CAR T Cell Receptor Immunotherapy for Patients With B-cell Lymphoma

BACKGROUND:

- We have constructed a retroviral vector that encodes an anti-cluster of differentiation
19 (CD19) chimeric antigen receptor (CAR) that recognizes the CD19 antigen. This
chimeric receptor also contains the signaling domains of cluster of differentiation 28
(CD28) and cluster of differentiation 3 (CD3)-zeta. The retroviral vector can be used to
mediate genetic transfer of this CAR to T cells with high efficiency (> 50%) without the
need to perform any selection.

- In co-cultures with CD19-expressing target cells, anti-CD19-CAR-transduced T-cells
secreted significant amounts of interferon gamma (IFN-y) and interleukin 2 (IL-2).

- We have developed a process for cryopreserving the cell product which may lead to the
ability for this product to be manufactured at a central location and shipped to other
institutions for treatment of a broader patient population

OBJECTIVE:

- Primary objective:

--With the approval of amendment S, to determine the safety and feasibility of the
administration of cryopreserved anti-CD19-CAR engineered peripheral blood lymphocytes with a
non-myeloablative conditioning regimen in patients with Bcell lymphomas.

ELIGIBILITY:

Patients of 18 years of age or older must:

- Have a CD19-expressing B-cell lymphoma

- Be a non-responder to, or recurred after one or more standard chemotherapy-containing
regimens for their malignancy

- Currently require treatment due to progressive malignancy

- Be deemed to be incurable by standard therapy

Patients may not have:

- A history of allogeneic stem cell transplantation

- Central nervous system (CNS) disease

DESIGN:

- Peripheral blood mononuclear cells (PBMC) obtained by leukapheresis (approximately 5.0x
10^9 cells) will be cultured in the presence of anti-CD3 (muromonab-CD3 (OKT3)) and
aldesleukin in order to stimulate T-cell proliferation.

- Transduction is initiated by exposure of approximately 1.0x 10^8 to 5.0x 10^8 cells to
retroviral vector supernatant containing the anti-CD19 CAR.

- With the approval of Amendment S, patients will receive fludarabine and cyclophosphamide
chemotherapy (NMA) for lymphodepletion, followed by cryopreserved
anti-CD19-CAR-transduced T-cells.

- Patients will be followed until disease progression.

- Patients who have responded to treatment and then progress may receive one retreatment.

- INCLUSION CRITERIA:

1. Patient must have a cluster of differentiation 19 (CD19)-expressing B-cell
lymphoma. Patients with diffuse large B-cell lymphoma, primary mediastinal B-cell
lymphoma, and diffuse large B-cell lymphoma transformed from follicular lymphoma
must have measurable disease after at least two prior chemotherapy regimens one
of which must have contained doxorubicin and rituximab.

2. Confirmation of diagnosis of B-cell malignancy and positivity for CD19 confirmed
by the Laboratory of Pathology of the National Cancer Institute (NCI). The choice
of whether to use flow cytometry or immunohistochemistry will be determined by
what is the most easily available tissue sample in each patient.
Immunohistochemistry will be used for lymph node biopsies, flow cytometry will be
used for peripheral blood, fine needle aspirates and bone marrow samples.

3. Patients must have indications for treatment for their B-cell malignancy at the
time of enrollment on this trial.

4. Greater than or equal to 18 years of age and less than or equal to age 70.

5. Willing to sign a durable power of attorney.

6. Able to understand and sign the Informed Consent Document.

7. Clinical performance status of Eastern Cooperative Oncology Group (ECOG) 0 or 1.

8. Life expectancy of greater than three months.

9. Patients of both genders must be willing to practice birth control from the time
of enrollment on this study and for four months after treatment.

10. Women of child bearing potential must have a negative pregnancy test because of
the potentially dangerous effects of the treatment on the fetus.

11. Serology:

- Seronegative for human immunodeficiency virus (HIV) antibody. (The
experimental treatment being evaluated in this protocol depends on an intact
immune system. Patients who are HIV seropositive can have decreased immune
-competence and thus be less responsive to the experimental treatment and
more susceptible to its toxicities.).

- Seronegative for hepatitis B antigen and hepatitis C antibody unless antigen
negative. If hepatitis C antibody test is positive. Then patients must be
tested for the presence of antigen by reverse transcription-polymerase chain
reaction (RT-PCR) and be hepatitis C virus ribonucleic acid (HCV RNA)
negative.

12. Hematology:

- Absolute neutrophil count greater than or equal to 1000/mm^3 without the
support of filgrastim.

- Platelet count greater than or equal to 50,000/mm^3.

- Hemoglobin greater than 8.0 g/dl.

- Lymphocyte count less than or equal to 4,000/ mm^3

13. Chemistry:

- Serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) less
or equal to 5 times the upper limit of normal.

- Serum creatinine less than or equal to 1.6 mg/dl

- Total bilirubin less than or equal to 1.5 mg/dl, except in patients with
Gilbert's Syndrome who must have a total bilirubin less than 3.0 mg/dl

14. More than three weeks must have elapsed since any prior systemic therapy at the
time the patient receives the preparative regimen, and patient toxicities must
have recovered to a grade 1 or less (except for toxicities such as alopecia or
vitiligo).

15. Normal cardiac ejection fraction and no evidence of pericardial effusion as
determined by an echocardiogram.

EXCLUSION CRITERIA:

1. Patients that require urgent therapy due to tumor mass effects such as bowel
obstruction or blood vessel compression.

2. Patients that have active hemolytic anemia.

3. Patients with active brain metastases, or with a history of any central nervous system
(CNS) metastases or cerebrospinal fluid malignant cells.

Note: patients who are asymptomatic but are found to have malignant cells in the
cerebrospinal fluid (CSF) on lumbar puncture prior to treatment will be considered
eligible.

4. Women of child-bearing potential who are pregnant or breastfeeding because of the
potentially dangerous effects of the treatment on the fetus or infant.

5. Active systemic infections, coagulation disorders or other major medical illnesses of
the cardiovascular, respiratory or immune system, myocardial infarction, cardiac
arrhythmias, obstructive or restrictive pulmonary disease.

6. Any form of primary immunodeficiency (such as Severe Combined Immunodeficiency
Disease).

7. Concurrent opportunistic infections (The experimental treatment being evaluated in
this protocol depends on an intact immune system. Patients who have decreased immune
competence may be less responsive to the experimental treatment and more susceptible
to its toxicities).

8. Concurrent systemic steroid therapy.

9. History of severe immediate hypersensitivity reaction to any of the agents used in
this study.

10. History of allogeneic stem cell transplantation

11. Patients with cardiac atrial or cardiac ventricular lymphoma involvement.

Screening Evaluation:

Within 4 weeks prior to starting the chemotherapy regimen:

1. Complete history and physical examination, including, weight and vital signs, noting
in detail the exact size and location of any lesions that exist. (Note: patient
history may be obtained within 8 weeks.)

2. Chest x-ray

3. Electrocardiography (EKG)

4. Baseline computed tomography (CT) of the chest, abdomen and pelvis, positron emission
tomography (PET) scan, and brain magnetic resonance imaging (MRI) to evaluate the
status of disease. Additional scans and x-rays may be performed if clinically
indicated based on patient signs and symptoms.

5. HIV antibody titer and Hepatitis B surface antigen (HbsAG) determination, and anti
HCV, (Note: May be performed within 3 months of the chemotherapy start date).

6. Anti cytomegalovirus (CMV) antibody titer, herpes simplex virus (HSV) serology, and
Epstein-Barr virus (EBV) panel (Note: patients who are known to be positive for any of
the above do not need to be retested; may be performed within 3 months of chemotherapy
start date)

7. Patients with a left ventricular ejection fraction (LVEF) of less than or equal to 55%
will not proceed to treatment (Note: may be performed within 8 weeks of treatment).

8. Cluster of differentiation 19 (CD19) staining of malignant cells by
immunohistochemistry or flow cytometry (testing is permitted to be conducted at any
time prior to this point).

9. All patients must have a T cells, B cells, and natural killer cells (TBNK) for
Peripheral blood cluster of differentiation 3 (CD3) count and CD19#.

10. Patients with a history of leptomeningeal disease, or signs/symptoms suggestive of
leptomeningeal involvement, or with symptoms of central nervous system malignancy such
as new onset severe headaches, neck stiffness, or any focal neurologic findings on
physical exam will have lumbar puncture for examination of cerebral spinal fluid.

11. Patients may undergo lumbar puncture (LP) for flow cytometry of the CSF in order to
assess the presence of CD19 positive lymphocytes for potential correlation with
neurologic toxicity. Patients who have no neurologic symptoms at the time of LP will
be eligible for enrollment regardless of the results of the flow cytometry.

Within 14 days prior to starting the chemotherapy regimen:

12. Chem 20: (Sodium (Na), Potassium (K), Chloride (Cl), Total carbon dioxide (CO2)
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin, Calcium total,
Magnesium total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/glutamic pyruvic
transaminase (GPT), AST/glutamic oxaloacetic (GOT), Total Bilirubin, Direct Bilirubin,
lactate hydrogenase (LD), Total Protein, Total creatine kinase (CK), Uric Acid)

13. Thyroid panel

14. Complete blood count (CBC) with differential and platelet count

15. Prothrombin time (PT)/partial thromboplastin time (PTT)

16. Urinalysis and culture, if indicated

Within 7 days prior to starting the chemotherapy regimen:

17. Beta-human chorionic gonadotropin (βHCG) pregnancy test (serum or urine) on all women
of child-bearing potential

18. ECOG performance status of 0 or 1