine), proton pump inhibitors, or narcotics for pain.

[1198] In the unlikely event that radiation must be permanently discontinued, patients will

[1199] discontinue all treatment.

[1200] ADVERSE EVENTS REPORTING REQUIREMENTS

[1201] Adverse Events for Investigational Study Agent: OBP-301 (07-DEC-2020)

[1202] Identified Risks of OBP-301

[1203] • Administration site reaction

[1204] • Lymphocyte count decrease

[1205] Potential Risks of OBP-301

[1206] • Accidental exposure of a third party to OBP-301

[1207] • Spread of OBP-301 to close contacts or healthcare providers after direct contact

[1208] with patients

[1209] • Adenovirus infection affecting the entire body in patients with impaired immune function

[1210] • Inflammatory lesions in virus-infected organs (liver, kidney, lung)

[1211] • Influenza-like symptoms

[1212] For more details see Investigator’s Brochure.

[1213] DRUG INFORMATIONAttorney Docket No. 4456-0340PWO1

[1214] Investigational Study Agent: OBP-301 (19-MAY-2023)

[1215] To supplement the toxicity information contained in this document, investigators must

[1216] obtain the current version of the Investigator Brochure for comprehensive pharmacologic and safety information

[1217] The Investigator Brochure can be obtained from the protocol specific page of the NRG website.

[1218] Adverse Events

[1219] See Section 7.3 and consult the Investigator Brochure for comprehensive information.

[1220] The OBP-301 clinical study material and product is generated according to current Good Manufacturing Practice (cGMP). Below shows an overview of OBP-301. OBP-301 will

[1221] be provided by Oncolys BioPharma Inc.

[1222] Investigational OBP-301

[1223] product code:

[1224] Content: 2 mL / vial (1×1012Viral Particles / mL)

[1225] Dosage form: Solution; formulated in 20 mM Tris pH 8.0, 25 mM NaCl with

[1226] 2.5% glycerin, USP by volume, filled in each 5 mL glass vial

[1227] Storage method for

[1228] the investigational Store at < -60°C

[1229] product:

[1230]

[1231] Agent Specific Information: OBP-301

[1232] Refer to the Pharmacy Manual (Appendix I) and the Investigator Brochure for

[1233] detailed information

[1234] Other Names: Suratadenoturev (pending trade name Telomelysin ™), hereafter referred to as OBP-301

[1235] Classification: Adenovirus

[1236] Description: Adenoviral vector containing the adenoviral E1A and E1B gene linked with an internal ribosome entry site (IRES) under the control of the human Telomerase

[1237] Reverse Transcriptase (hTERT) promoter.

[1238] Mode of Action: The normal transcriptional regulatory element of the Ad5 E1A gene is replaced by the human Telomerase Reverse Transcriptase gene (hTERT) promoter, and

[1239] the normal transcriptional element of the E1B gene is replaced by an IRES (Internal Ribosomal Entry Site) sequence.

[1240] How Supplied: OBP-301 will be provided as a frozen viral suspension formulated in 20

[1241] mM Tris pH 8.0, 25 mM NaCl with 2.5% Glycerin, USP by volume. The product is

[1242] packaged using a 5 mL glass vial, siliconized gray butyl-rubber stoppered vials, an

[1243] aluminum seal, and yellow flip-top plastic cap. Each vial contains a volume of 2.0 mL atAttorney Docket No. 4456-0340PWO1

[1244] a concentration of 1 x 1012viral particles (vp) / mL. The product in this configuration is

[1245] stored at < -60° C prior to administration.

[1246] Preparation: OBP-301 should be prepared according to Biosafety Level 2 Guidelines,

[1247] unless local regulations require a more rigorous containment level. See Appendix I

[1248] Storage: See Appendix I.

[1249] Route of Administration: Injection into the tumor

[1250] Method of Administration: OBP-301 is administered as intratumoral injection into a

[1251] primary esophageal tumor mass that is suitable for injection, e.g. greater than 1 cm2in

[1252] size and in a location amenable for endoscopic injection.

[1253] Administration Instructions can be found in the pharmacy manual (Appendix I)

[1254] Commercial Agent: Paclitaxel

[1255] Sites must refer to the package insert for detailed pharmacologic and safety information.

[1256] Product Description: Paclitaxel injection is a clear colorless to slightly yellow viscous

[1257] solution.

[1258] Solution Preparation: Paclitaxel injection should be diluted in_0.9% Sodium Chloride Injection, USP; 5% Dextrose Injection, USP; 5% Dextrose and 0.9% Sodium Chloride Injection, USP; or 5% Dextrose in Ringer’s Injection to a final concentration of 0.3 to 1.2 mg / mL.

[1259] Route of Administration: Intravenous

[1260] Agent Ordering / Availability: Commercially available

[1261] Please see Section 5.2.2 for administration instructions. Please refer to the current FDA-approved package insert provided with each drug and the site-specific pharmacy for

[1262] toxicity information and instructions for drug preparation, handling, and storage.

[1263] Adverse Events

[1264] Please refer to the package insert.Attorney Docket No. 4456-0340PWO1

[1265] Commercial Agent: Carboplatin

[1266] Sites must refer to the package insert for detailed pharmacologic and safety information.

[1267] Product Description: Carboplatin injection is a premixed aqueous solution of 10 mg / mL carboplatin.

[1268] Solution Preparation: Carboplatin aqueous solution can be further diluted to

[1269] concentrations as low as 0.5 mg / mL with 5% Dextrose in Water (D 5W) or 0.9% Sodium Chloride Injection.

[1270] Route of Administration; Intravenous

[1271] Agent Ordering / Availability; Commercially available

[1272] Please see Section 5.2.2 for administration instructions. Please refer to the current FDA- approved package insert provided with each drug and the site-specific pharmacy for

[1273] toxicity information and instructions for drug preparation, handling, and storage.

[1274] Adverse Events

[1275] Please refer to the package insert.

[1276] STATISTICAL CONSIDERATIONS

[1277] Study Design (11 -JAN-2022)

[1278] This is a limited multicenter phase 1 study of OBP-301 and chemoradiation (CRT) with carboplatin / paclitaxel for patients with locally advanced esophageal / GEJ cancer, for

[1279] whom surgery is not planned. Up to 2 OBP-301 dose regimens will be assessed. If either dose regimen is determined to be safe, additional patients will be accrued to that dose

[1280] regimen.

[1281] Study Endpoints

[1282] Primary Endpoint: Dose limiting toxicity to assess the safety of OBP-301 given in

[1283] combination with chemoradiation.

[1284] Secondary Endpoints:

[1285] ■ Adverse events as measured by CTCAE v5.0

[1286] ■ Clinical complete response (cCR) - if one of the OBP-301 regimens is declared to be

[1287] safe.

[1288] ■ Number of patients alive without progression and number of patients alive at 1 and 2

[1289] years - if one of the OBP-301 regimens is declared to be safe.

[1290] Primary Objectives Study Design

[1291] Primary Hypothesis and Endpoints

[1292] The primary hypothesis is that OBP-301 given in combination with chemoradiation using carboplatin and paclitaxel is safe.Attorney Docket No. 4456-0340PWO1

[1293] How Primary Endpoint Will Be Analyzed

[1294] To assess the safety endpoint, the initial cohort of patients to be assessed will receive 3

[1295] doses of OBP-301 at 1 x1012vp / mL in conjunction with chemoradiation.

[1296] The dose-limiting toxicity (DLT) assessment period is from start of protocol treatmentAttorney Docket No. 4456-0340PWO1

[1297] until 30 days after the completion of CRT (total of ~10 weeks).

[1298] DLT is defined as the following that are definitely or probably attributed to OBP-301:

[1299] • Any grade >3 toxicity EXCEPT for the following:

[1300] o Grade 3 nausea / vomiting

[1301] o Grade 3 esophagitis or dehydration

[1302] o The first occurrence of grade 3 / 4 neutropenia

[1303] o Grade 3 / 4 lymphopenia (since this is a known toxicity of chemoradiation and

[1304] OBP-301)

[1305] • Any toxicity that leads to a >14-day cumulative delay in chemoradiation

[1306] Evaluable patients for the safety endpoint will be defined as (i) patients who started all

[1307] study treatment (OBP-301 and CRT) and had a DLT and (ii) patients who started all

[1308] study treatment (OBP-301 and CRT) and completed DLT evaluation period without a

[1309] DLT. If <1 of 6 evaluable patients in the 3 doses of OBP-301 at 1 x1012vp / mL cohort

[1310] have a dose limiting toxicity, then the regimen will be declared safe and an expansion

[1311] cohort of 9 more patients will be treated on this regimen to further evaluate toxicity and

[1312] to get preliminary data on cCR.

[1313] If >2 of these 6 evaluable patients have a dose limiting toxicity, then the 3 doses of OBP-301 at 1 x1012vp / mL will be deemed too toxic and a second cohort will assess 3 doses of

[1314] OBP-301 at 1 x1011vp / mL in conjunction with chemoradiation. If <1 of 6 evaluable

[1315] patients in the 3 doses of OBP-301 at 1 x1011vp / mL cohort have a dose limiting toxicity, then the regimen will be declared safe and an expansion cohort of 9 more patients will be treated on this regimen to further evaluate toxicity and to get preliminary data on cCR. If

[1316] >2 of 6 evaluable patients in this cohort have a dose limiting toxicity, then the 3 doses of

[1317] OBP-301 at 1 x1011vp / mL will be deemed too toxic, no more patients will be accrued,

[1318] and the results will be reported.

[1319] If a grade 5 AE and / or 2 or more grade 4 AEs occur within 30 days of OBP-301 administration, there will be a pause in accrual in order for NRG to discuss these

[1320] events with the FDA via teleconference.

[1321] If a safe OBP-301 dose is determined, then cCR will be evaluated and reported for the

[1322] cohort of 15 patients treated at that dose, including the 6 used for the DLT assessment.

[1323] Sample Size and Power Calculations:

[1324] The DLT portion of the trial will accrue 6 or 12 evaluable patients, depending on whether or not the OBP-301 doses needs to be reduced. If a regimen is determined to be safe,

[1325] based on the DLT endpoint, then the total number of evaluable patients accrued to the

[1326] trial will be 15 patients, if OBP-301 3 doses at 1 x1012vp / mL is the DLT safe dose, or 21 patients, if OBP-301 3 doses at 1 xlO11vp / mL is the DLT safe dose.

[1327] With a cohort of 6 patients, the probability of the treatment being judged to be too toxicAttorney Docket No. 4456-0340PWO1

[1328] when the true toxicity rate is >42% is at least 80%. If the true toxicity rate is <18%, the probability that the treatment will be deemed to be safe is at least 70%.

[1329] Study reports focusing on accrual and adverse event data will be prepared regularly and

[1330] the PI / NRG study team will have regular conference calls (biweekly or at most monthly) to review the accrual / safety data. Information from these calls will be reviewed regularly by the NRG Oncology Early Phase Oversight Committee.

[1331] Secondary or Exploratory Endpoints (including correlative science aims)

[1332] Secondary Hypotheses and Endpoints:

[1333] ■ Adverse events as measured by CTCAE v5.0

[1334] ■ Clinical complete response (cCR) - if one of the OBP-301 regimens is declared to be

[1335] safe.

[1336] ■ Number of patients alive without progression (PFS) and number of patients alive

[1337] (OS) at 1 and 2 years - if one of the OBP-301 regimens is declared to be safe.

[1338] Definitions of Secondary Endpoints and How These Will Be Analyzed

[1339] Adverse events

[1340] Adverse events (AE) will be evaluated using the CTCAE v 5.0. Counts of all AEs by grade will be provided by treatment arm. Counts and frequencies will be provided for the worst grade AE experienced by the patient by treatment arm and within the subset of AEs related to treatment. Severe AEs will be any grade 4 or greater non-hematologic toxicities and will be summarized by frequency tables by treatment arm. No formal statistical testing will be performed on these summary data.

[1341] Clinical Complete Response (cCR)

[1342] For the regimen that is determined to be safe, out of all patients accrued to that

[1343] regimen, the number of cCRs will be reported. No formal statistical testing will be

[1344] performed on these summary data.

[1345] Progression-free Survival (PFS)Attorney Docket No. 4456-0340PWO1

[1346] For the regimen that is determined to be safe, out of all patients accrued to that

[1347] regimen, the number of patients that are alive without progression at 1 and 2 years

[1348] will be reported. No formal statistical testing will be performed on these summary

[1349] data.

[1350] Overall Survival (OS)

[1351] For the regimen that is determined to be safe, out of all patients accrued to that

[1352] regimen, the number of patients that are alive at 1 and 2 years will be reported. No formal statistical testing will be performed on these summary data.

[1353] .7.5 Expected Sample Size or Patient Cohorts: 6 evaluable patients will be accrued for up to 2

[1354] dose levels. If a dose level is determined to be safe, an additional 9 patients will be

[1355] accrued at that dose level.

[1356] .5 Exploratory Hypothesis and Endpoints

[1357] Tumor and blood immune and virus-based correlative assays will be performed (see

[1358] Section 10).

[1359] These analyses will allow for the following exploratory hypotheses to be evaluated:

[1360] • Correlate qualitative and semi-quantitative in the tumor micro-environment and

[1361] persistence of adenovirus infection with outcomes to identify biomarkers of response • Correlate changes in immune cell subsets via multi -parameter flow cytometry with outcomes to identify biomarkers of response

[1362] • Correlates changes in cytokine profile via multiplex ELISA with outcomes to identify biomarkers of response

[1363] .6 Gender / Ethnicity / Race Distribution

[1364] DOMESTIC PLANNED ENROLLMENT REPORT

[1365] Ethnic Categories

[1366] Racial Categories Not Hispanic or

[1367] Hispanic or Latino

[1368] Latino

[1369] Total

[1370] Female Male Female Male

[1371] American Indian / Alaska 0 1 0 0 1

[1372] Native

[1373] Asian 0 1 0 0 1

[1374] Native Hawaiian or Other 0 0 0 0 0

[1375] Pacific Islander

[1376] Black or African American 1 2 0 0 3

[1377] White 2 12 0 2 16

[1378] More Than One Race 0 0 0 0 0

[1379] Total 3 16 0 2

[1380]

[1381] Attorney Docket No. 4456-0340PWO1

[1382] APPENDIX I: PHARMACY MANUAL (19-M AY-2023)

[1383] 1. Telomelysin (OBP-301)

[1384] Summary of Investigational Product

[1385] OBP-301 (Telomelysin, hereafter referred to as OBP-301) is an adenoviral vector containing the adenoviral El A and E1B gene linked with an internal ribosome entry site (IRES) under the

[1386] control of the human telomerase reverse transcriptase (hTERT) promoter.

[1387] Packaging and Labeling

[1388] OBP-301 will be provided as a frozen viral suspension formulated in 20 mM Tris pH 8.0, 25 mM NaCl with 2.5% Glycerin, USP by volume. The product is packaged using a 5 mL glass vial, siliconized gray butyl-rubber stoppered vials and an aluminum seal with a plastic cap. Each vial contains a volume of 2.0 mL at a concentration of 1 x 1012viral particles (vp) / mL. The product in this configuration is stored at < -60° C prior to administration

[1389] Vials will be labeled with the following information:

[1390] Vial label:

[1391] Name of the Sponsor, Protocol No., Contents, Nominal Concentration, Content,

[1392] Route of Administration, Batch No., Lot No., Storage Condition

[1393] Label for storage box:

[1394] Caution: For Clinical Trial Use Only,

[1395] Name of the Sponsor, Protocol No., Contents, Nominal Concentration, Content,

[1396] Route of Administration, Batch No., Lot No., Storage Condition

[1397] STUDY AGENT RECEIPT PROCEDURES

[1398] Shipping Conditions

[1399] Each vial is boxed in an upright position within a small box. Each storage box is placed in a

[1400] plastic self-sealing leak-proof bag with an adequate number of absorbent pads in the event of

[1401] leakage during shipping. The storage boxes are shipped to the clinical sites in leak-proof foam-lined cardboard boxes.

[1402] Due to product specific requirements, the shipments are made on dry ice. Within each shipment there will be an official shipping form to be completed by the study investigator or designee (e.g. pharmacist).

[1403] Receipt Procedures

[1404] The study investigator or designee is responsible for receiving each OBP-301 shipment. Prior to

[1405] each shipment, the drug distributor, Catalent, will contact the assigned personnel with the

[1406] delivery information.

[1407] Upon receipt the following should be performed:

[1408] 1. Always wear disposable gloves when handling OBP-301.Attorney Docket No. 4456-0340PWO1

[1409] 2. Examine the outside of the shipment package(s) for integrity. If the shipment package or

[1410] contents are not intact:

[1411] • The outer surfaces of all containers should be decontaminated with a virucidal

[1412] disinfectant such as CaviCide.

[1413] • If a vial is damaged in any way, it should be placed in a biohazardous waste container for sharps, and destroyed immediately according to institutional and local regulations for biohazardous waste management. Be sure to record the destruction of any clinical

[1414] material on the Clinical Trial Material Destruction Certificate (located on the NRG

[1415] website).

[1416] • Contact the drug distributor, Catalent, to report the damage.

[1417] 3. Open the shipment package(s).

[1418] 4. Confirm the package still contains dry-ice.

[1419] 5. Transfer the leak-proof package to a Biological Safety Cabinet (BSC).

[1420] 6. Remove the storage box from the plastic bag(s).

[1421] 7. OBP-301 will be provided in glass, chlorobutyl rubber stoppered vials, with an aluminum seal as a frozen viral suspension. It will be transported to the pharmacy, following the current International Airway Transport Association (IATA) regulations.

[1422] 8. Promptly, inventory the vials of OBP-301 contained in the storage box and examine the

[1423] integrity of the vials. If the shipment package or contents are not intact, follow the steps listed above in step 2. If the shipment does not match the detailed description, the site should

[1424] contact the drug distributor, Catalent.

[1425] 9. Transfer vials to the appropriate location in the assigned < -60°C freezer. The vials must be

[1426] stored in the original storage box to facilitate supply tracking.

[1427] 10. Wearing gloves, dispose of the dry ice, according to institutional procedures.

[1428] 11. Decontaminate the biological safety cabinet with a virucidal disinfectant.

[1429] 12. Complete the lower portion of the official shipping form, email it to the originator of the form

[1430] as instructed and retain the original document in the site file.

[1431] 13. Enter the number of vials of clinical trial material received on the NCI’s Investigational

[1432] Agent Accountability Record (located on CTEP / PMB website).

[1433] Storage Conditions

[1434] Prior to dilution, the vials of OBP-301 must be stored in a < -60°C freezer, in an upright position.

[1435] The vials must be stored in the original vial storage boxes to facilitate supply tracking. It is recommended to leave the vial storage boxes in the original leak-proof bag or in a substituteleakproof container, particularly if the supplies are not segregated (i.e. in a separate section of the freezer) from other products or biological samples.

[1436] The freezer should be in a locked or secured area, with limited access. Additionally, the freezer should have a temperature recorder and an alarm system. The temperature plot should be properly maintained and checked. Documentation for calibration of the freezer at installation and at each maintenance inspection is required, (at least annually or as per manufacturer’s recommendations).

[1437] A biohazard symbol should be affixed to the outside of the storage compartment.Attorney Docket No. 4456-0340PWO1

[1438] PREPARATION NOTES OBP-301 should be prepared according to Biosafety Level 2 Guidelines, unless local regulations require a more rigorous containment level.

[1439] Disinfectant

[1440] A disinfectant that has been qualified as an effective virucide for adenoviruses should be used for any decontamination procedure. The recommended exposure time should be at least 5 to 15 minutes. However, if the handling precautions recommended by the manufacturer differ, the manufacturer’s instructions should be followed.

[1441] Study Material Expiration After Thaw

[1442] The duration that OBP-301 remains at room temperature should be minimized. It is recommended that OBP-301 be administered as soon as possible (within 4 hours) after reconstitution.

[1443] Immediately prior to injection, it may be warmed by rolling the syringe between the palms of the hands, which may facilitate comfort of administration.

[1444] Aseptic Procedures

[1445] Aseptic techniques must be followed at all times during the preparation and transport to the

[1446] treatment area.

[1447] Biosafety Level 2

[1448] As there is the potential of creating infectious aerosol during the preparation procedure, all work involving OBP-301 must be performed inside a properly maintained class II Biological Safety Cabinet (BSC). Biosafety Level 2 guidelines should be adhered to, unless local regulations require a more rigorous containment level.

[1449] Universal Precautions

[1450] Universal precautions should always be adhered to, as defined by federal, state and institutional

[1451] and local regulations.

[1452] Biohazardous Waste Management

[1453] All OBP-301 wastes are considered biohazardous and consequently must be handled by the site according to federal, state and local regulations on management of biohazardous materials.

[1454] Syringe Label

[1455] According to local practice, a label will be affixed either to each injection syringe containing the final OBP-301 suspension or to the leak-proof plastic bags used in the transfer of the syringes.

[1456] Care should be taken not to cover the graduation markings on the syringe with the label.

[1457] The label should include but is not limited to the following information:Attorney Docket No. 4456-0340PWO1

[1458] - Patient name or initials

[1459] - Patient study number

[1460] - Study investigational product: OBP-301

[1461] - Dose (vp) and volume of injection

[1462] - Expiration date

[1463] - Lot number

[1464] MATERIALS REQUIRED

[1465] 1. Vials of OBP-301

[1466] 2. Labels for injection syringes

[1467] 3. Disposable needles with bore 22-25 Gauge

[1468] 4. Self-sealing leak-proof plastic bags

[1469] 5. Puncture resistant leak-proof container for transport from pharmacy to treatment area

[1470] 6. Virucidal Disinfectant

[1471] 7. Biological Safety Cabinet (Class II) for OBP-301 preparation

[1472] 8. Polypropylene syringe(s)

[1473] PREPARATION PROCEDURES

[1474] 1. Prior to assembly and placement of the OBP-301 preparation material, wipe Biological

[1475] Safety Cabinet work surface with an appropriate disinfectant, following manufacturer’s instructions. Allow to dry.

[1476] 2. Wipe all outside surfaces of materials with an appropriate disinfectant, following

[1477] manufacturer’s instructions, prior to placement of study material inside the biological safety cabinet. Arrange materials for preparation toward the back of the cabinet in order to protect

[1478] from contamination.

[1479] 3. Remove the vial(s) of OBP-301 from the < -60°C freezer on the day of administration,

[1480] following confirmation that the patient will be treated on that day. Transfer the vials in a

[1481] puncture resistant leak-proof container to the preparation area.

[1482] 4. Prepare labels.

[1483] 5. Assemble the appropriate needles and syringes.

[1484] 6. Thaw vial containing OBP-301 by rolling the vial between the palms of the hands and

[1485] mixing 10 times by inverting the vial. The OBP-301 should be allowed to thaw completely

[1486] prior to removing the aluminum seal. Before proceeding, visually inspect the solution in the

[1487] vials to ensure there are no ice particles. Remove the seal on the OBP-301 vials.Attorney Docket No. 4456-0340PWO1

[1488] 7. As there is a “dead space” of up to 1.5 ml in the tubing and syringe, it will be necessary to

[1489] utilize 2 vials of OBP-301, drawing up 2 mL from vial 1 and 1.5 mL from vial 2. At the end

[1490] of the endoscopy procedure, any OBP-301 in the syringe, tubing and the 2nd vial that has not been administered should be disposed of per biosafety protocols.

[1491] 8. Using polypropylene syringe(s), aseptically pierce the rubber cap(s) with the needle(s) and

[1492] draw up the required volume. Caution should be used to avoid excessive shear forces in

[1493] aspirating and discharging through needles.

[1494] 9. According to local practice, label either the OBP-301 syringe and / or the plastic transfer bag

[1495] with a patient label.

[1496] 10. Place the syringe into the primary self-sealing leak-proof plastic bag and seal tightly. Place

[1497] this bag into the second transfer bag. Seal outer transfer bag tightly, assuring that the labels

[1498] can easily be read.

[1499] 11. When the syringe is to be transported to the treatment area, the transfer bags should be placed

[1500] in a puncture resistant leak-proof container, marked with a biohazard label. Effort should be

[1501] made to minimize the time that the OBP-301 is placed at room temperature.

[1502] 12. Immediately after use in the preparation procedure, the OBP-301 vials should be placed in a biohazardous waste container. Thereafter, the local procedures for handling of biohazardous wastes must be followed.

[1503] 13. Immediately dispose of all materials used in the preparation of the syringe, according to

[1504] practices established by local health authorities and / or hospital for disposal of biohazardous waste.

[1505] 14. Clean and disinfect the Biological Safety Cabinet with a virucidal disinfectant.

[1506] 15. Record dispensing of study material on the NCI Investigational Agent Accountability Record.

[1507] 16. If you have any questions concerning the preparation procedure, please contact NRG

[1508] Oncology.

[1509] INJECTION TECHNIQUE

[1510] Pre-inj ection planning is the key to success, therefore, a sketch of the tumor and the planned

[1511] injection sites should be made. (The sketch will be helpful in determining the total quantity of

[1512] study drug to inject.)

[1513] Irrespective of the size of the tumor, every effort should be made to inject 1.0-2.0 mL of OBP-301; the maximal volume of 2.0 mb is preferred and should be injected as long as this is

[1514] technically possible

[1515] Example of Mapping into up to 5 Tracks shows in Figure 4.Attorney Docket No. 4456-0340PWO1

[1516] Specific endoscopic instructions are provided in a separate training document and video.

[1517] Pain Management for Intratumoral Injections with OBP-301

[1518] Injection site pain is the one of the more common adverse events in connection with intratumoral injections. Therefore, the following pain control measures are suggested to improve the comfort of the patient.

[1519] 1. Review the analgesics already prescribed for the patient and determine the level of pain

[1520] control.

[1521] Additional pain medication will with all likelihood be needed during the day of the

[1522] intratumoral injections and for a couple of days afterwards. Local anesthesia or nerve block

[1523] may very well be required just before or during the intratumoral injection.

[1524] DISPENSING AND ACCOUNTABILITY

[1525] The study investigator, pharmacist, or designee may only dispense clinical supplies in

[1526] accordance with the protocol.

[1527] The study investigator, pharmacist, or designee, is responsible for maintaining an accurate and

[1528] current record of all clinical supplies received from the sponsor, dispensed to the investigator or destroyed. Accountability records must be maintained throughout the course of the study,

[1529] showing receipt and disposition of the clinical supplies. At the end of the study, it must be

[1530] possible to reconcile delivery records with that of used, unused and destroyed clinical supplies.Attorney Docket No. 4456-0340PWO1

[1531] The NCI’s Investigational Agent Accountability Record (located on CTEP / PMB website) must

[1532] be used for accountability and destruction certificate to be completed upon destruction (located

[1533] on the NRG website).

[1534] During site monitoring visits, the drug accountability forms will be verified and checked for

[1535] accuracy and consistency.

[1536] DESTRUCTION OF EMPTY OR PARTIALLY USED VIALS

[1537] Immediately after use, the used vials should be placed in a biohazardous waste container. Empty or partially used vials should be destroyed by the study pharmacy, in accordance with institutional biosafety policy and procedures for biohazardous waste management. Proper accounting of the vials must be performed immediately using the Investigational Agent Accountability Record.

[1538] DESTRUCTION OF UNUSED VIALS

[1539] Written authorization must be obtained from Oncolys before any destruction of unused

[1540] clinical material supplies.

[1541] This will occur at the completion of the clinical study or when the expiration date has been

[1542] reached and could not be prolonged by the sponsor based on updated stability information. In addition, destruction of unused supplies may only be performed after product reconciliation by

[1543] the pharmacist.

[1544] The unused vials must be destroyed by the study pharmacy, in accordance with institutional Biosafety policy and procedures for biohazardous waste management.

[1545] The investigator or designee must fill in, sign and date the appropriate section of Clinical Trial Material Destruction Certificate (located on the study-specific page on the NRG website).

[1546] Discrepancies in vial accountability are to be explained in writing by the investigator or

[1547] designee. The original will be retained for the site study file.Attorney Docket No. 4456-0340PWO1

[1548] APPENDIX V: CARBOPLATIN DOSE CALCULATION INSTRUCTIONS (07-DEC-2020)

[1549] 1) The Cockcroft-Gault formula will be used in NRG Oncology trials.

[1550] Dosing of Carboplatin:

[1551] 1) The carboplatin dose will be calculated to reach a target area under the curve (AUC)

[1552] according to the Calvert formula using creatinine clearance (mL / min) from the Cockcroft-Gault formula.

[1553] 2) In patients with an abnormally low serum creatinine (less than 0.7 mg / dl), the creatinine

[1554] clearance should be estimated using a minimum value of 0.7 mg / dL.

[1555] 3) The initial dose of carboplatin must be calculated using GFR. In the absence of renal toxicity greater than or equal to CTCAE Grade 2 (serum creatinine >1.5 x ULN) or toxicity requiring

[1556] dose modification, the dose of carboplatin will not need to be recalculated for subsequent cycles, but will be subject to dose modification for toxicity as noted in the protocol.

[1557] 4) Carboplatin doses are required to be recalculated if the patient has a weight change of greater than or equal to 10%. Patients are permitted to have chemotherapy doses recalculated for < 10% weight changes.

[1558] 5) At the time of dose modification, if the patient’s age had changed (the patient has had a

[1559] birthday), the site can use the current age.

[1560] CALVERT FORMULA:

[1561] Carboplatin dose (mg) = target AUC x (GFR [or estimated CrCl] + 25)

[1562] NOTE: the GFR used in the Calvert formula should not exceed 125 ml / min.

[1563] Maximum carboplatin dose (mg) = target AUC (mg / ml x min) x 150 ml / min.

[1564] The maximum allowed doses of carboplatin are:

[1565] AUC 2 = 300 mg

[1566] For the purposes of this protocol, the GFR is considered to be equivalent to the estimated

[1567] creatinine clearance. The estimated creatinine clearance (mL / min) is calculated by the method of Cockcroft-Gault using the following formula:

[1568] Creatinine Clearance (mL / min) = [140-Age (years)] x actual body Weight* (kg) {x 0.85 if female}

[1569] 72 x serum creatinine (mg / dl)

[1570] Notes:

[1571] 1) Weight in kilograms (kg):

[1572] a. Body Mass Index (BMI) should be calculated for each patient.

[1573] b. Actual weight should be used for estimation of GFR for patients with BMI of less than 25.Attorney Docket No. 4456-0340PWO1

[1574] c. Adjusted weight should be used for estimation of GFR for patients with BMI of greater

[1575] than or equal to 25

[1576] d. Adjusted weight calculation:

[1577] Ideal weight (kg) = ((Height (cm) / 2.54) - 60) x 2.3) (+ 45.5 females) or (+ 50 for men) Adjusted weight (kg) = ((Actual weight - Ideal weight) x 0.40) + Ideal weight

[1578] At the time of a dose modification for toxicity:

[1579] If the creatinine at the time of a dose modification is lower than the creatinine used to

[1580] calculate the previous dose, use the previous (higher) creatinine; if the creatinine at the time of a dose modification is higher than the creatinine used to calculate the previous dose, use the current (higher) creatinine. This will ensure that the patient is actually receiving a dose

[1581] reduction

Claims

1. Attorney Docket No. 4456-0340PWO1CLAIMS1. A method for treating a tumor in a patient comprising the steps of:(i) administering an oncolytic virus and one or more anticancer drugs to the patient:and(ii) irradiating the tumor thereafter.

2. A method for treating a tumor, comprising administering to a patient an oncolytic virus and two types of anticancer drugs, and irradiating the patient with radiation.

3. The method according to claim 1, wherein the oncolytic virus is an oncolytic adenovirus.

4. The method according to claim 1, wherein the tumor is at least one selected from the group consisting of stomach cancer, gastroesophageal junction cancer, esophageal cancer, duodenal cancer, pancreatic cancer, colon cancer, head and neck cancer, anal cancer, rectal cancer, small intestine cancer, lung cancer, liver cancer, brain tumors, cervical cancer, jaw cancer, maxillary cancer, submandibular gland cancer, oral cancer, salivary gland cancer, sublingual gland cancer, parotid gland cancer, nasal cavity cancer, paranasal sinus cancer, laryngeal cancer, breast cancer, biliary tract cancer, bladder cancer, kidney cancer, prostate cancer, uterine cancer, ovarian cancer, thyroid cancer, pharyngeal cancer, sarcoma, malignant lymphoma, leukemia, skin cancer, and melanoma.Attorney Docket No. 4456-0340PWO15. The method according to claim 1, wherein the oncolytic adenovirus is a virus having incorporated into its genome a polynucleotide comprising a human telomerase promoter (hTERT promoter), an El A gene, an IRES sequence, and an E1B gene in this order (OBP-301), or a virus having incorporated into the E3 region of OBP-301 a polynucleotide comprising an Egr-1 promoter and a p53 gene in this order (OBP-702).

6. The method according to claim 1, wherein the anticancer drug is selected from the group consisting of alkylating agents, platinum agents, topoisomerase inhibitors, metabolic antagonists, microtubule inhibitors, antibiotic anticancer agents, and molecular targeted drugs.

7. The method according to claim 6, wherein the platinum agent is selected from the group consisting of cisplatin, carboplatin, nedaplatin, and oxaliplatin.

8. The method according to claim 6, wherein the microtubule inhibitor is selected from the group consisting of eribulin, paclitaxel, docetaxel, vinblastine, vinorelbine, and vincristine.

9. The method according to claim 1, wherein the anticancer drug is a combination of the platinum agent and microtubule inhibitor.Attorney Docket No. 4456-0340PWO110. The method according to claim 1, wherein the anticancer drug is a combination of carboplatin and paclitaxel.

11. The method according to claim 1, wherein the oncolytic virus is administered at least three times at intervals of 6 to 10 days.

12. The method according to claim 1, wherein the anticancer drug is administered at least five times, with each type of agent being administered once a week.

13. The method according to claim 1, wherein the oncolytic virus is administered at a concentration of 1x1010vp / mL to IxlO13vp / ml in a dose of 1-2 ml.

14. The method according to claim 10, wherein the carboplatin is administered at a dose of AUC1-6.

15. The method according to claim 10, wherein the paclitaxel is administered at a dose of 25-100 mg / m2.

16. The method according to claim 1, wherein the radiation is administered at a dose of 1.5-2 Gy in at least 28 doses.Attorney Docket No. 4456-0340PWO117. The method according to claim 1, wherein the oncolytic virus is administered at a concentration of 1 x 1012vp / mL and in a dose of 1 to 2 ml on days 3, 12, and 26 after the start of treatment, carboplatin is administered at a dose of AUC1-3 once a week for 5 doses, paclitaxel is administered at a dose of 25-70 mg / m2once a week for 5 doses, and radiation is administered at a dose of 1.8 Gy for 28 doses.

18. A pharmaceutical composition for treatment of tumor to be used in combination with one or more anticancer drugs and radiation therapy, comprising an oncolytic virus as an active ingredient.

19. A pharmaceutical composition for treatment of tumor to be used in combination with one or more anticancer drugs, oncolytic virus, and radiation therapy, comprising a platinum agent an active ingredient.

20. A pharmaceutical composition for treatment of tumor to be used in combination with one or more anticancer drugs, oncolytic virus, and radiation therapy, comprising a microtubule inhibitor an active ingredient.

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