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11.2.2 filgrastim (granulocyte colony-stimulating factor

Filgrastim will be obtained commercially by the Clinical Center Pharmacy Department and is supplied in 300 ug/mL and 480 ug/1.6 mL vials. G-CSF should be refrigerated and not allowed to freeze. The product bears the expiration date. The product should not be shaken. It is generally stable for at least 10 months when refrigerated. The appropriate dose is drawn up into a syringe. G-CSF will be given as a daily subcutaneous injection. The side effects of G-CSF are skin rash, myalgia and bone pain, an increase of preexisting inflammatory conditions, enlarged spleen with occasional associated low platelet counts, alopecia (with prolonged use) elevated blood chemistry levels.
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11.2.3 trimethoprim and sulfamethoxazole double

TMP/SMX DS will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used for the prevention of PCP pneumonia. The oral dose is 1 tablet PO daily three times a week (MUST be on non-consecutive days) beginning on day -7 and continuing for at least 6 months and until the CD4 count is greater than 200 on 2 consecutive lab studies. Like other sulfa drugs, TMP/SMX DS can cause allergies, fever, photosensitivity, nausea, and vomiting. Allergies typically develop as a widespread itchy red rash with fever eight to fourteen days after beginning the standard dose. Neutropenia, a reduction in the number of neutrophils, can also occur.
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11.2.3.1 aerosolized pentamidine in place

Patients with sulfa allergies will receive aerosolized Pentamidine 300 mg per nebulizer with one week prior to admission and continued monthly until the CD4 count is above 200 on two consecutive follow up lab studies and for at least 6 months post chemotherapy. Pentamidine Isethionate will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used to prevent the occurrence of PCP infections. It is supplied in 300 mg vials of lyophilized powder and will be administered via nebulizer. Toxicities reported with the use of Pentamidine include metallic taste, coughing, bronchospasm in heavy smokers and asthmatics; increased incidence of spontaneous pneumothorax in patients with previous PCP infection or pneumatoceles, or hypoglycemia.
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11.2.4 herpes virus prophylaxis 11.2.4.1

Valacyclovir will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used orally to prevent the occurrence of herpes virus infections in patients with positive HSV serology. It is supplied in 500 mg tablets. Valcyclovir will be started the day after the last dose of fludarabine at a dose of 500 mg orally daily if the patient is able to tolerate oral intake. See package insert for dosing adjustments in patients with renal impairment. Common side effects include headache, upset stomach, nausea, vomiting, diarrhea or constipation. Rare serious side effects include hemolytic uremic syndrome and thrombotic thrombocytopenic purpura.
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11.2.4.2 acyclovir

Acyclovir will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used to prevent the occurrence of herpes virus infections in patients who cannot take oral medications. It is supplied as powder for injection in 500 mg/vials. Reconstitute in 10 mL of sterile water for injection to a concentration of 50 mg/mL. Reconstituted solutions should be used within 12 hours. IV solutions should be diluted to a concentration of 7mg/mL or less and infused over 1 hour to avoid renal damage. Reversible renal insufficiency has been reported with IV but not oral acyclovir. Neurologic toxicity including delirium, tremors, coma, acute psychiatric disturbances, and abnormal EEGs have been reported with higher doses of acyclovir. Should this occur, a dosage adjustment will be made or the drug will be discontinued. Stomach upset, headache or nausea, rash or hives; peripheral edema; pain, elevated liver function tests; and leukopenia, diarrhea, lymphadenopathy, myalgias, visual abnormalities and elevated creatinine have been reported. Hair loss from prolonged use has been reported. Acyclovir will not be used concomitantly with other nucleoside analogs which interfere with DNA synthesis, e.g. ganciclovir. In renal disease, the dose is adjusted as per product labeling.
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11.2.5 fluconazole

Fluconazole will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used to prophylax against fungal infections. It is available in 200 mg tablets. It can cause headache, nausea, vomiting, diarrhea or abdominal pain, and liver damage which may be irreversible. It can cause rashes and itching, which in rare cases has caused Stevens Johnson Syndrome. It has several significant drug interactions. The package insert should be consulted prior to prescribing. For IV administration in patients who cannot tolerate the oral preparation, Fluconazole comes in 2 mg/mL solution for injection, and prepared according to Clinical Center Pharmacy standard procedures. It should be administered at a maximum IV rate of 200 mg/hr. 11.2.6 Ondansetron Hydrochloride Ondansetron hydrochloride will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used to control nausea and vomiting during the chemotherapy preparative regimen. It can cause headache, dizziness, myalgias, drowsiness, malaise, and weakness. Less common side effects include chest pain, hypotension, pruritis, constipation and urinary retention. Consult the package insert for specific dosing instructions.
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11.2.7 allopurinol

Allopurinol will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used as prophylaxis or treatment of patients with or at high risk for Tumor Lysis Syndrome. Dosage is approximately 100 mg/m2/dose po TID (maximum dose 200 mg TID). The most common side effects include hypersensitivity, rash, nausea, vomiting, renal insufficiency, and hepatic dysfunction. Allopurinol should be stopped immediately if rash develops. Consult the package insert for a complete list of all side effects.
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12 references

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44. Nicholson IC, Lenton KA, Little DJ, Decorso T, Lee FT, Scott AM, et al. Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma. Mol Immunol. 1997;34(16-17):1157-65. 45. Maher J, Brentjens RJ, Gunset G, Riviere I, Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor. Nat Biotechnol. 2002;20:70-5. 46. Hermans IF, Silk JD, Yang J, Palmowski MJ, Gileadi U, McCarthy C, et al. The VITAL assay: a versatile fluorometric technique for assessing CTL- and NKT-mediated cytotoxicity against multiple targets in vitro and in vivo. J Immunol Methods. 2004;285(1):25-40. 47. Cooper LJ, Topp MS, Serrano LM, Gonzalez S, Chang WC, Naranjo A, et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versusB lineage leukemia effect. Blood. 2003;101:1637-44. 48. Jensen MC, Popplewell L, DiGiusto D, Kalos M, Raubitschek A, Forman SJ. A First-InHuman Clinical Trial of Adoptive Therapy Using CD19-Specific Chimeric Antigen Receptor Re-Directed T-Cells for Recurrent/Refractory Follicular Lymphoma. ASH Annual Meeting Abstracts. 2007;110(11):288. 49. Cheadle EJ, Hawkins RE, Batha H, Rothwell DG, Ashton G, Gilham DE. Eradication of established B-cell lymphoma by CD19-specific murine T cells is dependent on host lymphopenic environment and can be mediated by CD4+ and CD8+ T cells. J Immunother. 2009;32(3):20718. 50. Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U, et al. Adoptive cell therapy for patients with metastatic melanoma: Evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol. 2008;26:5233-9.  
51. James SE, Orgun NN, Tedder TF, Shlomchik MJ, Jensen MC, Lin Y, et al. Antibodymediated B-cell depletion before adoptive immunotherapy with T cells expressing CD20-specific chimeric T-cell receptors facilitates eradication of leukemia in immunocompetent mice. Blood.  
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13 tables figures and appendices

| Data as of 09/30/2008  Cellular  Study #/                                                                                                                                                               | Arms (n)                                                                                                          | Chemotherapy, Cytokines,                                |               |           |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------|---------------|-----------|
| Product                                                                                                                                                                                                 | and Immunizations                                                                                                 | # of Cells                                              | Response      | Reference |
| Disease                                                                                                                                                                                                 | Administered                                                                                                      | 1. Cloned PBL/TIL  intravenously (12)                   | None          |           |
| Cloned  Peripheral  Blood  Lymphocytes  (PBL)/ Tumor  infiltrating  lymphocytes  (TIL)                                                                                                                  | 2. IV Cloned                                                                                                      | SQ IL-2 (125,000 IU/kg/d X                              |               |           |
| PBL/TIL  intravenously (6a )                                                                                                                                                                            | 12 d)                                                                                                             |                                                         |               |           |
| 98-C-0095  in patients  with  metastatic  melanoma                                                                                                                                                      | 1.5 to  34.8x109                                                                                                  | NRd                                                     | Dudley, ME,   |           |
| cells                                                                                                                                                                                                   | et al. 2001                                                                                                       |                                                         |               |           |
| 3. IV Cloned                                                                                                                                                                                            | HD IL-2 (720,000 IU/kg                                                                                            |                                                         |               |           |
| PBL/TIL                                                                                                                                                                                                 | 3X/d to tolerance (max 12)                                                                                        |                                                         |               |           |
| intravenously (6b )  4. IV Cloned  PBL/TIL (3c )                                                                                                                                                        | Gp100:209-217(210M)                                                                                               |                                                         |               |           |
| 1. In vitro expanded  cloned T-cells  intravenously (3)                                                                                                                                                 | 30 mg/kg cyclophosphamide  X2 days, 25 mg/kg  fludarabine X5 days                                                 | 0.9x109 to 24.2x109  cells (avg.  10.4x109)             | NR            |           |
| 2. In vitro expanded  cloned T-cells  intravenously (3)                                                                                                                                                 | 60 mg/kg cyclophosphamide  X2 days, 25 mg/kg                                                                      | NR                                                      |               |           |
| fludarabine X5 days 60 mg/kg cyclophosphamide  X2 days, 25 mg/kg  fludarabine X5 days plus IV  IL-2 (72,000 IU/kg 3X/day  X5 days)                                                                      | 0.9x109 to 24.2x109  cells (avg.  10.4x109)                                                                       |                                                         |               |           |
| 3. In vitro expanded  cloned T-cells  intravenously (3)                                                                                                                                                 | Dudley, ME,  et al. 2002                                                                                          |                                                         |               |           |
| NR                                                                                                                                                                                                      |                                                                                                                   |                                                         |               |           |
| [60 mg/kg  cyclophosphamide X2 days,  25 mg/kg fludarabine X5  days]g plus IV IL-2 (720,000  IU/kg 3X/day to tolerance  (max 12)                                                                        |                                                                                                                   |                                                         |               |           |
| 99-C-0158  in patients  with  metastatic  melanoma                                                                                                                                                      | Tumor  infiltrating  lymphocytes  (TIL)                                                                           | 4. In vitro expanded  cloned T-cells  intravenously (6) | NR            |           |
| Chemotherapyg plus IV HD  IL-2 [(720,000 IU/kg 3X/day  to tolerance (max 15)]h with  or without immunization  with gp100:209-217(210M)  or MART-1:26-35(27L) in  Montanide ISA-51 QD X5,  then Qwk X3. | 18/35  (51%)f  3 CRs 15 PRs                                                                                       |                                                         |               |           |
| 5. Autologous tumor  reactive REP'de bulk  TIL cells  intravenously (35)                                                                                                                                | 1.1 to  16.0x1010  cells (avg.  6.3x1010)                                                                         | Dudley, ME,  et al. 2003,  Dudley, ME,  et al. 2005     |               |           |
| 6. Autologous tumor  reactive REP'de bulk  TIL cells  intravenously (6)                                                                                                                                 | Chemotherapyg plus Low  Dose IL-2k with or without  immunization with  gp100:209-217(210M)  peptide or MART-1:26- | 8.3x109 to 52.9x109  cells (avg.  33.7x109)             | 3PR 2NR 1 TEj |           |  
| Version Date: August 23, 2018                                                                                     | 35(27L) peptide in  Montanide ISA-51 QD X5,  then Qwk X3. Chemotherapyg plus IV HD  IL-2h with 1mg gp100:209- 217(210M) in Montanide  ISA-51 QD X5, then Qwk  X3   |                                                 |               |       |
|-------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------|---------------|-------|
| 1. Escalating doses  of anti-gp100 TCR  transduced PBL (3)                                                        | Up to  30x109                                                                                                                                                        | NR                                              |               |       |
| 04-C-0181  in patients  with  metastatic  melanoma                                                                | Chemotherapyg plus IV HD  IL-2h with 6X109 pfu  rFgp100P209 IV;  rFgp100P209 IV and IL-2  repeated 28 days later.                                                    |                                                 |               |       |
| Gp100 TCR  engineered Tcells (PBL or  TIL)                                                                                                                   | 2. Anti-gp100 TCR  CD8+ enriched PBL                                                                                                                                 | NA                                              |               |       |
| (8)                                                                                                               | Up to  30x109                                                                                                                                                        | NR                                              |               |       |
| Chemotherapyg plus IV HD  IL-2h with 6X109 pfu  rFgp100P209 IV;  rFgp100P209 IV and IL-2  repeated 28 days later. |                                                                                                                                                                      |                                                 |               |       |
| 3. Escalating doses  of anti-gp100 TCR  transduced TIL (3)                                                        | Up to  30x109                                                                                                                                                        | 1 PRi                                           |               |       |
| 1. Escalating doses  of anti-MART-1  TCR transduced  PBL (18)                                                     | Chemotherapyg plus IV HD  IL-2h with MART-1:26-                                                                                                                      | Up to  30x109                                   | 2PRsi         |       |
| 35(27L) in Montanide ISA51 QD X5, then Qwk X3.                                                                                                                   |                                                                                                                                                                      |                                                 |               |       |
| 2. Escalating doses  of anti-MART-1  CD8+ TCR  transduced PBL (6)                                                 | Chemotherapyg plus IV HD  IL-2h with MART-1:27-35 in  Montanide ISA-51 QD X5,  then Qwk X3                                                                          | Up to  30x109                                   | 1 PRi         |       |
| 04-C-0251  in patients  with  metastatic  melanoma                                                                | 3. Escalating doses  of anti-MART-1  CD8+ TCR  transduced PBL (8)                                                                                                    | Chemotherapyg plus IV HD  IL-2h with MART-1:26- | Up to  30x109 | 1 PRi |
| 35(27L) in Montanide ISA51 QD X5, then Qwk X3                                                                                                                   |                                                                                                                                                                      |                                                 |               |       |
| MART-1 TCR  engineered Tcells (PBL or  TIL)                                                                                                                   | Morgan, et  al. Science,  2006 Oct  6;314(5796):  126-9                                                                                                              |                                                 |               |       |
| 4. Escalating doses  of anti-MART-1  TCR transduced TIL  (3)                                                      | Chemotherapyg plus IV HD  IL-2h with MART-1:26-                                                                                                                      | Up to  30x109                                   | NR            |       |
| 35(27L) in Montanide ISA51 QD X5, then Qwk X3                                                                                                                   |                                                                                                                                                                      |                                                 |               |       |
| 5. Escalating doses  of anti-MART-1  TCR transduced  PBL plus 1200 TBI  (4)                                       | Chemotherapyg and 1200  TBI plus IV HD IL-2h with  MART-1:26-35(27L) in  Montanide ISA-51 QD X5,  then Qwk X3                                                       | Up to  30x109                                   | NR            |       |
| 1. Melanoma or  renal cell cancer: Up  to 50 X 109 cells  (PBL) (2)                                               |                                                                                                                                                                      |                                                 |               |       |
| 07-C-0003  in  melanoma/ renal cell  or other  histologies                                                        | Chemotherapyg plus IV HD                                                                                                                                             | Up to  50x109                                   | NR            |       |
| IL-2h                                                                                                             |                                                                                                                                                                      |                                                 |               |       |
| Anti-p53 TCR  engineered Tcells (PBL)                                                                                                                   | 2. Other histologies:                                                                                                                                                | NA                                              |               |       |
| Chemotherapyg plus IV HD                                                                                          | 1 PRi                                                                                                                                                                |                                                 |               |       |
| Up to 50 X 109 cells                                                                                              | Up to                                                                                                                                                                |                                                 |               |       |
| IL-2h                                                                                                             | 50x109                                                                                                                                                               |                                                 |               |       |
| (PBL) (10)                                                                                                        |                                                                                                                                                                      |                                                 |               |       |  
| Anti-gp100(154)  TCR transduced  PBL at a dose  ranging from 0.5 X  109 cells up to 300  x109 (19)      |                                              |                                                |       |    |
|---------------------------------------------------------------------------------------------------------|----------------------------------------------|------------------------------------------------|-------|----|
| 07-C-0174 in patients  with  metastatic  melanoma                                                       | Antigp100(154)  TCR  engineered Tcells (PBL)                                              | 1.7x109 to 110.0x109  cells (avg.  31.5x109)   | 3 PR  |    |
| Chemotherapyg plus IV HD  IL-2h                                                                         | 14 NR                                        | NA                                             |       |    |
| 2 TEj                                                                                                   |                                              |                                                |       |    |
| Anti-MART-1 F5  TCR transduced  PBL at a dose  ranging from 0.5 X  109 cells up to 300  x109 cells (21) |                                              |                                                |       |    |
| 07-C-0175 in patients  with  metastatic  melanoma                                                       | Anti-MART-1  F5 TCR  engineered Tcells (PBL or  TIL)                                              | 1.45x109  to 110.0x109  cells (avg.  34.2x109) | 6 PR  |    |
| Chemotherapyg plus IV HD  IL-2h                                                                         | 12 NR                                        | NA                                             |       |    |
| 3 TEj                                                                                                   |                                              |                                                |       |    |
| Anti-MART-1 F5  TCR transduced TIL  at a dose ranging from 0.5 X 109 cells  up to 300 x109 cells        | Chemotherapyg plus IV HD                     | 84x109  cells                                  | 1 TE  | NA |
| IL-2h                                                                                                   | 3 PR                                         |                                                |       |    |
| 07-C-0176 in patients  with  metastatic  melanoma                                                       | 11.1x109  to 124x109  cells (avg.  55.6x109) |                                                |       |    |
| Young TIL at a dose  ranging from 1.0 X                                                                 |                                              |                                                |       |    |
| Young TIL                                                                                               | 109 cells up to 300  x109 cells (26)         | Chemotherapyg plus IV HD  IL-2h                | 19 NR | NA |
| 2 TEj  2 NTj                                                                                            |                                              |                                                |       |    |
| a Previously treated with cells alone                                                                   |                                              |                                                |       |    |  
b Five previously treated with cells alone, one new patient for first cell treatment c All three patients had previously been treated with cell alone and cells with IL-2 d NR: No Response e Rapid Expansion Protocol f Responses defined: CR (complete response) is disappearance of all clinical evidence of disease; PR
(partial response) defined as > 50% reduction in the sum of the products of the perpendicular diameters for at least one month and no increase in any lesion and no new lesions.  
g Chemotherapy at maximum dose: 60 mg/kg cyclophosphamide X2 days, 25 mg/kg fludarabine X5 days h High dose (HD) IL-2: 720,000 IU/kg 3X/day to tolerance (max 15)
i Responses defined according to RECIST criteria j TE: Too early to evaluate, NT: not treated k Low dose IL-2: 250,000 IU/kg subcutaneously daily for 5 days. After a two day rest, IL-2 will be administered at a dose of 125,000 IU/kg subcutaneously daily for 5 days for the next five weeks (2 days rest per week).  
13.2 TABLE 2: TIME IN HOSPITAL AND NON-HEMATOLOGICAL GRADE 3 AND 4 **TOXICITIES**
RELATED **TO LYMPHODEPLETING CHEMOTHERAPY AND CELL TRANSFER**  
| Attribute measured                                          | Duration, Number or Type   | Number of Patients (%)   |
|-------------------------------------------------------------|----------------------------|--------------------------|
| Days in Hospital1                                           | 6-10                       | 6 (17%)                  |
| 11-15                                                       | 18 (51%)                   |                          |
| 16-20                                                       | 4 (11%)                    |                          |
| 21-25                                                       | 7 (20%)                    |                          |
| pRBC Transfusions                                           | 0                          | 2 (6%)                   |
| 1-5                                                         | 18 (51%)                   |                          |
| 6-10                                                        | 13 (37%)                   |                          |
| 11-15                                                       | 2(6%)                      |                          |
| Platelet Transfusions                                       | 0                          | 6 (17%)                  |
| 1-5                                                         | 21 (60%)                   |                          |
| 6-10                                                        | 5 (14%)                    |                          |
| 11-15                                                       | 2 (6%)                     |                          |
| 16-20                                                       | 1 (3%)                     |                          |
| Autoimmunity                                                | Uveitis                    | 5 (14%)                  |
| Vitiligo                                                    | 13 (37%)                   |                          |
| Opportunistic Infections                                    | Herpes zoster              | 3 (9%)                   |
| Pneumocystis pneumonia                                      | 2 (6%)                     |                          |
| EBV-B cell lymphoma                                         | 1 (3%)                     |                          |
| RSV pneumonia                                               | 1 (3%)                     |                          |
| Other                                                       | Febrile neutropenia        | 13 (37%)                 |
| Intubated for dyspnea                                       | 3 (9%)                     |                          |
| Cortical blindness                                          | 1 (3%)                     |                          |
| 1 Measured from the day of cell administration to discharge |                            |                          |  
  
  
13.4 F IGURE 1: N ORMAL PBMC CAN BE T RANSDUCED WITH AN A NTI - CD19 CAR
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13.5 f igure 2 pbmc

| CD19+ targets         | CD19 negative targets   | Effectors   |       |      |      |     |      |       |    |
|-----------------------|-------------------------|-------------|-------|------|------|-----|------|-------|----|
| SupB15                | NALM6                   | Toledo      | 624   | A549 | TC71 | CEM | K562 | alone |    |
| Effector cells        |                         |             |       |      |      |     |      |       |    |
| Anti-CD19 CAR         | 14800                   | 15150       | 15150 | 38   | 19   | 63  | 8    | 24    | 4  |
| SP6 Control CAR       | 23                      | 35          | 36    | 83   | 124  | 46  | 22   | 184   | 14 |
| Donor 1 Nontransduced | 13                      | 54          | 39    | 7    | 5    | 7   | 16   | 8     | 3  |  
Figure 3. PBMC that were transduced with an anti-CD19 CAR specifically recognize CD19-expressing target cells. 100,000 effector cells were cultured overnight with 100,000 target cells, and an interferon- γ ELISA was performed. All values are pg/mL of interferon- γ
(mean of duplicate wells). Effector cells were either anti-CD19-CAR-transduced T-cells, T-cells transduced with the control CAR SP6, or non-transduced T-cells from the same patient stimulated with OKT3 in the same manner. SupB15 and NALM6 are CD19-expressing leukemia cell lines, Toledo is a CD19-expressing large cell lymphoma cell line. 624, A549, TC71, CEM
and K562 are immortalized CD19 negative cell lines. The control CAR SP6 is specific for the chemical 2,4,6-TNP.  
13.6 F IGURE 3: T-C ELLS FROM A CLL P ATIENT P OST F LUDARABINE /R ITUXIMAB T HERAPY  
| A              | CD19+ targets   | CD19 negative targets   | Effectors         |     |      |       |
|----------------|-----------------|-------------------------|-------------------|-----|------|-------|
| Effector cells | CLL1            | CLL2                    | Allogeneic T cell | CEM | K562 | alone |
| anti-CD19      | 72,000          | 47,000                  | 1945              | 32  | 490  | 24    |
| CR-transduced  |                 |                         |                   |     |      |       |
| Nontransduced  | 132             | 345                     | 305               | 36  | 52   | 18    |
| B              |                 |                         |                   |     |      |       |  
  
Figure 4. T-cells from a CLL patient post fludarabine/rituximab therapy can be transduced with an anti-CD19 CAR and then produce interferon-γ in response to a primary allogeneic CLL cells and kill primary allogeneic CLL cells. A. 100,000 effector cells were cultured overnight with 100,000 target cells, and an interferon- γ ELISA was performed. All values are pg/mL of interferon- γ (mean of duplicate wells). Effector cells were either anti-CD19- CAR-transduced T-cells from a CLL patient or non-transduced T-cells from the same patient stimulated with OKT3 in the same manner. CLL1 and CLL2 are primary CLL cells from two different patients. Allogeneic T-cell targets were 7 days post OKT3 stimulation. CEM and K562 are immortalized CD19- negative leukemia cell lines. B. The same effector cell cultures were tested for their ability to kill allogeneic primary CLL cells. Effector cells transduced with the anti-CD19 CAR killed the CLL cells, while non-transduced T-cells from the same donor that were cultured identically did not kill CLL cells.
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13.7 f igure 4 a

| A               | CD19+ targets   | CD19 negative targets   | Effectors   |        |     |      |      |     |       |
|-----------------|-----------------|-------------------------|-------------|--------|-----|------|------|-----|-------|
| Effector cells  | bv173           | SupB15                  | CLL3        | MDA231 | 624 | TC71 | A549 | CEM | alone |
| Anti-CD19 CAR   | 16070           | 7758                    | 108         | 37     |     |      |      |     |       |
| 2321            | 343             | 65                      | 20          | 20     |     |      |      |     |       |
| SP6 Control CAR | 78              | 16                      | 27          | 3044   | 342 | 60   | 198  | 15  | 10    |  
  
Figure 5. Following rapid expansion to a number of cells sufficient for clinical adoptive T-cell transfer, anti-CD19-transduced T-cells maintain function. A.  
100,000 effector cells that were 9 days post REP initiation were cultured overnight with 100,000 target cells, and an interferon- γ ELISA was performed. All values are pg/mL of interferon- γ (mean of duplicate wells). Effector cells were either anti-CD19-CAR-
transduced T-cells or T-cells transduced with the control CAR SP6 from the same patient stimulated with OKT3 in the same manner. SupB15 and bv173 are CD19-expressing leukemia cell lines, CLL3 are primary CLL cells. MDA231, A549, TC71, CEM and K562 are immortalized CD19 negative cell lines. The control CAR SP6 is specific for the chemical 2,4,6-TNP. B Twenty-one days after the initiation of rapid expansion, anti- CD19 CAR transduced T-cells can kill allogeneic primary CLL cells while T-cells transduced with the control CAR SP6 cannot.  
13.8   FIGURE 5: T-CELLS PRODUCE IFN-GAMMA AND IL-2 IN RESPONSE TO CD-19-EXPRESSING TARGET CELLS FOLLOWING
RAPID EXPANSION  
  
  
Figure 6. Fourteen days after initiation of rapid expansion a large percentage of T-cells produce IFN7 (Figure 6A)
and IL-2 (Figure 6B) specifically in response to CD19-expressing target cells. Anti-CD19-CAR-transduced or controltransduced T-cells were cultured with K562 cell transduced with either full-length CD19 or nerve growth factor receptor
(NGFR) for 5 hours and then intracellular cytokine staining was performed. Control-transduced T-cells were transduced with the SP6 CAR. The SP6 CAR is described in the legend for Figure 3. The K562 target cells were identical except that CD19-
K562 cells were transduced with a retrovirus encoding full-length CD19 and NGFR-K562 cells were transduced with retroviruses encoding NGFR.  
B
13.9 F IGURE 6: A NTI -CD19 -CAR -T RANSDUCED T -C ELLS D EPENDENT U PON E XOGENOUS IL -
2 FOR IN VITRO SURVIVAL
A
Donor 1
+/- IL-2 comparison was started on dav B  
  
  
staining on the indicated days.
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13.11 f igure 8 c

13.12 FIGURE 9: TOTAL CELL NUMBER OF PBMC FOLLOWING TRANSDUCTION IN CLOSED VS
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open system

13.13 F IGURE 10: C ELL E XPANSION OF PBMC F OLLOWING T RANSDUCTION IN C LOSED VS
OPEN SYSTEM  
  
  
13.14 F IGURE 11: C OMPARISON OF IFN - GAMMA R ELEASE OF C ELLS T RANSDUCED AND
EXPANDED IN CLOSED VS OPEN SYSTEM  
  
Figure 12: Comparison of T-Cell Phenotype for PBMC Transduced and Expanded in Closed vs Open System  
  
13.15 F IGURE 13: C OMPARISON OF P ERCENTAGE AND V IABILITY OF CAR+ T-C ELLS
MANUFACTURED IN 6-DAY CLOSED PROCESS  
  
Figure 14. Comparison of the percentage and viability of CAR+ T cells manufactured in a 6 day closed process. PBMC were stimulated, transduced and expanded in bags for a total of 6 days and then cryopreserved in cryobags at a density of 8.0 x 106 cells/ml in 50ml saline-2.5% HSA-5% DMSO. A) The percentage of CD19 CAR+ T cells was measured by FACS on day 6 prior to cryopreservation and then again on day 3 post-thaw for two patients. There was no significant difference in the level of CAR+ T cells after cryopreservation (patient 1, 37.6% pre and 48.3% post; patient 2, 38.5% pre and 51.0% post).  B) The 13.16 F IGURE 14: C OMPARISON OF IFN - GAMMA R ELEASE OF CAR+ T - C ELLS
MANUFACTURED IN 6-D AY C LOSED P ROCESS AFTER C RYOPRESERVATION  
  
13.17 F IGURE 15: C OMPARISON OF V IABILITY AND F OLD E XPANSION OF CAR+ T - C ELLS
MANUFACTURED IN 6-D AY C LOSED P ROCESS AFTER C RYOPRESERVATION  
  
13.18 F IGURE 16: P HENOTYPE OF CD19 CAR+ T-C ELLS M ANUFACTURED IN 6-D AY C LOSED
PROCESS BEFORE AND AFTER CRYOPRESERVATION  
  
Figure 17. Phenotype of CD19 CAR+ T cells manufactured in a 6 day closed process before and after cryopreservation. PBMC were stimulated, transduced and expanded in bags for a total of 6 days and cryopreserved.  After thaw (day 0), the cells were expanded in Optimizer plus 2.5% TCSR and 50 IU/ml IL-2 for 3 days. T cell phenorype was measured by FACS following staining with anti-CD45RA and anti-CCR7 antibodies. While there are slight changes in the percentages of the cell subsets represent pre- and post-
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13.19 appendix 1 certificate of

Date of preparation of final product: Patient:  
| Tests performed on final product:                                                                                                | Number of days in culture:   |                                                     |        |                |
|----------------------------------------------------------------------------------------------------------------------------------|------------------------------|-----------------------------------------------------|--------|----------------|
| Test                                                                                                                             | Method                       | Limits                                              | Result | Initials/ Date |
| Cell viability1                                                                                                                  | trypan blue exclusion        | >70%                                                |        |                |
| CAR positive  cells/patient weight  (kg)                                                                                         | Calculated Value             | Between 0.2x107 and  1.0x107 CAR positive  cells/kg |        |                |
| Tumor reactivity2                                                                                                                | γ-IFN release vs. CD19-      | >200 pg/mL                                          |        |                |
| expressing target cell lines                                                                                                     |                              |                                                     |        |                |
| Microbiological                                                                                                                  | gram stain1                  | no micro-organisms                                  |        |                |
| studies                                                                                                                          | seen                         |                                                     |        |                |
| aerobic culture3                                                                                                                 | no growth                    |                                                     |        |                |
| fungal culture3                                                                                                                  | no growth                    |                                                     |        |                |
| anaerobic culture3                                                                                                               | no growth                    |                                                     |        |                |
| mycoplasma test4                                                                                                                 | negative                     |                                                     |        |                |
| Endotoxin                                                                                                                        | limulus assay1               | <5 E.U./kg                                          |        |                |
| RCR                                                                                                                              | S+L- Assay3                  | negative                                            |        |                |
| RCR-PCR5                                                                                                                         |                              |                                                     |        |                |
| 1 Performed on sample of the final product immediately prior to cryopreservation. Results are available at the time of infusion. |                              |                                                     |        |                |  
1 Performed on sample of the final product immediately prior to cryopreservation. Results are available at the time of infusion.  
Total viable cell number is dependent on CAR+ cells patient's weight.  
2 Performed 2-10 post transduction. Results are available at the time of infusion.  
3 Sample collected from the final product prior to cryopreservation. Results will not be available before cells are infused into the patient.  
4 Performed 2-10 days prior to infusion. Results are available at the time of infusion.  
5 A sample will be saved from the final product prior to cryopreservation. If the culture period after retroviral transduction is less than 96 hours the sample will be archived. If the culture period exceeds 96 hours after retroviral transduction, the assay will be performed and results will be available at the time of cell infusion.  
Prepared by: _________________________________________ Date: ____________________ QC sign-off: __________________________________________ Date: ____________________
Qualified laboratory or Clinical Supervisor
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13.20 appendix 2 description of

This trial will consist of two phases. In phase I, a dose escalation of the number of anti-CD19- CAR-transduced T-cells will be carried out. All patients in this phase of the trial will be lymphodepleted with fludarabine and cyclophosphamide, and all patients will receive aldesleukin (720,000 IU/kg every 8 hours for a maximum of 15 doses). Patients with platelet counts between 75,000 and 99,999 cells/mm3 will receive half the cyclophosphamide dose that patients with platelet counts of 100,000/mm3 or greater will receive. After a maximum tolerated cell dose is determined in phase I of the trial, phase II of the trial will begin. Starting with approval of Amendment E (to be discontinued with approval of Amendment F), this trial is being refocused to evaluate the safety and feasibility of administering between 1.0x109 and 1.0x1010 anti CD19-CAR transduced cells and aldesleukin (720,000 IU/kg every 8 hours for a maximum of 15 doses) following a preparative conditioning regimen of fludarabine and cyclophosphamide. We have accrued 4 patients at this dose level with one patient experiencing a DLT. If we observe no more DLTs in the next 2 patients treated for a total of 1/6 patients treated, accrual will continue up to 18 patients. If at any time during accrual a cumulative total of 3 evaluable patients have experienced a DLT, then no further patients will be enrolled. If DLTs are encountered in up to 3/18 patients, the trial will be considered safe and feasible. A one-week safety assessment period will follow regimen completion (defined as the last dose of aldesleukin) between each patient in the first 6 patients and a two-week safety assessment period will follow between the 6th and 7th patient before expansion of the trial to 18 patients. With approval of Amendment F (to be discontinued with the approval of Amendment H), the trial is being refocused to evaluate the safety and toxicity of the regimen studied at lower escalating dose levels since 3 DLTs were observed at dose level 1 (between 1.0x109 and 1.0x1010 cells). The dose of cells administered will be on a CAR positive cells/kg basis. If a DLT is observed at the revised dose levels, the dose of aldesleukin may be reduced to 72,000 IU/kg every 8 hours for a maximum of 15 doses.
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phase i - dose escalation

The initial portion of this protocol will be a phase I dose escalation design with three cohorts of a minimum of 3 patients per cohort. The number of anti-CD19-transduced T-cells transferred for each cohort will be:  
| Cohort 1   | between      | 1.0x109  and 1.0x1010   |
|------------|--------------|-------------------------|
| Cohort 2   | greater than | 1.0x1010 up to 3.0x1010 |
| Cohort 3   | greater than | 3.0x1010 up to 1.0x1011 |  
Patients will be enrolled sequentially, therefore enrollment will not proceed to a higher dose level until all patients have been treated in the prior cohort. If sufficient cells cannot be grown to meet the criteria for the assigned cohort, the patient will be enrolled in the appropriate cohort for the number of cells infused. If a DLT occurs in an additional patient entered at a lower dose due to cell growth limitations, accrual will continue at this level as described in the dose-escalation scheme below. Accrual will be halted at the higher level until the cohort at the lower level is complete as described below. Should a single patient experience a dose limiting toxicity at a particular dose level, three more patients would be treated at that dose to confirm that no greater than 1/6 patients have a DLT prior to proceeding to the next higher level. If a level with 2 or more DLTs in 3-6 patients has been identified, three additional patients will be accrued at the next-lowest dose for a total of 6, in order to further characterize the safety of the maximum tolerated dose prior to starting the phase II portion of the trial. After the maximum tolerated dose is determined, the phase II portion of the trial will begin. A one-week safety assessment period will follow regimen completion (defined as the last dose of aldesleukin) between each patient in a cohort except for the last patient in each cohort for which a two-week safety assessment period will follow regimen completion (defined as the last dose of aldesleukin) before a patient will be accrued to the next cohort. Upon completion of the Phase I portion of the study, the adverse events observed in this cohort will be reported to and evaluated by the IRB before proceeding to the Phase II portion.
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phase ii - randomization to

In phase II of the trial, patients will be randomized to two treatment arms. Patients assigned treatment arm 1 will receive fludarabine and cyclophosphamide lymphodepleting chemotherapy prior to receiving the engineered PBL cells while patients assigned to treatment arm 2 will not receive chemotherapy. Randomization will also be stratified so that an equal number of patients with circulating malignant cells (leukemia) will be entered on each arm of the phase II part of the trial. One to four days after completion of chemotherapy, anti-CD19 CAR-transduced T-cells will be infused. After cell infusion, IV aldesleukin therapy will be initiated (720,000 IU/kg every 8 hours for a maximum of 15 doses). The randomization performed during phase II of this trial will be stratified so that each arm will receive an equal number of patients with a total peripheral blood CD3 count greater than or equal to 500 cells/microliter, and each arm will receive an equal number of patients with a CD3 count less than 500 cells/microliter. In addition, the randomization will be stratified so that each arm will receive an equal number of patients with peripheral blood involvement with malignant cells. Patients with peripheral blood involvement with malignant cells are defined as patients with pre-treatment peripheral blood lymphocyte counts greater than 4000 lymphocytes per microliters. During both phases multiple immunologic assays will be carried out on peripheral blood T-cells to determine persistence of anti-CD19-CAR-transduced T-cells as well as their function. During phase II an assessment of the anti-malignancy effect of anti-CD19-CAR-transduced T-cells will be made. All patients will be followed for anti-malignancy response, survival and toxicity.
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dose escalation and cohort expansion

With approval of Amendment F, the trial is being refocused to evaluate the safety and toxicity of the regimen studied at lower escalating dose levels since 3 DLTs were observed at dose level 1
(between 1.0x109 and 1.0x1010 cells). For cohort 2, 3 patients will be treated with NMA, 0.5x107 CAR positive cells/kg (± 20%), and high dose aldesleukin (720,000 IU/kg). This starting dose of cells has been selected due to PK modeling of interferon gamma levels and toxicity data from the first 8 patients treated on this study. If one DLT is observed in a patient treated in Cohort 2, after analysis of the IFN-gamma cytokine data, 3 additional patients will be treated at this dose level
(0.5x107 CAR positive cells/kg (± 20%) after receiving NMA followed by a reduced dose of aldesleukin (72,000 IU/kg every 8 hours for a maximum of 15 doses) (Cohort 3). This 10-fold reduction of aldesleukin was previously investigated in a study of patients with renal cancer. The incidence of grade 3 and 4 aldesleukin related toxicities was less in patients treated with 72,000 IU/kg every 8 hours than those treated with the higher dose of 720,000 IU/kg aldesleukin, every 8 hours (Yang, J.C., et al., J Clin Oncol. 2003, 21(16):3127-32). If no DLTs are observed in the 3 patients in Cohort 2, after analysis of the IFN-gamma cytokine data, 3 additional patients will be treated with NMA, 1.0x107 CAR positive cells/kg (± 20%) and high dose aldesleukin (Cohort 4). If one DLT is observed in a patient treated in Cohort 4, 3 additional patients will be treated at this dose level (1.0x107 CAR positive cells/kg (± 20%) after receiving NMA followed by a reduced dose of aldesleukin (72,000 IU/kg every 8 hours for a maximum of 15 doses) (Cohort 5).  
| Cohort                                                        | Treatment Regimen NMA Chemotherapy Regimen   | Cell Dose                     | IL-2 Dose        |
|---------------------------------------------------------------|----------------------------------------------|-------------------------------|------------------|
| (as specified in protocol)                                    |                                              |                               |                  |
| 1 (Closed with                                                | Yes                                          | between 1.0x109  and 1.0x1010 | High dose        |
| Amendment F)                                                  | cells                                        | (720,000 IU/kg)               |                  |
| 2                                                             | Yes                                          | 0.5x107 CAR positive cells/kg | High dose        |
| (± 20%)                                                       | (720,000 IU/kg)                              |                               |                  |
| 31                                                            | Yes                                          | 0.5x107 CAR positive cells/kg | Low dose (72,000 |
| (± 20%)                                                       | IU/kg)                                       |                               |                  |
| 4                                                             | Yes                                          | 1.0x107 CAR positive cells/kg | High dose        |
| (± 20%)                                                       | (720,000 IU/kg)                              |                               |                  |
| 52                                                            | Yes                                          | 1.0x107 CAR positive cells/kg | Low dose (72,000 |
| (± 20%)                                                       | IU/kg)                                       |                               |                  |
| 1  Cohort 3 will be skipped if there are no DLTs in Cohort 2. |                                              |                               |                  |  
2 Cohort 5 will be skipped if there are no DLTs in Cohort 4.  
The highest dose cohort evaluated with no DLTs in 3 patients (either 3, 4 or 5) will be expanded to a total of 18 evaluable patients, subject to not exceeding 3 patients in total with a DLT.
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safety assessment

Cytokine (IFN-gamma levels) and toxicity data must be analyzed prior to dose escalation. A oneweek safety assessment period will follow regimen completion (defined as the last dose of aldesleukin) between each patient in a cohort except for the last patient in each cohort for which a two-week safety assessment period will follow regimen completion (defined as the last dose of aldesleukin) before a patient will be accrued to the next cohort. A two-week safety assessment period will follow between the 3th and 4th patient before expansion of the trial to 18 patients.
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dose escalation and cohort expansion

With Amendment H, cohorts 2-5 will be closed and two new cohorts will be opened at the same cell dose level as cohorts 2 and 4 but without aldesleukin. These cohorts will be designated cohort 6 and 7. For Cohort 6, 3 patients will be treated with NMA, and 0.5x107 CAR positive cells/kg (± 20%). If one DLT is observed in a patient treated in Cohort 6, after analysis of the IFN-gamma cytokine data, accrual will be halted pending discussions with the FDA and IRB. In November 2011, the first patient in Cohort 6 experienced a DLT of grade 3 confusion (possible aphasia), and grade 3 neuropathy (facial droop) possibly related to the cell therapy. The serum IFN-gamma levels were low in this patient (range 9-44 pg/mL) following treatment with cells. These events were discussed with the FDA, and the FDA indicated that we can treat an additional patient in Cohort 6 (with approval of Amendment I). If this additional patient does not experience at DLT, Cohort 6 will be expanded to a total of 6 patients. If any additional patients treated in Cohort 6 experiences a DLT, after analysis of the IFN-gamma cytokine data, accrual will be halted pending discussions with the FDA and IRB. If no additional DLTs are observed in the 6 patients in Cohort 6, after analysis of the IFN-gamma cytokine data, 3 patients will be treated with NMA, and 1.0x107 CAR positive cells/kg (± 20%)
in Cohort 7. If one DLT is observed in a patient treated in Cohort 7, 3 additional patients will be accrued to Cohort 7. If 2 DLTs occur in 6 or less patients in Cohort 7, no additional patients will be accrued at this dose level.  
| Cohort                                                                                       | Treatment Regimen NMA Chemotherapy Regimen   | Cell Dose                           | IL-2 Dose          |
|----------------------------------------------------------------------------------------------|----------------------------------------------|-------------------------------------|--------------------|
| (as specified in protocol)                                                                   |                                              |                                     |                    |
| 1 (Closed with                                                                               | Yes                                          | between 1.0x109  and 1.0x1010 cells | High dose (720,000 |
| Amendment F)                                                                                 | IU/kg)                                       |                                     |                    |
| 2 (Closed with                                                                               | Yes                                          | 0.5x107 CAR positive cells/kg (±    | High dose (720,000 |
| Amendment H)                                                                                 | 20%)                                         | IU/kg)                              |                    |
| 3 (Closed with                                                                               | Yes                                          | 0.5x107 CAR positive cells/kg (±    | Low dose (72,000   |
| Amendment H)                                                                                 | 20%)                                         | IU/kg)                              |                    |
| 4 (Closed with                                                                               | Yes                                          | 1.0x107 CAR positive cells/kg (±    | High dose (720,000 |
| Amendment H)                                                                                 | 20%)                                         | IU/kg)                              |                    |
| 5 (Closed with                                                                               | Yes                                          | 1.0x107 CAR positive cells/kg (±    | Low dose (72,000   |
| Amendment H)                                                                                 | 20%)                                         | IU/kg)                              |                    |
| 6 (Closed with                                                                               | Yes                                          | 0.5x107 CAR positive cells/kg (±    | None               |
| Amendment J)                                                                                 | 20%)                                         |                                     |                    |
| 7 (Closed with                                                                               | Yes                                          | 1.0x107 CAR positive cells/kg (±    | None               |
| Amendment J)                                                                                 | 20%)                                         |                                     |                    |
| The highest dose cohort evaluated with no DLTs in 3 or more patients, or 1 DLT in 6 patients |                                              |                                     |                    |  
The highest dose cohort evaluated with no DLTs in 3 or more patients, or 1 DLT in 6 patients will be expanded to a total of 18 evaluable patients, subject to not exceeding 3 patients in total with a DLT.
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safety assessment

Cytokine (IFN-gamma levels) and toxicity data must be analyzed prior to dose escalation. A oneweek safety assessment period will follow regimen completion between each patient in a cohort except for the last patient in each cohort for which a two-week safety assessment period will follow regimen completion before a patient will be accrued to the next cohort. A two-week safety assessment period will follow the last patient treated in the dose escalation cohort before expansion of the trial to 18 patients.
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dose de-escalation and cohort expansion

In February 2012, the second patient in Cohort 6 experienced DLTs. The SAEs consisted of grade 3 hypotension and grade 3 confusion (possibly aphasia) related to the cell therapy. These toxicities have resolved and the patient has been discharged. After discussions with the FDA, cohort 6 and 7 were closed and two new dose de-escalation cohorts were opened as follows:
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13.21 appendix 3 description of

PBMC will be obtained by leukapheresis (approximately 1.0x1010 cells). In most patients, whole PBMC will be cultured in the presence of anti-CD3 (OKT3) and aldesleukin in order to stimulate T-cell growth. In some patients the peripheral blood might be contaminated with large numbers of CD19-expressing malignant cells. In patients with peripheral blood lymphocytes counts greater than 4000 cells per microliter, CD19-expressing cells will be depleted using the Miltenyi Biotec CliniMacs system with anti-CD19 microbeads and then CD19-depleted PBMC will be stimulated with OKT3. In all patients, 2 days after OKT3 stimulation transduction will be initiated by exposure of approximately 1.0x107 to 5.0x108 cells to supernatant containing the anti-CD19 CAR retroviral vector. These transduced cells will be expanded and tested for their anti-tumor activity. Successful TCR gene transfer will be determined by FACS analysis for the CAR and specific anti-tumor reactivity will be tested by cytokine release measured against CD19+ cell lines and CD19-negative cell lines. Successful CAR gene transfer for each transduced PBL population will be defined as >30% CAR positive cells and for biological activity, gamma-interferon secretion against CD19 targets must be at least 200 pg/mL. The cells administered vary depending on their growth characteristic. In prior protocols over 3.0x1011 Tcells have been safely infused to cancer patients.  
Due to toxicities experienced during the dose escalation phase of this trial, the protocol has been amended several times to decrease the dose and eliminate the administration of IL-2. The description of these changes has been moved to **Appendix 2**.  
Beginning with Amendment M patients will be lymphodepleted with fludarabine and a reduced dose of cyclophosphamide - 30mg/kg/day for 2 days and will receive 1.0x106CAR positive cells/kg (± 20%) as noted below.  
| Cohort                 | Treatment Regimen NMA Chemotherapy Regimen (as   | Cell Dose                        | IL-2 Dose   |
|------------------------|--------------------------------------------------|----------------------------------|-------------|
| specified in protocol) |                                                  |                                  |             |
| 8                      | Yes                                              | 2.5x106 CAR positive cells/kg (± | None        |
| (De-escalation Dose 1) | 20%)                                             |                                  |             |
| 9                      | Yes                                              | 1.0x106 CAR positive cells/kg (± | None        |
| (De-escalation Dose 2) | 20%)                                             |                                  |             |
| 10                     | Yes - dose of both agents reduced                | 1.0x106 CAR positive cells/kg (± | None        |
| (De-escalation Dose 2) | 20%)                                             |                                  |             |  
Cohort 8 will initially accrue 3 patients. If no DLTs are observed in these 3 patients, three additional patients will be accrued. If DLTs are observed in ≤ 1 patient out of 6 patients treated, this cohort will be expanded as described below. If a DLT is observed in one of the first 3 patients, after analysis of the cytokine data, 3 additional patients can be accrued to this cohort. If more than 1 out of 6 patients in this cohort experience a DLT, this cohort will be closed, and patients will be accrued to cohort 9. Cohort 8 was closed with Amendment M. Cohort 9 will initially accrue 3 patients. If no DLTs are observed in these 3 patients, three additional patients will be accrued. If DLTs are observed in ≤ 1 patient out of 6 patients treated, this cohort will be expanded as described below. If a DLT is observed in one of the first 3 patients, after analysis of the cytokine data, 3 additional patients can be accrued to this cohort. If more than 1 out of 6 patients in this cohort experience a DLT, this cohort will be closed.  
Subsequent dose reductions will be discussed with the FDA prior to initiation. If ≤ 1 patient in 6 patients treated develops a DLT then the 12 remaining patients will be enrolled in cohort 10 for a total of 18 evaluable patients at a dose level of 1.0x106 CAR positive cells/kg (± 20%), subject to not exceeding 3 patients in total with a DLT. Following administration of the cell product, neurological status will be closely monitored and urgently managed as described in Section **3.4.3**.  
In addition, we will be evaluating stored serum samples in an effort to identify the particular cytokines which may be contributing to these toxicities.
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safety assessment

Cytokine (IFN-gamma levels) and toxicity data must be analyzed prior to dose de-escalation. A one-week safety assessment period will follow regimen completion between each patient in a cohort except for the last patient in each cohort for which a two-week safety assessment period will follow regimen completion before a patient will be accrued to the next cohort. Beginning with Amendment R, the one week safety assessment period between patients will no longer be required during the expansion phase of the protocol for the remaining patients to be enrolled in Cohort 10. The study will be halted pending discussions with the FDA and IRB if the following conditions are met:
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prior to amendment e

- If two DLTs occur in the first cohort of the Phase I portion of this study.  
- Two or more patients develop a grade 3 or greater toxicity at any point in the study not attributable to the chemotherapy preparative regimen (or circumstances unrelated to this study).  
- If 1 of the first 3 patients (OR 2 of the first 6 patients, OR 3 of the first 9 patients, OR 4 of the first 12 patients) develop DLT due to autoimmune toxicity.  
- Development of EBV lymphoma or polyclonal lymphoproliferative disease (PLPD) in an EBV negative subject on this or any other SB adoptive cell therapy study. Accrual of EBV negative subjects will be halted only.
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with approval of amendment e

- If two DLTs occur in the first 6 patients treated on this study.  
- Two or more patients develop a grade 3 or greater toxicity at any point in the study not attributable to the chemotherapy preparative regimen (or circumstances unrelated to this study).  
- If 1 of the first 3 patients (OR 2 of the first 6 patients, OR 3 of the first 9 patients, OR 4 of the first 12 patients) develop DLT due to autoimmune toxicity.  
- Development of EBV lymphoma or polyclonal lymphoproliferative disease (PLPD) in an EBV negative subject on this or any other SB adoptive cell therapy study. Accrual of EBV negative subjects will be halted only.
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with approval of amendments f

As stated above, the study will be halted pending discussions with the FDA and IRB if the following conditions are met:
- If cohorts 2-7 or 9 cannot be expanded due to DLTs.  
- During the expansion phase, if at any time during accrual, a cumulative total of 3 evaluable patients have experienced a DLT.  
- Two or more patients develop a grade 3 or greater toxicity at any point in the study not attributable to the chemotherapy preparative (or circumstances unrelated to this study).  
- If 1 of the first 3 patients (OR 2 of the first 6 patients, OR 3 of the first 9 patients, OR 4 of the first 12 patients) in cohorts 2-5 develop DLT due to autoimmune toxicity.  
- Development of EBV lymphoma or polyclonal lymphoproliferative disease (PLPD) in an EBV negative subject on this or any other SB adoptive cell therapy study. Accrual of EBV negative subjects will be halted only.
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13.22 appendix 4 modification of

Beginning with Amendment M, actual body weight will be used for dose calculations of all treatment agents.  
1. BMI Determination:
BMI = weight (kg) / [height (m)]2 2. Calculation of ideal body weight:
Male = 50 kg + 2.3 (number of inches over 60 inches)
Example: Ideal body weight of 5'10" male 50 + 2.3 (10) = 73 kg Female = 45.5 kg + 2.3 (number of inches over 60 inches)
Example: Ideal body weight of 5'3" female 45.5 + 2.3 (3) = 57 kg 3. Calculation of "practical weight":
Calculate the average of the actual and the ideal body weights. This is the practical weight to be used in calculating the doses of chemotherapy and associated agents designated in the protocol.  
*Practical weight will NOT be used in the calculation of dose for aldesleukin.