Selective Antigen Specific T Cells and CAR T Cells in Subjects With Relapsed/Refractory Embryonal Tumors (SABRE)

This protocol is designed as a phase I dose-escalation study. Procurement phase: During the procurement phase of this protocol, upon SABRE Procurement Consent and procurement eligibility confirmation, participants will undergo a non-mobilized apheresis for collection of mononuclear cells to be used for the CAR-TA T cell product manufacturing.

Treatment phase: Once the CAR-TA T cell products are released and patients are confirmed eligible for CAR-TA T cell product infusion, participants will undergo protocol therapy at participating site(s), consisting of a standard lymphodepleting chemotherapy preparative regimen with fludarabine and cyclophosphamide, followed by intravenous infusion of the combined CAR-TA T cell product. The DNR-TA T cells and B7-H3 CAR T cells will be generated and combined into a final product comprised of the two T cell components combined at a 1:1 ratio.

Patients will be enrolled to one of three CAR-TA T cell product dose levels (dose levels 1, 2 and 3). There are provisions in place to dose de-escalate for safety concerns (dose level -1).

Fludarabine will be administered intravenously once daily over 30 minutes, days -5 through -2 (4 doses in total). The dose of fludarabine will be 30 mg/m2 /day. Cyclophosphamide will be administered intravenously once daily over 30 minutes, days -5 and -4 (2 doses in total). The dose of cyclophosphamide will be 500 mg/m2 /day.

The first 3 patients enrolled on study will be ≥ 12 years of age at enrollment and treated at dose level 1 (1 x 10e6/kg). If no DLTs are observed in this cohort, enrollment at dose levels 2 (3 x 10e6/kg) and 3 (10 x 10e6/kg) will expand to include patients aged ≥ 1 year and < 24 years.

Patients will remain admitted for at least 7 days following the CAR-TA T cell infusion. All infused patients will be followed with weekly visits during the 28-day dose-limiting toxicity monitoring period where they will be clinically assessed, and safety and research blood draws will be performed.

Ideally, patients should not receive other systemic or local cancer-directed therapies for at least 28 days after the CAR-TA T cell infusion.

Participants will be followed closely for 1 year following the CAR-TA T-cell infusion. After 1 year, yearly assessments will be done up to 15 years. The visits will consist of labs and examinations as well as talking to participants about how they are feeling. Participants will be followed for toxicity until the last follow-up post CAR-TA T cell product administration.

This study will be conducted at Children's National Hospital (CNH). Cell culture manipulations will be carried out in the CETI Good Manufacturing Practice (GMP) facility within Children's National Hospital using current standard operating procedures (SOPs).

Up to 18 toxicity-evaluable participants will be treated on this protocol to meet the primary objective over an estimated accrual period of 5 years

Inclusion criteria

Recipient Inclusion Criteria for Procurement:

• Diagnosis of relapsed/refractory rhabdomyosarcoma, Ewing sarcoma, neuroblastoma, or Wilms tumor
• Refractory disease, residual detectable disease or relapsed disease following available standard of care therapies with known clinical benefit for their specific tumor type, or unable to receive such therapies due to unacceptable toxicity or contraindication
• Measurable or evaluable disease by imaging, as determined following most recent therapy
• Age ≥ 1 year and < 24 years
• Weight ≥ 10 kg
• No systemic steroid exposure within 1 week of procurement
• Karnofsky/Lansky score of ≥ 60 (See Appendix 3)
• Participants of childbearing potential or capable of fathering a child must agree to use effective contraceptive measure/s (as described in Appendix 5) during study protocol participation through 6 months following the administration of the CAR-TA T cells
• ANC > 500/µL
• ALC > 1000/µL
• Platelet count > 50,000/uL (level can be achieved with transfusion)
• Bilirubin ≤ 2.5 mg/dL
• Aspartate aminotransferase (AST)/Alanine transaminase (ALT) ≤ 5x the upper limit of normal for age
• Serum creatinine Maximum serum creatinine (mg/dL) Age Male Female

• to < 2 years 0.6 0.6
• to < 6 years 0.8 0.8

6 to < 10 years 1 1 10 to < 13 years 1.2 1.2 13 to < 16 years 1.5 1.2

≥ 16 years 1.7 1.4 OR Creatinine clearance or glomerular filtration rate (GFR) ≥ 70 mL/min/1.73 m for patients with levels above

• For FOCBP: Negative pregnancy test
• Pulse oximetry of > 90% on room air
• Adequate cardiac function defined as:

• Shortening fraction of ≥ 27% by echocardiogram, or
• Ejection fraction of > 50% by echocardiogram or radionuclide angiogram (i.e., MUGA).
• No acute neurological toxicity > grade 1 (with the exception of peripheral sensory neuropathy or controlled seizure disorder on anti-epileptics).
• The following time frames must have elapsed between prior therapy completion and apheresis cell collection:

• Myelosuppressive chemotherapy/immunomodulatory medications: At least 3 weeks, or 6 weeks if prior nitrosourea.
• Hematopoietic growth factors: At least 7 days since the completion of therapy with a growth factor. At least 14 days after receiving pegfilgrastim.
• Biological agent, tyrosine kinase inhibitor, targeted agent, metronomic chemotherapy: At least 7 days since the completion of therapy with a biologic agent, tyrosine kinase inhibitor, targeted agent, or metronomic non-myelosuppressive regimen.
• Monoclonal antibodies and checkpoint inhibitors: At least 3 weeks or 5 half-lives (whichever is shorter) since the last dose of a monoclonal antibody or checkpoint inhibitor.
• Radiotherapy (XRT): At least 3 weeks since XRT, and at least 6 weeks if radiation involved the CNS or lung fields. Exception: There is no time restriction for palliative radiation with minimal bone marrow involvement and the patient has measurable/evaluable disease outside the radiation port or the site of radiation has documented progression.
• Autologous stem cell transplant/infusion: At least 6 weeks from their infusion after an autologous stem cell infusion following myeloablative therapy. Patients who received an autologous stem cell infusion following non-myeloablative therapy do not have a wash-out period; they are eligible once they meet all other eligibility requirements, including recovery from acute side effects.
• Investigational agent: at least 28 days since receiving an investigational agent.
• Adult participant or the legally authorized representative (LAR) of a minor (defined as <18 years of age) must be capable of providing informed consent. When appropriate, pediatric participants ≥7 years of age will participate in an age-appropriate discussion and provide assent, unless an IRB-approved waiver of assent applies and documentation of the participant's eligibility for the waiver is maintained.

Recipient Inclusion Criteria for CAR-TA T cell product Infusion:

• No systemic steroid exposure within 1 week prior to protocol therapy initiation
• Karnofsky/Lansky score of ≥ 60
• ANC > 750/uL
• Platelet count > 75,000/uL
• Bilirubin ≤ 2.5 mg/dL
• AST/ALT ≤ 5x the upper limit of normal for age
• Serum creatinine Maximum serum creatinine (mg/dL) Age Male Female

1 to < 2 years 0.6 0.6 2 to < 6 years 0.8 0.8 6 to < 10 years 1 1 10 to < 13 years 1.2 1.2 13 to < 16 years 1.5 1.2

≥ 16 years 1.7 1.4 OR Creatinine clearance or glomerular filtration rate (GFR) ≥ 70 mL/min/1.73 m for patients with levels above

• For FOCBP: Negative pregnancy test
• Participants of childbearing potential or capable of fathering a child must agree to use effective contraceptive measure/s through 6 months following the administration of the CAR-TA T cells
• Adequate respiratory function defined as oxygen saturation 90% or higher on room air
• For participants who underwent prior mediastinum-directed therapies (e.g., post radiation to chest): resolution of any respiratory symptoms
• Adequate respiratory rate, defined as <30 breaths per minute for patients aged <18 years, and <25 breaths per minute for patients aged ≥18 years (respiratory rate may be repeated if initial value is thought to be temporarily abnormal; if repeated, 2 consecutive readings obtained ≥30 minutes apart must be adequate to be eligible)
• No acute neurological toxicity > grade 1 (with the exception of peripheral sensory neuropathy or controlled seizure disorder on anti-epileptics).
• Adequate cardiac function defined as:

• Shortening fraction of ≥ 27% by echocardiogram, or
• Ejection fraction of > 50% by echocardiogram or radionuclide angiogram
• The following time frames must have elapsed between completion of prior therapy and the initiation of SABRE protocol therapy:

• Myelosuppressive chemotherapy: At least 2 weeks from last dose of chemotherapy.
• Hematopoietic growth factors: At least 7 days since the completion of therapy with a growth factor. At least 14 days after receiving pegfilgrastim.
• Biological agent, tyrosine kinase inhibitor, targeted agent, metronomic chemotherapy: At least 7 days since the completion of therapy with a biologic agent, tyrosine kinase inhibitor, targeted agent, or metronomic non-myelosuppressive regimen.
• Monoclonal antibodies and checkpoint inhibitors: At least 3 weeks or 5 half-lives (whichever is shorter) since the last dose of a monoclonal antibody or checkpoint inhibitor.
• Radiotherapy (XRT): At least 3 weeks since XRT, and at least 6 weeks if radiation involved CNS or lung fields. Exception: There is no time restriction for palliative radiation with minimal bone marrow involvement and the patient has measurable/evaluable disease outside the radiation port or the site of radiation has documented progression.
• Investigational agent: At least 28 days since receiving an investigational agent.
• Adult participant or the legally authorized representative (LAR) of a minor (defined as <18 years of age) must be capable of providing informed consent. When appropriate, pediatric participants ≥7 years of age will participate in an age-appropriate discussion and provide assent, unless an IRB-approved waiver of assent applies and documentation of the participant's eligibility for the waiver is maintained.

Exclusion criteria

Recipient Procurement Exclusion Criteria:

• Patients with known CNS disease.
• Patients with uncontrolled infection/s or known HIV infection
• Pregnant or lactating females.
• Patients who have undergone previous allogeneic stem cell transplant.
• Inability to tolerate leukapheresis (including any contraindication to the use of Anticoagulant Citrate Dextrose solution).

Recipient Exclusion Criteria for CAR-TA T cell product Infusions:

• Patients with uncontrolled infections or known HIV infection.
• Pregnant or lactating females
• Whole lung/mediastinal radiation within 12 weeks
• Clinically significant systemic illness or medical condition likely to interfere with assessment of safety or efficacy
• Patients who have received any live vaccine in the six weeks prior to planned initiation of lymphodepletion
• Any contraindication to lymphodepletion or to the use of Cyclophosphamide or Fludarabine, as per the clinical judgment of the PI or treating Sub-I
• History of allergy or hypersensitivity to study product excipients (e.g., DMSO)