Immunotherapy for Malignant Pediatric Brain Tumors Employing Adoptive Cellular Therapy (IMPACT)

TSA-T products will be manufactured at the cell therapy Good Manufacturing Practice (GMP) facility at Children's National Hospital (CNH). Patients enrolled in the study will receive their infusions at CNH.

This is an open-label phase 1 safety and feasibility study that will employ multi-tumor antigen specific cytotoxic T lymphocytes (TSA-T) directed against proteogenomically determined personalized tumor-specific antigens (TSA), derived from a patient's primary brain tumor tissues. Participants in this study will receive TSA-T after completion of standard-of-care/salvage therapy for either their newly diagnosed embryonal brain tumor or their recurrent ependymoma. Participants will be enrolled into one of two Groups:

• Group A: Children younger than 5 years of age with newly diagnosed embryonal brain tumors - including medulloblastoma (MB), atypical teratoid/rhabdoid tumor (ATRT), embryonal tumor with multilayered rosettes (ETMR), and embryonal brain tumor not otherwise specified (NOS).
• Group B: Children, adolescents and young adults greater than 1 year and less than 30 years of age with recurrent ependymoma (EPN).

Group A Standard-of-Care Backbone Therapy: Patients will undergo surgical resection, then be treated with standard-of-care therapy as required, which may include up to 3 induction chemotherapy cycles (vincristine, cyclophosphamide, cisplatin, etoposide with or without methotrexate) and up to 3 consolidation cycles (carboplatin and thiotepa, each followed by an infusion of autologous peripheral blood stem cells (PBSCs)). Please note: Methotrexate may be part of the induction chemotherapy, and radiation may be added during backbone therapy, as per treating physician's discretion.

Group B Salvage Backbone Therapy: Patients will undergo surgical re-resection - aiming for gross total resection when feasible - followed by re-irradiation. Re-irradiation may be delivered as a conventional fractionated course, hypofractionated stereotactic radiotherapy, or proton therapy, depending on prior treatments, institutional guidelines and treating physician's discretion. Bridging chemotherapy may be administered between re-irradiation and TSA-T cell infusion, at the treating physician's discretion.

Patients will receive the TSA-T product at assigned dose levels as per the table and details below until the highest planned dose level or the MTD is reached. The MTD will be declared when prespecified stopping/escalation rules and DLT data allow a determination, and the recommended phase 2 dose (RP2D) will be established using the MTD together with additional safety, tolerability and pharmacodynamic data.

Dose Levels TSA-T cell dose -1 1x107 cells 1* 2.5x107 cells 2 5x107 cells

*Starting dose

The MTD and RP2D determinations, as well as all dose escalation and de-escalation decisions, will be based on pooled safety data from Groups A and B combined. However, the secondary objectives pertaining to objective tumor response and clinical survival, will be analyzed separately for Group A and Group B.

Safety will be assessed at two planned TSA-T dose levels, 2.5×10⁷ and 5×10⁷ cells, with the option to de-escalate to 1×10⁷ cells if warranted. Dose escalation will follow a Bayesian optimal interval (BOIN) design with a 42-day DLT monitoring period. An interim safety analysis will be conducted after the first six patients.

Each patient will receive at least one TSA-T infusion and may receive a maximum of 8 total infusions if they remain eligible and sufficient TSA-T cells are available. All infusions will be intracerebroventricularly (ICV) administered through the protocol-required Rickham reservoir.

For any infusion following the first infusion, if a patient's remaining cryopreserved TSA-T product is insufficient to meet the dose at the enrollment dose level, the final infusion may be administered at a lower available dose at the discretion of study PI.

If patients have a response of stable disease or better by RAPNO criteria at the evaluation after the second infusion OR if they have clinical stability and a clinical assessment of possible pseudoprogression (per iRANO) on MRI despite the appearance of radiographic progression (with subsequent serial MRI findings most consistent with pseudoprogression), they may be eligible to receive additional infusions of TSA-T.

The first and second infusions will be administered at least 42 days apart and additional infusions will be spaced at least 28 days apart. Once a patient begins TSA-T treatment, and until they are taken off treatment, they are expected to adhere to the protocol-defined treatment schedule as closely as possible, with approximately two weeks of flexibility beyond the minimum required infusion interval. The exception is in cases of suspected pseudoprogression (PsP), where an extended interval of up to eight weeks beyond the minimum interval may be permitted to allow for repeat imaging prior to additional TSA-T infusion(s).

Prior to the first infusion, if a patient's cryopreserved TSA-T product is insufficient for the dose level that the patient is assigned to, the patient may receive TSA-Ts at a lower dose level with a minimum of 1x107 cells. These patients will not count towards the overall safety objective, although they will count towards the feasibility objective of identifying TSAs and producing adequate TSA-T products.

RECIPIENT SCREENING INCLUSION CRITERIA

• Diagnosis (select one group):

• Group A: New diagnosis of CNS embryonal tumors: medulloblastoma, embryonal tumor with multilayered rosettes, pineoblastoma, atypical teratoid/rhabdoid tumor, and embryonal tumor, not otherwise specified (NOS).
• Group B: Radiographic evidence consistent with recurrent ependymoma, with planned or recent re-resection.
• Age:

• Group A: <5 years of age at enrollment
• Group B: >1 year and <30 years of age at enrollment
• Tissue:

o Group A: Availability of sufficient fresh or frozen tumor tissue (approximately 50 mg).

o Group B: Expectation of sufficient fresh or frozen tumor tissue, in the opinion of study PI or sub-I (based upon radiographic evidence of disease).

• Non-pregnant:

• Group A: N/A
• Group B: For female of childbearing potential, must have negative pregnancy test.

Common to both groups:

• Karnofsky or Lansky score of ≥60%.
• Adequate organ function, defined below:

i. ANC ≥750/µL. ii. Absolute lymphocyte count (ALC) >500/μL. iii. Platelets ≥75K. iv. Bilirubin ≤3xULN. v. Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) <5x upper limit of normal (ULN).

vi. Serum creatinine ≤1.0 mg/dL or 1.5x ULN for age (whichever is higher). vii. Pulse oximetry >90% on room air.

• The patient (if ≥18 years old), or the patient's parent(s)/legal guardian(s) (if the patient is a minor), is capable of providing informed consent.
• Patient deemed to be of sufficient size to undergo MNC apheresis for TSA-T generation (Groups A and B) and PBSC rescue (Group A only).
• Patient is a surgical candidate for placement of a Rickham reservoir in the opinion of study PI or medically licensed sub-I.

RECIPIENT INCLUSION CRITERIA FOR PROCUREMENT

• Karnofsky or Lansky score of ≥60%. 2. Adequate organ function, defined below: i. ANC ≥750/µL. ii. Absolute lymphocyte count (ALC) >500/μL. iii. Platelets ≥75K. iv. Bilirubin ≤3xULN. v. Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) <5x upper limit of normal (ULN).

vi. Serum creatinine ≤1.0mg/dL or 1.5x ULN for age (whichever is higher). vii. Pulse oximetry >90% on room air. 3. Non-pregnant:

• Group A: N/A
• Group B: For female of childbearing potential (if applicable), must have negative pregnancy test.

RECIPIENT INCLUSION CRITERIA FOR INITIAL TSA-T ADMINISTRATION AND FOR ADDITIONAL INFUSIONS

• Applicable to TSA-T infusion #1 only: Group B participants must have histopathologic confirmation of recurrent ependymoma.
• Karnofsky or Lansky score of ≥60%.
• Adequate organ function, defined as below:

i. Bilirubin ≤3x ULN. ii. AST and ALT ≤5x ULN. iii. Serum creatinine ≤1.0mg/dL or 1.5x ULN for age (whichever is higher). iv. Pulse oximetry >90% on room air.

• Applicable to TSA-T Infusion #1 only: Adequate count recovery, as described below, from prior therapies:

i. Absolute Neutrophil Count (ANC) >1000/μL ii. Absolute Lymphocyte Count (ALC) >500/μL

• Patients must have received their last dose of:

a. Myelosuppressive chemotherapy (if applicable) ≥14 days prior to TSA-T infusion b. Focal radiation (if applicable) ≥14 days prior to TSA-T infusion c. Craniospinal irradiation (if applicable) ≥28 days prior to TSA-T infusion

• Patients must have recovered from all acute effects of prior surgical intervention/s.
• Group B female of childbearing potential or male capable of fathering a child (if applicable): Agree to use contraceptive measures during TSA-T treatment participation through 6 months following last administration of TSA-Ts
• Group B female of childbearing potential (if applicable), must have negative pregnancy test.
• Neurologic status: Patient must have a stable neurologic exam for 2 weeks, on a stable or decreasing dose of steroids, prior to administration of the first dose of TSA-T cells, and stability for 1 week prior to all subsequent infusions. The exams demonstrating stability must be performed by the study team, although these may occur via telemedicine if necessary. Patient must agree to a brief (<72 hours) course of steroids if the PI or medically-licensed sub-I deems it clinically necessary in the context of clinical deterioration.
• Presence of a Rickham reservoir and catheter for intracerebroventricular administration of TSA-T therapy, placed >7 days prior to TSA-T infusion.
• For patients with programmable VP shunts: Able to tolerate the shunt being closed for at least 4 hours, in the opinion of study PI or medically licensed sub-I.

Exclusion criteria

RECIPIENT SCREENING EXCLUSION CRITERIA

• Patients with uncontrolled infections. 2. Patients with known HIV infection. 3. Group A patients with medulloblastoma of the SHH subtype.

RECIPIENT EXCLUSION CRITERIA FOR PROCUREMENT

• Patients with a fever above 38.0°C. 2. Patients with known HIV infection. 3. Prior immunotherapy with an investigational agent within the 28 days prior to planned date of procurement collection for TSA-T manufacturing.

• Patients who will be unable to tolerate the apheresis procedure, including inability to tolerate placement of apheresis line (if applicable), in the opinion of PI or medically licensed sub-I.

• Patients who have overly bulky tumors on imaging are ineligible. These include the following: i. Tumor with any evidence of herniation or significant midline shift. ii. Tumor with a significant brainstem component. iii. Patients who are deemed to have overly bulky tumor by the PI of the study.

If, due to complications during apheresis or subsequent manufacturing, procurement is repeated at a later date using peripheral whole blood collection, exclusion criterion #4 does not apply.

RECIPIENT EXCLUSION CRITERIA FOR INITIAL AND SUBSEQUENT TSA-T INFUSIONS

• Patients with progressive disease based on most recent evaluation (for subsequent infusions).

a. Patients with progressive disease based on most recent evaluation may receive initial TSA-T infusion but would be ineligible if the tumor is found to be progressive before subsequent infusions

• Patients with uncontrolled infections.
• Patients who have overly bulky tumors on imaging are ineligible. These include the following:

i. Tumor with any evidence of herniation or significant midline shift. ii. Tumor with a significant brainstem component. iii. Patients who are deemed to have overly bulky tumor by the PI of the study.

• Patients who received ATG, Campath or other immunosuppressive T cell monoclonal antibodies within 28 days of TSA-T infusion.
• Patients receiving steroids (e.g., dexamethasone) at a dose of >0.05 mg/kg/day.
• Patients who have non-programmable VP shunts.