• Maximum tolerated dose [ Time Frame: Day 28 post CD33CART infusion ]
  This a dose level immediately below the level at which the enrollment is stopped due to a Dose Limiting Toxicity (DLT)
• Morphologic remission [ Time Frame: Day 28 post CD33CART infusion ]
  Percentage of subjects treated with CD33CART who achieve morphologic remission (<5% blasts in marrow)

• Feasibility of CD33CART manufacture [ Time Frame: 2 weeks post start of CD33CART manufacture ]
  Percentage of subjects for whom the desired dose of CD33CAR-T cells can be successfully manufactured
• Feasibility of CD33CART infusion [ Time Frame: 6 weeks post apheresis ]
  Number of subjects able to proceed to infusion of CD33CART 6 weeks post apheresis
• Molecular remission [ Time Frame: Day 28 post CD33CART infusion ]
  Percentage of subjects who receive CD33CART infusion who achieve molecular remission.
• Cytokine release syndrome (CRS), sinusoidal occlusion syndrome (SOS), or other CD33CART related toxicities [ Time Frame: 8 weeks post CD33CART infusion ]
  CRS, or neurotoxicity, SOS, development of any toxicity that precludes proceeding to HCT
• Allogeneic hematopoietic stem cell transplantation [ Time Frame: 6 weeks post CD33CART infusion ]
  Percentage of subjects able to proceed to allogeneic hematopoietic stem cell transplantation following treatment with CD33CART
• Overall survival [ Time Frame: 1 year post HCT ]
  Overall survival will be determined as time from the start of CD33CART infusion until death
• Progression free survival [ Time Frame: I year post HCT ]
  Measured from infusion of CD33CART cells until the documentation of disease progression or death due to any cause, whichever occurs first.
• Treatment related mortality [ Time Frame: I year post HCT ]
  Treatment related mortality is defined as death occurring in a patient from causes other than disease relapse or progression.
• Post HCT time to engraftment [ Time Frame: Day 42 post HCT ]
  Time to engraftment is calculated from the day of HCT to the first day when the absolute neutrophil count (ANC) was > 500/mcL for three consecutive days.

This study consists of two phases. The objectives of Phase 1 and Phase 2 are:

Phase 1: To determine the maximum tolerated dose of lentivirally-transduced CD33-redirected CAR-T cells (CD33CART) in children and young adults with relapsed/refractory AML

Phase 2: To determine the percentage of subjects treated with CD33CART who achieve morphologic remission (<5% blasts in marrow) at Day 28 post-CD33CART cell infusion

Inclusion Criteria:

1. Subjects must have CD33+ AML in second or greater relapse, post-transplant relapse, or have demonstrated chemotherapy-refractory disease (definitions in criteria 2c) to be eligible to participate in this trial.

2. Disease status at the time of enrollment:

   1. Subjects in second or greater relapse will be eligible with relapse defined as >5% blasts (bone marrow) after second documented complete remission
   2. Any degree of detectable disease post-transplant relapse will be eligible (with flow cytometric confirmation of CD33+ myeloid leukemia of at least 0.1%)
   3. Refractory disease is defined as persistent bone marrow involvement with >5% blasts after two courses of induction chemotherapy for patients at initial presentation or >5% bone marrow blasts after one course of re-induction chemotherapy for patients in relapse
3. CD33 expression must be detected on greater than 50% of the malignant cells by immunohistochemistry or greater than 80% by flow cytometry
4. Age: Greater than or equal to 1 year of age (and at least 15 kg) and less than or equal to 35 years of age at time of enrollment. (NOTE: The first three subjects treated on this trial must be ≥ 16 years of age)
5. All subjects must have an allogeneic HCT donor identified with a plan to proceed to HCT conditioning within 6 weeks of CD33CART cell infusion
6. Performance status: > 50% (for subjects > 16 years of age use Karnofsky ≥ 50%; subjects < 16 years of age: Lansky scale ≥ 50%) Subjects who are unable to walk because of paralysis, but who are upright in a wheelchair will be considered ambulatory for the purpose of calculating the performance score;

7. Adequate organ function as defined by:

   1. Cardiac function: left ventricular ejection fraction (LVEF) ≥ 45% or fractional shortening ≥28%
   2. Pulmonary function: baseline oxygen saturation > 92% on room air at rest

   3. Hepatic function:

      • Total bilirubin < grade 2 bilirubin CTCAE version 5 (<3 x ULN) (except in case of subjects with documented Gilbert's disease > 3 x ULN)
      • AST (SGOT)/ALT (SGPT) < 5 x institutional ULN (< grade 3)
   4. Renal function: Serum creatinine must be < 1.2 x institutional upper limit of normal (ULN) according to age. If the serum creatinine is greater than 1.2 x ULN, the patient must have a creatinine clearance (CrCl) > 70mL/min/1.73 m2 (measured by 24 hour- urine specimen or radioisotope GFR).
8. Subjects > 18 years of age must have the ability to give informed consent according to applicable regulatory and local institutional requirements. Legal guardian permission must be obtained for subjects < 18 years of age. Pediatric subjects will be included in age appropriate discussion in order to obtain assent; Adults with cognitive impairment who are unable to consent and those with Down Syndrome are also eligible for this protocol
9. Enrollment in the NMDP protocol: Protocol for a Research Database for Hematopoietic Cell Transplantation, Other Cellular Therapies and Marrow Toxicity Injuries.

Exclusion Criteria:

1. Subjects with radiologically-detected CNS chloromas or CNS 3 disease (presence of ≥ 5/μL white blood cells (WBCs) in cerebral spinal fluid (CSF) and cytospin positive for blasts [in the absence of a traumatic lumbar puncture] and/or clinical signs of CNS leukemia such as a cranial nerve palsy from active disease). Subjects with adequately treated CNS leukemia are eligible
2. Hyperleukocytosis (≥ 50,000 blasts/μL) or rapidly progressive disease that in the estimation of the investigator and sponsor would compromise ability to complete study therapy
3. Pregnancy (negative serum or urine pregnancy test must be obtained at time of enrollment for females of childbearing potential and to be repeated 72 hours prior to lymphodepleting chemotherapy regimen)
4. Breast feeding
5. Sexually active female subjects of childbearing potential and male subjects who are of childbearing potential and are unwilling to practice birth control at time of enrollment and for four months after receiving the lymphodepletion preparative regimen
6. Active or uncontrolled viral, bacterial or fungal infection. May be receiving ongoing therapy for controlled infection
7. Treatment with any prior CAR-T therapy product

8. Recent prior therapy:

   1. Systemic chemotherapy ≤ 2 weeks

   2. TKI ≤ 2 weeks

      • Exceptions: 6 weeks for gemtuzumab or other CD33-targeted antibody, clofarabine or nitrosoureas;
      • Exception: Subjects who are on azacytidine/decitabine may be enrolled provided that chemotherapy is discontinued at least 1 week prior to apheresis)
      • There is no time restriction in regard to prior intrathecal chemotherapy for initial enrollment, however, IT therapy should be performed > 72 hours prior to apheresis:
      • Steroid therapy is not allowed, unless at or below physiologic replacement doses;
      • Subjects receiving hydroxyurea may be enrolled provided there has been no increase in dose for at least 2 weeks prior to apheresis or study enrollment if an apheresis product was previously collected;
      • Any anti-neoplastic investigational agents or monoclonal antibody therapy should not be given within 30 days prior to apheresis (i.e. start of protocol therapy);
   3. For radiation therapy: Radiation therapy must have been completed at least 3 weeks prior to apheresis or enrollment if an apheresis product was previously collected, with the exception that there is no time restriction if the volume of bone marrow treated is less than 10% and also the subject has measurable/evaluable disease outside the radiation port; Any CNS radiation will require a 3-week washout prior to enrollment
   4. For prior stem cell transplant: Subjects with a history of more than one prior stem cell transplant will not be eligible.

9. Subjects with a history of a single allogeneic stem cell transplantation are excluded if:

   1. Subjects are less than 100 days post-transplant OR
   2. Subjects have evidence of ongoing active GVHD and are taking immunosuppressive agents (>0.5 mg/kg/methylprednisolone equivalents or other immunosuppression for GVHD treatment) OR
   3. Subjects have received DLI within 30 days prior to enrollment OR
   4. Subjects are on active immunosuppression for GVHD prophylaxis (must be off for 30 days prior to enrollment)

10. HIV/HBV/HCV Infection:

    1. Seropositive for HIV 1 or 2 (Subjects with HIV are at increased risk of lethal infections when treated with marrow-suppressive therapy. Appropriate studies will be undertaken in subjects receiving combination antiretroviral therapy in the future should study results indicate effectiveness)
    2. Seropositive for Hepatitis C or positive for Hepatitis B surface antigen (HbsAG)
11. Uncontrolled, symptomatic, intercurrent illness including but not limited to infection, congestive heart failure, unstable angina pectoris, cardiac arrhythmia, psychiatric illness, or social situations that would limit compliance with study requirements or in the opinion of the site PI would pose an unacceptable risk to the subject

12. Active second malignancy will not be eligible with the following exceptions:

    1. Treatment-related or secondary CD33+ myeloid malignancy which may potentially benefit from CD33CART (which may be considered for enrollment),
    2. Carcinoma in situ of the cervix (which may be considered for enrollment),
    3. Subject is in remission from a prior second malignancy (which may be considered for enrollment).
13. History of severe, immediate hypersensitivity reaction attributed to compounds of similar chemical or biologic composition to any agents used in study or in the manufacturing of the cells (i.e. gentamicin).

• National Marrow Donor Program
• St. Baldrick's Foundation