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7.1.9 unanticipated problems any incident

x Is unexpected in terms of nature, severity, or frequency in relation to
(a) the research risks that are described in the IRB-approved research protocol and informed consent document; Investigator's Brochure or other study documents, and (b) the characteristics of the subject population being studied; AND
x Is related or possibly related to participation in the research; AND
x Places subjects or others at a g*reater risk of harm* (including physical, psychological, economic, or social harm) than was previously known or recognized
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7.2 nci-irb reporting 7.2.1 nci-irb

The PI will provide a summary report of adverse events that have occurred on the protocol since the previous continuing review and in aggregate, and interpreted in relation to the risk:benefit of study participants in the narrative. The protocol PI will report to the NCI-IRB:
1. All Grade 2 **unexpected** events that are possibly, probably or definitely related to the research; 2. All Grade 3 and 4 events that are possibly, probably or definitely related to the research; 3. All Grade 5 events regardless of attribution; 4. All Serious Events regardless of attribution. NOTE: Grade 1 events are not required to be reported.
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7.2.3 nci-irb reporting of ind

Only IND Safety Reports that require a sponsor recommended change to the protocol or the consent form or in the opinion of the PI increases risks to study participants will need to be reported to the NCI IRB.
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7. 3. 1 seri o

T he Pri nci pal I n vesti gat or will n otif y O B A/I B C vi a e mail of a n y u n e x pecte d fatal or life-t hreat e ni n g e x perie nce ass o ciate d wit h t he use of a nti- N Y- E S O 1 T C R-tra ns d uce d P B L as s o o n as p ossi ble b ut i n n o e ve nt later t ha n 7 cale n dar da ys of i nitial recei pt of t he i nf or mati o n. Seri o us a d verse e ve nts t hat are u ne x pecte d a n d ass ociate d wit h t he use of t he a nti- N Y- E S O 1 T C R-tra ns d uce d P B L, b ut are n o fatal or life-t hreat e ni n g, m uc h be re p orte d t o NIH O B A/I B C as s o o n as p ossi ble, b ut n ot later t ha n 1 5 cale n dar da ys after t h e s p o ns or's i nitial recei pt of t he i nf or mati o n.
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7. 3. 2 a n

T he st u d y Pri nci pal I n vesti gat or will s u b mit a brief re p ort a n n uall y of t he pr o gr ess of t he tri al wit hi n 6 0 da ys of t he a n ni versar y dat e t hat t he I N D we nt i nt o effect u nless t he I N D s p o ns or has bee n a ut h orize d t o s u b mit t his re p ort, as per NI H G ui deli nes, A p pe n di x M.
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7. 4 seri o us

re p orti n g a n y seri o us a n d u ne x pecte d a d vers e e v e nts t o t he U. S. F o o d a n d Dr u g A d mi nistrati o n  
( F D A) wit h a p pr o pri ate dea dli nes defi n e d b y t he re g ulati o ns:  
T he F D A, O B A a n d I B C will be i nf or me d b y p h o ne or fa x of a n y u n e x pecte d fatal or life t hreate ni n g s us pect e d a d verse r eacti o n as s o o n as p ossi ble b ut i n n o e ve nt, later t ha n 7 cale n dar da ys after t h e s p o ns or's i nitial recei pt of t he i nf or mati o n; T he F D A, O B A a n d I B C will be i nf or me d of a n y ot her u ne x pecte d a n d s eri o us s us pecte d a d verse reacti o n b y writte n n otificati o n wit hi n 1 5 cale n dar da ys after t he s p o ns or's i nitial recei pt of t he i nf or mati o n.  
All a d verse e ve nts, i ncl u di n g seri o us a d vers e e ve nts, will be re p orte d i n t he a n n ual r e p ort t o t he F D A, O B A, a n d I B C as i n dicate d i n 2 1 C F R 3 1 2. 3 3.
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h o w s u

Cli nical P har mac y De p art me nt.  
F or m ulati o n/ Rec o nstit uti o n: Al desl e u ki n, N S C \# 3 7 3 3 6 4, is pr o vi de d as si n gle- us e vials c o ntai ni n g 2 2 milli o n I U (  
off- w hite l y o p hilize d ca ke pl us 5 0 m g ma n nit ol a n d 0. 1 8 m g s o di u m d o dec yl s ulf ate, b uffer e d wit h a p pr o xi matel y 0. 1 7 m g m o n o basic a n d 0. 8 9 m g di basic s o di u m p h os p hate t o a p H of 7. 5 (ra n ge 7. 2 t o 7. 8). T he vial is rec o nstit ute d wit h 1. 2 m L of Sterile Water f or I njecti o n, U S P, a n d t he res ulta nt c o nce ntr ati o n is 1 8 milli o n I U/ ml or 1. 1 m g/ m L. Dil ue nt s h o ul d be directe d a g ai nst t he si de of t he vial t o a v oi d e x cess f oa mi n g. S wirl c o nt e nts ge ntl y u ntil c o m pletel y diss ol ve d. D o n ot s ha ke. Si nce vials c o ntai n n o preser vati ve, rec o nstit ute d s ol uti o n s h o ul d be use d wit h 2 4 h o urs.  
St ora ge: I ntact vials are st ore d i n t he refri ger at or ( 2 - 8 ° C) pr otecte d fr o m li g ht. Eac h vial b ears a n e x pirati o n date.  
Dil uti o n/ Sta bilit y: Rec o nstit ute d al desle u ki n s h o ul d be f urt her dil ute d wit h 5 0 m L of 5 % H u ma n Ser u m Al b u mi n ( H S A). T he H S A s h o ul d be a d de d t o t he dil ue nt prior to the addition of aldesleukin. Dilutions of the reconstituted solution over a 1000-fold range (i.e., 1 mg/mL to 1 mcg/mL) are acceptable in either glass bottles or polyvinyl chloride bags. Aldesleukin is chemically stable for 48 hours at refrigerated and room temperatures, 20 - 30°C.  
Administration: The final dilution of aldesleukin will be infused over 15 minutes. Aldesleukin will be administered as an inpatient.  
Toxicities: Expected toxicities of aldesleukin are listed in the product label and in Appendix 1 and 2. Grade 3 toxicities common to aldesleukin include diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or constitutional symptoms and laboratory changes as detailed in Appendix 1. Additional grade 3 and 4 toxicities seen with aldesleukin are detailed in Appendix 2.  
8.2. Fludarabine Description: (Please refer to package insert for complete product Information)
Fludarabine phosphate is a synthetic purine nucleoside that differs from physiologic nucleosides in that the sugar moiety is arabinose instead of ribose or deoxyribose. Fludarabine is a purine antagonist antimetabolite.  
How Supplied: It will be purchased by the NIH Clinical Pharmacy Department from commercial sources. Fludarabine is supplied in a 50 mg vial as a fludarabine phosphate powder in the form of a white, lyophilized solid cake.  
Stability: Following reconstitution with 2 mL of sterile water for injection to a concentration of 25 mg/ml, the solution has a pH of 7.7. The fludarabine powder is stable for at least 18 months at 2-8°C; when reconstituted, fludarabine is stable for at least 16 days at room temperature. Because no preservative is present, reconstituted fludarabine will typically be administered within 8 hours. Specialized references should be consulted for specific compatibility information. Fludarabine is dephosphorylated in serum, transported intracellularly and converted to the nucleotide fludarabine triphosphate; this 2-fluoro-ara-ATP molecule is thought to be required for the drug's cytotoxic effects. Fludarabine inhibits DNA polymerase, ribnucleotide reductase, DNA primase, and may interfere with chain elongation, and RNA and protein synthesis.  
Storage: Intact vials should be stored refrigerated (2-8°C).  
Administration: Fludarabine is administered as an IV infusion in 100 ml 0.9% sodium chloride, USP over 15 to 30 minutes. The doses will be based on body surface area (BSA). If patient is obese (BMI > 35) drug dosage will be calculated using practical weight as described in Table 10.  
Toxicities: At doses of 25 mg/m2/day for 5 days, the primary side effect is myelosuppression; however, thrombocytopenia is responsible for most cases of severe and life-threatening hematologic toxicity. Serious opportunistic infections have occurred in CLL patients treated with fludarabine. Hemolytic anemia has been reported after one or more courses of fludarabine with or without a prior history of a positive Coomb's test; fatal hemolytic anemia has been reported. In addition, bone marrow fibrosis has been observed after fludarabine therapy. Other common adverse effects include malaise, fever, chills, fatigue, anorexia, nausea and vomiting, and weakness. Irreversible and potentially fatal central nervous system toxicity in the form of progressive encephalopathy, blindness, and coma is only rarely observed at the currently administered doses of fludarabine. More common neurologic side effects at the current doses of fludarabine include weakness, pain, malaise, fatigue, paresthesia, visual or hearing disturbances, and sleep disorders. Adverse respiratory effects of fludarabine include cough, dyspnea, allergic or idiopathic interstitial pneumonitis. Tumor lysis syndrome has been rarely observed in fludarabine treatment of CLL. Treatment on previous adoptive cell therapy protocols in the Surgery Branch have caused persistently low (below 200) CD4 counts, and one patient developed polyneuropathy manifested by vision blindness, and motor and sensory defects.  
8.3. Cyclophosphamide
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(refer to fda-approved package insert

agent. Following conversion to active metabolites in the liver, cyclophosphamide functions as an alkyating agent; the drug also possesses potent immunosuppressive activity. The serum half-life after IV administration ranges from 3-12 hours; the drug and/or its metabolites can be detected in the serum for up to 72 hours after administration.  
How Supplied: Cyclophosphamide will be obtained from commercially available sources by the Clinical Center Pharmacy Department.  
Stability: Following reconstitution as directed with sterile water for injection, cyclophosphamide is stable for 24 hours at room temperature or 6 days when kept at 2-8°C.  
Administration: It will be diluted in 250 ml D5W and infused over one hour.  
The dose will be based on the patient's body weight. If patient is obese (BMI > 35) drug dosage will be calculated using practical weight as described in Table 10.  
Toxicities: Hematologic toxicity occurring with cyclophosphamide usually includes leukopenia and thrombocytopenia. Anorexia, nausea and vomiting, rash and alopecia occur, especially after high-dose cyclophosphamide; diarrhea, hemorrhagic colitis, infertility, and mucosal and oral ulceration have been reported. Sterile hemorrhagic cystitis occurs in about 20% of patients; severity can range from microscopic hematuria to extensive cystitis with bladder fibrosis. Although the incidence of hemorrhagic cystitis associated with cyclophosphamide appears to be lower than that associated with ifosfamide, mesna (sodium 2mercaptoethanesulfonate) has been used prophylactically as a uroprotective agent in patients receiving cyclophosphamide. Prophylactic mesna is not effective in preventing hemorrhagic cystitis in all patients. Patients who receive high dose cyclophosphamide may develop interstitial pulmonary fibrosis, which can be fatal. Hyperuricemia due to rapid cellular destruction may occur, particularly in patients with hematologic malignancy. Hyperuricemia may be minimized by adequate hydration, alkalinization of the urine, and/or administration of allopurinol. If allopurinol is administered, patients should be watched closely for cyclophosphamide toxicity (due to allopurinol induction of hepatic microsomal enzymes). At high doses, cyclophosphamide can result in a syndrome of inappropriate antidiuretic hormone secretion; hyponatremia with progressive weight gain without edema occurs. At high doses, cyclophosphamide can result in cardiotoxicity. Deaths have occurred from diffuse hemorrhagic myocardial necrosis and from a syndrome of acute myopericarditis; in such cases, congestive heart failure may occur within a few days of the first dose. Other consequences of cyclophosphamide cardiotoxicity include arrhythmias, potentially irreversible cardiomyopathy, and pericarditis. Other reported adverse effects of cyclophosphamide include headache, dizziness, and myxedema; faintness, facial flushing, and diaphoresis have occurred following IV administration. Mesna (sodium 2-mercaptoethanesulphonate; given by IV injection) is a synthetic sulfhydryl compound that can chemically interact with urotoxic metabolites of cyclophosphamide (acrolein and 4-hydroxycyclophosphamide) to decrease the incidence and severity of hemorrhagic cystitis.
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8.4. mesna (sodium 2-mercaptoethanesulfonate mesnum

Description: Mesna will be obtained commercially by the Clinical Center Pharmacy Department and is supplied as a 100 mg/ml solution. Storage: Intact ampoules are stored at room temperature.  
Stability: Diluted solutions (1 to 20 mg/mL) are physically and chemically stable for at least 24 hours under refrigeration. Mesna is chemically stable at room temperature for 48-72 hours in D5W, 48-72 hour in D5W/0.45% NaCl, or 24 hours in 0.9% NaCl.  
Administration: Dilute to concentrations less than or equal to 20 mg mesna/ml fluid in D5W or 0.9% NaCl and to be administered intravenously as a continuous infusion. If patient is obese (BMI > 35) drug dosage will be calculated using practical weight as described in Table 10. Toxicities include nausea, vomiting and diarrhea.
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8.5. 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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8.6. trimethoprim and sulfamethoxazole double

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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8.6.1. 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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8.7. herpes virus prophylaxis

8.7.1. Valacyclovir (Valtrex)
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.  
8.7.2. 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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8.8. 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.  
8.9. Anti-ESO TCR transduced PBL
The procedure for expanding the human PBL and the Certificate of Analysis are similar to those approved by the Food and Drug Administration, and used at the NCI in ongoing protocols evaluating cell therapy in the Surgery Branch (most recently 07-C-0003, 07-C-0174, 07-C-0175). The CoA is included in Appendix 4. The PBL will be transduced with retroviral supernatant containing the alpha chain and beta chain genes of the anti-ESO TCR.
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8.9.1. retroviral vector containing the

The retroviral vector supernatant (PG13-A2aB-IG4A-LY3H10) encoding a T
cell receptor directed against NY-ESO-1, was prepared and preserved following cGMP conditions in the Indiana University Vector Production Facility (IUVPF). The retroviral vector, MSGV1- A2aB-IG4A-LY3H10, consists of 7,417 bps including the 5' LTR from the murine stem cell virus (promoter), packaging signal including the splicing donor (SD) and splicing acceptor sites, alpha chain and beta chain genes of the anti-NY-ESO-1 TCR cloned from the peripheral blood lymphocytes of a patient with melanoma, and murine stem cell virus 3'LTR. The alpha and beta chains are linked by a T2A peptide. The physical titer will be determined by RNA dot blot according to sponsor certificate. The supernate has been stored in IUVPF upon the completion of production at –80 °C at IUVPF. Upon shipment on dry ice, the supernatant will be stored at –80 °C in Cryonix, Rockville, MD. This storage facility is equipped with around-the-clock temperature monitoring. Upon request, supernatant will be delivered on dry ice to be used in *in vitro* transduction. There will be no re-use of the same unit of supernate for different patients. Retroviral titer has been shown to be stable after immediate thawing and immediate administration (coating the tissue culture wells previously coated with Retronectin). Handling of the vector should follow the guidelines of Biosafety Level-2 (BSL-2). The specific guidelines for Biosafety Level-2 (BSL-2) can be viewed at http://bmbl.od.nih.gov/sect3bsl2.htm.  
8.9.2 OKT3 OKT3 will be obtained by the Surgery Branch Laboratory from commercial sources.  
Formulation: Muromonab-CD3 (Ortho), NSC \#618843, is provided as a sterile, clear, colorless solution at a concentration of 1 mg/ml in 5 ml ampoules. The solution may contain a few fine, translucent protein particles. The antibody is dissolved in a buffered solution at pH of 6.5 to 7.5. The solution contains 2.25 mg of monobasic sodium phosphate, 9 mg of dibasic sodium phosphate, 43 mg of sodium chloride and 1 mg of polysorbate 80 per 5 ml of water for injection.  
Storage/Stability: Ampules should be stored in a refrigerator at 2-8 °C.  
Solution should not be frozen or shaken. Each ampule bears an expiration date.
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8.10. alvac eso-1 vaccine the

- ALVAC(2)-NY-ESO-1(M)/TRICOM (vCP2292) The construct is provided in liquid formulation, and is administered by the subcutaneous route.  
Identity of the Investigational Product: NY-ESO-1 antigen therapeutic vaccine.  
Composition: The investigational vaccine uses ALVAC(2), a modified canarypox virus, as a vector for carrying the gene for the NY-ESO-1 tumorassociated antigen. The vaccine also contains the genes for three co-stimulatory molecules: B7.1, ICAM-1, and LFA-3, collectively referred to as TRICOM.  
The genes for NY-ESO-1 and the co-stimulatory molecules are contained in the same construct. Each vial contains a target dose of 107 CCID50 (with a range of approximately 106.4 to 107.9 /mL) of the ALVAC(2) virus.  
Other ingredients: The only excipient used in the final formulation of the drug product is 10 mM Tris pH 9.0 with 0.9% NaCl, which may be added if necessary to reduce titer. This type of titer adjustment has not been required for any of the clinical lots to date.  
Production Site: Sanofi Pasteur Ltd., BB-IND 13450 Batch/lot numbers: TBD Formulation: Liquid; 1 vial contains 0.72 mL of vaccine construct, with a target dose of 107 CCID50 (with a range of approximately 106.4 to 107.9 /mL) of the ALVAC(2) virus in a withdrawable volume of 0.5 mL.  
Appearance: Clear to opalescent solution with white to pink tint. Some settling may occur, which can be resuspended with shaking Storage: 2–8°C Injection route: Subcutaneous Preparation and Administration: Patients will receive 0.5 mL of the construct in each of the four extremities. If a patient has a compromised extremity due to lymphedema, prior radiation or other causes, vaccination may be given on the abdominal wall or chest wall proximal to the extremity. As the construct is provided in a 0.5 mL vial, one vial is therefore required for each injection. Each construct must be administered at the same injection site throughout the treatment. All injections will be subcutaneous, using a needle not more than 5/8 of an inch in length.  
In the event of overdose: Due to the nature of the product, there is no suggested treatment currently available.  
Precautions for Use: Prior to administration, all study products must be inspected visually for cracks, broken seals, correct label content and extraneous particulate matter and/or discoloration, whenever solution and container permit. If any of these conditions exists, the vaccine should not be administered and a replacement dose may be used.  
As after any vaccination, subjects must be kept under observation for 30 minutes after each injection to ensure their safety. Appropriate equipment must be available on site in the event of immediate allergic reactions.  
Dose Selection and Timing: The vaccine construct contains a target dose of 107 CCID50 (with a range of approximately 106.4 to 107.9 /mL) of the ALVAC(2) virus.  
Earlier studies with ALVAC vaccines have found lower doses to not yield satisfactory immune responses, while higher doses present a manufacturing challenge, so cannot be used at this stage of development. Patients will receive two sets of vaccinations, two weeks apart (+/- 2 days).  
8.11. Support Medications
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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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furosemide

Furosemide will be obtained by the Clinical Center Pharmacy Department from commercial sources. It will be used to enhance urine output during the chemotherapy preparative regimen with cyclophosphamide. Adverse effects include dizziness, vertigo, paresthesias, weakness, orthostatic hypotension, photosensitivity, rash and pruritis. Consult the package insert for a complete list of all side effects.
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9.0 references

1. Wunderlich, J. R. and R. J. Hodes. 1991. Principles of tumor immunity:Biology of cellur immune responses. *Biologic Therapy of Cancer, DeVita, Hellman and Rosenberg, eds.*3-21.  
2. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, L. Rivoltini, S. L. Topalian, T. Miki, and S. A. Rosenberg. 1994. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. *Proc Natl Acad Sci USA* 91:35153519.  
3. Kawakami, Y., S. Eliyahu, K. Sakaguchi, P. F. Robbins, L. Rivoltini, J. R. Yannelli, E.  
Appella, and S. A. Rosenberg. 1994. Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2 restricted tumor infiltrating lymphocytes. *J.Exp.Med* 180:347-352.  
4. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, K. Sakaguchi, E. Appella, J. R.  
Yannelli, Adema, GJ, T. Miki, and Rosenberg SA. 1994. Identification of a human melanoma antigen recognized by tumor infiltrating lymphocytes from a patient responding to immunotherapy. *Proc.Natl.Acad.Sci.* 54:3124-3126.  
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Schwarztentruber, P. Hwu, F. M. Marincola, R. Sherry, S. F. Leitman, and S. A. Rosenberg. 2001. Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treament of patients with metastatic melanoma. *J.Immunotherapy* 24:363-373.  
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Sherry, F. M. Marincola, S. F. Leitman, C. A. Seipp, L. Rogers-Freezer, K. E. Morton, S. A. Mavroukakis, D. E. White, and Rosenberg SA. 2002. A Phase I study of non-myeloablative chemotherapy and adoptive transfer of autologous tumor antigen-specific T lymphocytes in patients with metastatic melanoma. *J Immunother* 25:243-251.  
7. Dudley, M. E., J. R. Wunderlich, T. E. Shelton, J. Even, and S. A. Rosenberg. 2003.  
Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. *J.Immunother* 26:332-342.  
8. Dudley, M. E., J. R. Wunderlich, J. C. Yang, R. M. Sherry, S. L. Topalian, N. P. Restifo, R. E.  
Royal, U. Kammula, D. E. White, S. A. Mavroukakis, L. J. Rogers, G. J. Gracia, S. A. Jones, D. P. Mangiameli, M. M. Pelletier, J. Gea-Banacloche, M. R. Robinson, D. M. Berman, A. C.  
Filie, A. Abati, and S. A. Rosenberg. 2005. Adoptive cell transfer therapy following nonmyeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. *J.Clin Oncol* 23:2346-2357.  
9. Sun, J. C. and M. J. Bevan. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. *Science* 300:339-342.  
10. Khong, H. T. and N. P. Restifo. 2002. Natural selection of tumor variants in the generation of
"tumor escape" phenotypes. *Nature Immunology* 3:999-1005.  
11. Clay, T. M., M. C. Custer, J. Sachs, P. Hwu, S. A. Rosenberg, and M. I. Nishimura. 1999.  
Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. *J.Immunol.* 163:507-513.  
12. Hughes, M. S., Y. Y. L. Yu, M. E. Dudley, Z. Zheng, P. F. Robbins, Y. Li, J. Wunderlich, R.  
G. Hawley, M. Moayeri, S. A. Rosenberg, and R. A. Morgan. 2005. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. *Hum.Gene.Ther.* 16:457-472.  
13. Morgan, R. A., M. E. Dudley, Y. Y. L. Yu, Z. Zheng, P. F. Robbins, M. R. Theoret, J. R.  
Wunderlich, M. S. Hughes, N. P. Restifo, and S. A. Rosenberg. 2003. High efficiency TCR
gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J.Immunol. 171:3287-3295.  
14. Morgan, R. A., M. E. Dudley, J. R. Wunderlich, M. S. Hughes, J. C. Yang, R. M. Sherry, R. E.  
Royal, S. L. Topalian, U. S. Kammula, N. P. Restifo, Z. Zheng, A. Nahvi, C. R. de Vries, L. J. Rogers-Freezer, S. A. Mavroukakis, and S. A. Rosenberg. 2006. Cancer regression in patients after transfer of genetically engineered lymphocytes. *Science* 314:126-129.  
15. Riddell, S. R. and P. D. Greenberg. 1990. The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. *J Immunol Meth* 128:189-201.  
16. Kohn, D. B., M. S. Hershfield, D. Carbonaro, A. Shigeoka, J. Brooks, E. M. Smogorzewska, L.  
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17. Therasse, P., S. G. Arbuck, E. A. Eisenhauer, J. Wanders, R. S. Kaplan, L. Rubinstein, J.  
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19. Scanlan, M. J., A. O. Gure, A. A. Jungbluth, L. J. Old, and Y. T. Chen. 2002. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. *Immunol.Rev.* 188:22-32.  
20. Lethe, B., S. Lucas, and L. Michaux. 1998. A New gene with tumor specificity. *Int J Cancer* 76:903-908.  
21. Jager, E., Y. T. Chen, and J. W. Drijfhout. 1998. Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1:definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes. *J Exp Med* 187:265-270.  
22. Aarnoudse, C. A., P. B. van den Doel, B. Heemskerk, and P. I. Schrier. 1999. Interleukin-2induced, melanoma-specific T cells recognize CAMEL, an unexpected translation product of LAGE-1. *Int.J.Cancer* 82:442-448.  
23. Wang, R.-F., S. L. Johnston, G. Zeng, S. L. Topalian, D. J. Schwartzentruber, and S. A.  
Rosenberg. 1998. A breast and melanoma-shared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames. *J Immunol* 161:3596-3606.  
24. Zeng, G., C. E. Touloukian, X. Wang, N. P. Restifo, S. A. Rosenberg, and R.-F. Wang. 2000.  
Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules.  
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25. Jager, E., D. Jager, J. Karbach, Y. T. Chen, G. Ritter, Y. Nagata, S. Gnjatic, E. Stockert, M.  
Arand, L. J. Old, and A. Knuth. 2000. Identification of NY-ESO-1 epitopes presented by human histocompatibility antigen (HLA)-DRB4*0101-0103 and recognized by CD4(+) T lymphocytes of patients with NY-ESO-1-expressing melanoma. *J.Exp.Med.* 191:625-630.  
26. Chen, Q., H. Jackson, P. Parente, T. Luke, M. Rizkalla, T. Y. Tai, H. C. Zhu, N. A. Mifsud, N.  
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Immunodominant CD4+ responses identified in a patient vaccinated with full-length NY-ESO1 formulated with ISCOMATRIX adjuvant. *Proc.Natl.Acad.Sci.U.S.A* 101:9363-9368.  
27. Jager, E., E. Stockert, Z. Zidianakis, Y. T. Chen, J. Karbach, D. Jager, M. Arand, G. Ritter, L.  
J. Old, and A. Knuth. 1999. Humoral immune responses of cancer patients against "CancerTestis" antigen NY-ESO-1: correlation with clinical events. *Int.J.Cancer* 84:506-510.  
28. Stockert, E., E. Jager, and Y. T. Chen. 1998. A survey of the humoral immune response of cancer patients to a panel of human tumor antigens. *J Exp Med* 187:1349-1354.  
29. Valmori, D., V. Dutoit, D. Lienard, D. Rimoldi, M. J. Pittet, P. Champagne, K. Ellefsen, U.  
Sahin, D. Speiser, F. Lejeune, J. C. Cerottini, and P. Romero. 2000. Naturally occurring human lymphocyte antigen-A2 restricted CD8+ T-cell response to the cancer testis antigen NY-ESO-1 in melanoma patients. *Cancer Res.* 60:4499-4506.  
30. Jager, E., S. Gnjatic, Y. Nagata, E. Stockert, D. Jager, J. Karbach, A. Neumann, J.  
Rieckenberg, Y.-T. Chen, G. Ritter, E. Hoffman, M. Arand, L. J. Old, and A. Knuth. 2000. Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. *Proc Natl Acad Sci* 97:12198-12203.  
31. Shackleton, M., I. D. Davis, W. Hopkins, H. Jackson, N. Dimopoulos, T. Tai, Q. Chen, P.  
Parente, M. Jefford, K. A. Masterman, D. Caron, W. Chen, E. Maraskovsky, and J. Cebon. 2004. The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand. *Cancer Immun.* 4:9.  
32. Khong, H. T., J. C. Yang, S. L. Topalian, R. M. Sherry, S. A. Mavroukakis, D. E. White, and S. A. Rosenberg. 2004. Immunization of HLA-A*0201 and/or HLA-DP%1*04 patients with metastatic melanoma using epitopes from the NY-ESO-1 antigen. *J.Immunother.* 27:472-477.  
33. Davis, I. D., W. Chen, H. Jackson, P. Parente, M. Shackleton, W. Hopkins, Q. Chen, N.  
Dimopoulos, T. Luke, R. Murphy, A. M. Scott, E. Maraskovsky, G. McArthur, D. MacGregor, S. Sturrock, T. Y. Tai, S. Green, A. Cuthbertson, D. Maher, L. Miloradovic, S. V. Mitchell, G. Ritter, A. A. Jungbluth, Y. T. Chen, S. Gnjatic, E. W. Hoffman, L. J. Old, and J. S. Cebon. 2004. Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans. *Proc.Natl.Acad.Sci.U.S.A* 101:10697-10702.  
34. Rosenberg, S. A., J. C. Yang, and N. P. Restifo. 2004. Cancer immunotherapy: moving beyond current vaccines. *Nat Med* 10:909-915.  
35. Zhao, Y., A. D. Bennett, Z. Zheng, Q. J. Wang, P. F. Robbins, L. Y. Yu, Y. Li, P. E. Molloy, S. M. Dunn, B. K. Jakobsen, S. A. Rosenberg, and R. A. Morgan. 2007. High-Affinity TCRs Generated by Phage Display Provide CD4+ T Cells with the Ability to Recognize and Kill Tumor Cell Lines. *J.Immunol.* 179:5845-5854.  
36. Li, Y., R. Moysey, P. E. Molloy, A. L. Vuidepot, T. Mahon, E. Baston, S. Dunn, N. Liddy, J.  
Jacob, B. K. Jakobsen, and J. M. Boulter. 2005. Directed evoluation of human T-cell receptors with picomolar affinities by phage display. *Nat.Biotechnol.* 23:349-354.  
37. Peggs, K. S., S. Verfuerth, A. Pizzey, N. Khan, M. Guiver, P. A. Moss, and S. Mackinnon.  
2003. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. *Lancet* 362:1375-1377.  
38. Walter, E. A., P. D. Greenberg, M. J. Gilbert, R. J. Finch, K. S. Watanabe, E. D. Thomas, and S. R. Riddell. 1995. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. *N.Engl.J.Med.* 333:1038-1044.  
39. Kuball, J., F. W. Schmitz, and R. H. Voss. 2005. Cooperation of human tumor-reactive CD4+
and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR.  
Immunity 22:117-129.  
40. Morris, E. C., A. Tsallios, G. M. Bendle, S. A. Xue, and H. J. Stauss. 2005. A critical role of T
cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection. Proc.Natl.Acad.Sci.U.S.A 102:7934-7939.  
41. Overwijk, W. W., M. R. Theoret, S. E. Finkelstein, D. R. Surman, L. A. de Jong, F. A. VythDreese, T. A. Dellemijn, P. A. Antony, P. J. Spiess, D. C. Palmer, D. M. Heimann, C. A.  
Klebanoff, Z. Yu, L. N. Hwang, L. Feigenbaum, A. M. Kruisbeek, S. A. Rosenberg, and N. P. Restifo. 2003. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. *J.Exp.Med* 198:569-580.  
42. Gattinoni, L., D. J. Powell, S. A. Rosenberg, and N. P. Restifo. 2006. Adoptive immunotherapy for cancer: building on success. *Nat Rev Immunol* 6:383-393.  
43. Eglitis, M. A. and W. F. Anderson. 1988. Retroviral vectors for introduction of genes into mammalian cells. *Biotechniques* 6:608-614.  
44. Rosenberg, S. A., J. C. Yang, D. J. Schwartzentruber, P. Hwu, S. L. Topalian, R. M. Sherry, N.  
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untitled h1
10.0 tables figures and appendices

| Number of occurrences1                               |       |       |       |       |       |
|------------------------------------------------------|-------|-------|-------|-------|-------|
| Study group2                                         | Arm 1 | Arm 2 | Arm 3 | Arm 4 | Total |
| Toxicity Hypotension                                 | 0     | 0     | 0     | 1     | 1     |
| Malaise                                              | 0     | 0     | 0     | 0     | 0     |
| Nausea                                               | 2     | 0     | 0     | 2     | 4     |
| Neurologic                                           | 0     | 0     | 0     | 0     | 0     |
| Febrile neutropenia                                  | 0     | 2     | 1     | 3     | 6     |
| RBC transfusion                                      | 1     | 2     | 3     | 6     | 12    |
| Platelet transfusion                                 | 1     | 3     | 1     | 6     | 11    |
| 1 Counted once for each patient at the highest grade |       |       |       |       |       |
untitled h1
table 1. grade 3-4 non-hematologic

2 Arms defined:
Arm 1: 3 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 30 mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1, without IL-2. Arm 2: 3 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 60 mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1, without IL-2. Arm 3: 3 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 60 mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1 + IV IL-2 (72,000 IU/kg q8h for a maximum of 15 doses) Arm 4: 6 patients received up to 3 X 1011 cloned and/or uncloned lymphocytes plus cyclophosphamide 60 mg/kg d-7 & -6 and fludarabine 25 mg/m2 d-5 to -1+ IV IL-2 (720,000 IU/kg q8h for a maximum of 12 doses)  
| Table 2: Clinical Responses to treatment                                                                                                                                                                                          | 11/27/06                                     |          |                    |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------|----------|--------------------|
| Patient1                                                                                                                                                                                                                          | Sites of Disease                             | Response | Duration (months)2 |
| 1                                                                                                                                                                                                                                 | Axillary, Mesenteric, and Pelvic Lymph Nodes | PR       | 29                 |
| 2                                                                                                                                                                                                                                 | Subcutaneous and Skin                        | PR       | 8                  |
| 4                                                                                                                                                                                                                                 | Iliac and Inguinal Lymph Nodes, Skin         | PR       | 2                  |
| 6                                                                                                                                                                                                                                 | Intraperitoneal Lymph Nodes, Lungs,          | PR       | 28                 |
| Subcutaneous                                                                                                                                                                                                                      |                                              |          |                    |
| 9                                                                                                                                                                                                                                 | Subcutaneous and Skin                        | PR       | 12                 |
| 10                                                                                                                                                                                                                                | Inguinal Lymph Nodes, Subcutaneous and Skin  | PR       | 14                 |
| 16                                                                                                                                                                                                                                | Subcutaneous                                 | CR       | 52+                |
| 17                                                                                                                                                                                                                                | Bone, Liver, Lung, Subcutaneous              | PR       | 38+                |
| 19                                                                                                                                                                                                                                | Intramuscular                                | PR       | 13                 |
| 21                                                                                                                                                                                                                                | Lung, Subcutaneous                           | CR       | 45+                |
| 25                                                                                                                                                                                                                                | Lung, Subcutaneous                           | PR       | 2                  |
| 26                                                                                                                                                                                                                                | Inguinal Lymph Nodes, Liver, Subcutaneous    | PR       | 8                  |
| 28                                                                                                                                                                                                                                | Axillary Lymph Nodes, Brain                  | PR       | 4                  |
| 30                                                                                                                                                                                                                                | Axillary and Inguinal Lymph Nodes,           | PR       | 7                  |
| Intramuscular, Subcutaneous                                                                                                                                                                                                       |                                              |          |                    |
| 31                                                                                                                                                                                                                                | Liver, Lung                                  | CR       | 35+                |
| 32                                                                                                                                                                                                                                | Skin                                         | CR       | 34+                |
| 33                                                                                                                                                                                                                                | Lung, Subcutaneous                           | PR       | 4                  |
| 34                                                                                                                                                                                                                                | Intramuscular, Pelvis                        | PR       | 5                  |
| 1 Patients 2, 6, and 17 had minor (Patient 6) or mixed (Patient 2 and Patient 17) responses after a first course  of treatment, and were treated with a second course of treatment consisting of ablation, cell transfer and high |                                              |          |                    |  
31 Liver, Lung CR 35+ 32 Skin CR 34+ 33 Lung, Subcutaneous PR 4 34 Intramuscular, Pelvis PR 5  
1Patients 2, 6, and 17 had minor (Patient 6) or mixed (Patient 2 and Patient 17) responses after a first course  
of treatment, and were treated with a second course of treatment consisting of ablation, cell transfer and high  
dose IL-2 therapy prior to achieving an objective clinic response.  
2Measured from treatment date to time of first recurrence. Patients 16, 17, 21, 31 and 32 had ongoing  
responses at the time of this writing.
untitled h1
table 3

| Time in Hospital and Non-hematological Grade 3 and 4 Toxicities Related to  Lymphodepleting Chemotherapy and Cell Transfer Attribute measured Duration, Number or  Number of Patients  Type (%) 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 0 6 (17%) Transfusions 1-5 21 (60%) 6-10 5 (14%) 11-15 2 (6%) 16-20 1 (3%) Autoimmunity Uveitis 5 (14%) Vitiligo 13 (37%) Opportunistic Herpes zoster 3 (9%) Infections 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   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Table 4  
Patient demographics, treatments received and clinical outcome  
  
  
Sites of evaluable disease  
  
  
| Chilum   |  Bun
Bun
|                                  |       |    |
|----------|------|----------------------------------|-------|----|
| IL-2
doses*          | l of | თ → ∞ → ∞ ∞ ∞ ∞ ∞ ∞              | 10    | This patient was treated twice, separated by seven months
"Tolal cells infused multipled by %Vβ12
720,000 IU/kg every eight hours.  All patients were previously refractory to    |
| wissing)
SullenoD          | 0.
6 L 8      | t open be en                     | res
E       |    |
| companies.  
SABO          | மம   | 8+21.
6+81.
6+81.                                  |       |    |
| 61
61          |      |                                  |       |    |
| #(-01x)
pesnyl
MVL-1          | 8
2 1 1      | e and the sense of               | 00
1999
1999  1999       |    |
| %VB12    | 55
19      | d head of the search and the sea | 99    |    |
| %CD4/
%CD8          | CB/LL
GB/GB
EL/LL      | 82/11
1278/91
09/11                                  |       |    |
| 65/03
22/22 03/22
22/22 03/22 03/22
03/03          |      |                                  |       |    |
|          | 0 VL
0 ELL      | 00 100                           | 8     |    |
| Age/Sex  | W85
HELL
WRZ      | nels
MSC WEST
MSC WEST
MSC WEST                                  | 202
SELEN
WOS       |    |
| Patient  | ,,   | e do not be a sens               | n n n |    |
| Cohort   |      |                                  |       |    |
untitled h1
table 5 gene transfer efficiency

| 1               | 2a   | 3   | 4   | 5   | 6   | 7   | 8   | 9   | 10   | 11   | 12   | 13   | 2b   | 14   | 15   | 16   | 17   | Ave   |    |
|-----------------|------|-----|-----|-----|-----|-----|-----|-----|------|------|------|------|------|------|------|------|------|-------|----|
| VE12            | 58   | 63  | 21  | n/d | 18  | 36  | 45  | 38  | 43   | 52   | 51   | 66   | 43   | 66   | 58   | 47   | 18   | 41    | 45 |
| (%CD4) Tetramer | 8    | 15  | 1   | n/d | 2   | 10  | 15  | 8   | 15   | 21   | 22   | 33   | 15   | 15   | 7    | 4    | 10   | 7     | 12 |
| (%CD4) VE12     | 49   | 65  | 63  | 24  | 17  | 32  | 23  | 29  | 43   | 42   | 43   | 59   | 44   | 72   | 51   | 51   | 36   | 24    | 42 |
| (%CD8) Tetramer | 16   | 28  | 3   | 23  | 3   | 10  | 6   | 5   | 13   | 15   | 19   | 30   | 16   | 33   | 17   | 19   | 11   | 15    | 17 |
| (%CD8)          |      |     |     |     |     |     |     |     |      |      |      |      |      |      |      |      |      |       |    |
untitled h1
patient number

Samples were taken from transduced T lymphocytes cultures at or near time of infusion. FACS
analysis for CD4, CD8, VE12, and MART-1 Tetramer (M27L) was as described in methods.  
n/d, not determined.; Ave, average value.
untitled h1
table 6 q-rt pcr analysis

| 4        | 5   | 6   | 7    | 8    | 9    | 10   | 11   | 12   | 13   | 14   | 15   | 16   | 17   |      |
|----------|-----|-----|------|------|------|------|------|------|------|------|------|------|------|------|
| Infusion | 5.8 | 2.2 | 14.5 | 10.7 | 21.5 | 20.8 | 4.0  | 10.3 | 9.6  | 11.2 | 4.0  | 5.1  | 24   | 11.1 |
| Post     | 1.2 | 0.8 | 3.5  | 1.9  | 0.6  | 2.8  | 0.9  | 1.4  | 0.6  | 2.9  | 2.8  | 1.1  | 1.9  | 2.2  |
| Infusion |     |     |      |      |      |      |      |      |      |      |      |      |      |      |  
Amount of TCR vector-derived RNA normalized to cellular actin mRNA. Using RNA derived from a stable transduced human T cell line (Sup T1) as reference; total RNA from infused T cells or patient PBMC post-infusion were subjected to Q-RT PCR and then normalized to the amount of actin mRNA (values are vector/1 x 106 actin). Samples were 2-5 weeks post-infusion, with exception of patients 8, 9, and 11, which were 2 months, 3 months, and 2 weeks respectively.
untitled h1
table 7 elispot analysis

| 1                                                                                                     | 2a   | 3    | 4   | 5    | 6    | 8    | 9    | 13   | 2b   | 14   | 15   | 16   | 17   |     |
|-------------------------------------------------------------------------------------------------------|------|------|-----|------|------|------|------|------|------|------|------|------|------|-----|
| Infusion                                                                                              | 1523 | 1783 | 215 | 4527 | 1367 | 1203 | 1352 | 460  | 3380 | 4877 | 2023 | 2938 | 1393 | 190 |
| Pre                                                                                                   | 22   | 21   | 1   | 14   | 0    | 1    | 0    | 0    | 0    | 0    | 20   | 1    | 6    | 0   |
| treatment Post                                                                                        | 13   | 45   | 0   | 41   | 7    | 1    | 215  | 20   | 700  | 13   | 530  | 35   | 92   | 108 |
| infusion Number of interferon-J positive Eispots/100,000 cells. Post-infusion values derived from PBL |      |      |     |      |      |      |      |      |      |      |      |      |      |     |  
  
Number of interferon-J positive Eispots/100,000 cells. Post-infusion values derived from PBL
taken at one to four weeks post-infusion and rested overnight in medium (without cytokine) prior to assay.  
| Melanoma Cell Line A2- A2+   | Peptide pulsed T2 cells   |       |      |       |       |        |        |        |        |      |
|------------------------------|---------------------------|-------|------|-------|-------|--------|--------|--------|--------|------|
| 888                          | 938                       | 526   | 624  | None  | gp100 | MART-1 |        |        |        |      |
| 1.0                          | 0.1                       |       |      |       |       |        |        |        |        |      |
| None                         | A1,24                     | A1,24 | A2,3 | A2,3  | PM    | 10 PM  | 1.0 PM | PM     |        |      |
| Controls None                | 0                         | 0     | 0    | 0     | 0     | 0      | 0      | 0      | 0      | 0    |
| AK 1700-3                    | 181                       | 66    | 9    | 37    | 10    | 46     | 36     | 32     | 35     | 32   |
| JKF6                         | 0                         | 22    | 28   | 10250 | 10680 | 1      | 0      | >16910 | >15430 | 7770 |
| JR6C12                       | 0                         | 41    | 0    | 4190  | 4640  | 0      | 8000   | 0      | 8520   | 143  |
| Patient 4 Pretreatment                              | 130                       | 204   | 117  | 98    | 133   | 521    | 421    | 422    | 462    | 520  |
| Day 7                        | 40                        | 65    | 44   | 131   | 165   | 104    | 94     | >1255  | >1480  | 851  |
| Day 8                        | 87                        | 110   | 75   | 92    | 78    | 122    | 190    | 957    | 893    | 537  |
| Day 18                       | 42                        | 27    | 23   | 43    | 55    | 184    | 63     | 108    | 160    | 113  |  
Anti-melanoma properties of genetically engineered lymphocytes determined for patient 4 PBL
following overnight culture in IL-2 (300 IU/ml). The production of interferon-J (pg/ml)
following co-culture with peptide pulsed T2 cells (peptide reactivity) and anti-melanoma activity (tumor reactivity) for HLA-A2 + lines (526, 624) and HLA-A2- lines (888, 938). Controls included; non-peptide reactive TIL AK 1700-3, MART-1 reactive TIL JKF6, and gp100 reactive TIL JR6C12. JR6C12 recognition of 1.0PM MART-1 peptide was not reproducible in repeated assays. Values demonstrating specific release of cytokine are in bold.
untitled h1
table 8 (con't) patient 14

| Melanoma Cell Line A2- A2+   | Peptide pulsed T2 cells   |       |      |      |       |        |        |       |      |     |
|------------------------------|---------------------------|-------|------|------|-------|--------|--------|-------|------|-----|
| 888                          | 938                       | 526   | 624  | None | gp100 | MART-1 |        |       |      |     |
| 1.0                          | 0.1                       |       |      |      |       |        |        |       |      |     |
| None                         | A1,24                     | A1,24 | A2,3 | A2,3 | PM    | 10 PM  | 1.0 PM | PM    |      |     |
| Controls None                | 0                         | 0     | 43   | 0    | 0     | 0      | 23     | 70    | 0    | 18  |
| AK 1700-3                    | 35                        | 53    | 33   | 21   | 26    | 52     | 43     | 94    | 73   | 105 |
| 15                           | 11                        | 66    | 6750 | 9160 | 37    | 337    | >2168  | >2168 | >182 |     |
| JKF6                         | 8                         |       |      |      |       |        |        |       |      |     |
| JR6C12                       | 0                         | 0     | 0    | 4410 | 3760  | 0      | >1510  | 23    | 15   | 61  |
| Patient 14 Pretreatment                              | 368                       | 410   | 352  | 284  | 344   | 639    | 645    | 874   | 614  | 471 |
| Day 5                        | 66                        | 53    | 67   | 110  | 170   | 134    | 96     | 918   | 716  | 634 |
| Day 7                        | 69                        | 52    | 45   | 41   | 55    | 33     | 57     | 192   | 221  | 109 |
| Day 10                       | 79                        | 91    | 0    | 80   | 46    | 33     | 52     | 245   | 214  | 108 |
| Day 27                       | 52                        | 76    | 35   | 33   | 55    | 67     | 103    | 282   | 232  | 158 |
| Day 88                       | 26                        | 50    | 31   | 0    | 52    | 75     | 41     | 52    | 43   | 31  |
| Day 257                      | 105                       | 52    | 93   | 53   | 46    | 111    | 83     | 597   | 492  | 399 |  
Anti-melanoma properties of genetically engineered lymphocytes determined for patient 14 PBL
following overnight culture in IL-2 (300 IU/ml). The production of interferon-J (pg/ml)
following co-culture with peptide pulsed T2 cells (peptide reactivity) and anti-melanoma activity (tumor reactivity) for HLA-A2 + lines (526, 624) and HLA-A2- lines (888, 938). Controls included; non-peptide reactive TIL AK 1700-3, MART-1 reactive TIL JKF6, and gp100 reactive TIL JR6C12. Values demonstrating specific release of cytokine are in bold  
| 306                                                                                         | >1654                                                                        | 127      |                                                                |
|---------------------------------------------------------------------------------------------|------------------------------------------------------------------------------|----------|----------------------------------------------------------------|
| p53                                                                                         | 38                                                                           | 65       |                                                                |
| RNA electroporation of tumor antigen specific TCP                                           | gp100+), melanoma 526mel (HLA-A2+/MART-1+/gp100+/p53+),  non-small cell lung |          |                                                                |
| tured with tumor cell lines and after overnight co-culture, the supernatants were collected |                                                                              |          |                                                                |
| MART-1, gp100, NY-ESO-1, p53). Two hours post electroporation, the cells were co            |                                                                              |          |                                                                |
| CD8+ PBLs were electroporated with IVT GFP RNA or tumor antigen specific TCRs               | 2+/NY-ESO-1+/p53-), and breast cancer MDA 453S (HLA-A2-/NY-ESO-1+/p53-       |          |                                                                |
| subjected to ELISA detection of interferon-y secretion (in pg/ml). The know                 | ancer H2087 (HLA-A2+/NY-ESO-1-/p53+), breast cancer MDA 453S-A2 (HLA         |          |                                                                |
| NY-ESO-1                                                                                    | notypes of these tumor cell line were; melanoma 938mel (HLA-A2-/MART-        |          |                                                                |
| 163                                                                                         | 116                                                                          | 342      |                                                                |
| 47                                                                                          | 75                                                                           |          |                                                                |
| Effector TCR RNA                                                                            | gp100                                                                        | 317      | 125                                                            |
| 35                                                                                          | 96                                                                           | 73       | Values demonstrating specific release of cytokine are in bold. |
| MART-1                                                                                      | 1146                                                                         | 224      | 175                                                            |
| 68                                                                                          | 65                                                                           |          |                                                                |
| GFP                                                                                         | 130                                                                          |          |                                                                |
| 23                                                                                          | 62                                                                           | 96       |                                                                |
| 61                                                                                          | MDA 453S-A1                                                                  | IDA 453S |                                                                |
| Tumor                                                                                       | 938mel                                                                       | 526mel   | H2087                                                          |
| Table 9                                                                                     |                                                                              |          |                                                                |
untitled h1
table 10 modification of dose

Unless otherwise specified in this protocol, actual body weight is used for dose calculations of treatment agents. In patients who are determined to be obese (BMI > 35), the **practical weight** (see 3 below) will be used. 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.  
  
  
encoding control (GFP) or cloned TCRs reactive with HLA-A2 restricted epitopes from human tumor antigens MART-1, gp100, NY-
and MART-1 reactive TIL JKF6 (JKF6).  E.  Anti-melanoma properties of genetically engineered lymphocytes were determined for all patients prior to infusion.  The production of interferon- α (pg/ml) following co-culture with peptide pulsed T2 cells (Peptide Reactivity) and anti-melanoma activity (Tumor Reactivity) for HLA-A2 + lines (526, 624) and HLA-A2- lines (888, 938).  Values demonstrating specific release of cytokine are in bold.  
A
Gene Marled Cells  
  
Gene Marled Cells
%  
Gene Marled Cells f  
  
  
Figure 3:.
B  
  
08-C-0121  
Figure 4
untitled h1
figure 5

A B
CD4+ T cells CD8+ T cells  
  
C D 4 T c ell s C D 8 T c ell s A B  
  
Fi g ure 6. R a pi d scree ni n g of N Y- E S O- 1/ H L A- A * 0 2-re a cti ve T C R 1 G 4 C D R 3 v ari a nts i n C D 8 a n d C D 4 T cells. T cells wer e acti vat e d wit h a nti- C D 3 a nti b o d y a n d is olate d C D 4 or C D 8 ) T cells were c o-tra nsf ecte d wit h R N A 1 G 4 T C R  
A A Ss wit hi n t he C D R 3 re gi o n al o n g wit h a W T 1 G 4 T C R c hai n R N A c o nstr uct. Tra nsf ecte d T  
cells wer e i nc u bate d o v er ni g ht wit h H L A- A * 0 2 mela n o ma ( 3 9 7- A 2, 6 2 4. 3 8, 1 3 0 0, 5 2 6 a n d 8 8 8- A 2) or br east ca ncer ( M D A 4 3 5 S- A 2) tar get ce ll li nes t hat di d or di d n ot als o e x press N YE S O- 1 tra nscri pt a n d t he releas e of I F N- was meas ure d t he f oll o wi n g da y.  
  
Figure 7.  
Adoptive transfer of tumor-specific T cells combined with vaccination and IL-2 causes regression and cure of large, established tumors.  B16 tumor was implanted subcutaneously into C57BL/6 mice were treated by adoptive transfer of fresh pmel-1 splenocytes +/- vaccination with recombinant fowlpox virus (rFPV)hgp100 either seven
(A,B) or fourteen days (C,D) after inoculation with B16 melanoma.  IL-2 was administered twice daily for 6 doses.  Fresh or cultured splenocytes were effective I the treatment of large, established tumor.  Splenocytes derived from an identically constructed TCR transgenic mouse with specificity for  beta–galactosidase were used as a control in some experiments, (a representative of which is shown in panels C,D) were not therapeutic.  Statistically significant tumor regression was seen in mice treated with pmel-1 cells given in combination with rFPVhgp100 and IL-2 in over 20 independently performed experiments.  There were at least 5 mice/group in all experiments.  Mouse survival consistently correlated with tumor growth reduction.
untitled h1
figure 8.

Long –term (>1 yr) survival of mice bearing large, established B16 tumors after treatment with adoptive transfer of tumor-specific T cells combined with vaccination and IL-2 is associated with the development of vitiligo.  C57BL/6 mice were treated with adoptive transfer of fresh or cultured pmel-1 transgenic splenocytes fourteen days after inoculation with B16 melanoma, then vaccinated with rFPVhgp100.  IL-2 was administered twice dily for 6 doses.  Mice treated with fresh naïve or cultured transgenic T cells were cured of B16 and vitiligo was seen at the former tumor site.  At more than 1-year post therapy, these mice remain tumor-free.
untitled h1
appendix 1

| Toxicity of Treatment with Interleukin-2   |       |      |       |      |     |     |      |       |
|--------------------------------------------|-------|------|-------|------|-----|-----|------|-------|
| Interleukin-2 Plus                         | Alone | TNF  | a-IFN | MoAB | CYT | LAK | TIL  | Total |
| Number of Patients                         | 652*  |      |       |      |     |     |      |       |
| 155                                        | 38    | 128  | 32    | 19   | 214 | 66  |      |       |
| Number of Courses                          | 236   | 85   | 210   | 35   | 30  | 348 | 95   | 1039  |
| Chills                                     | 75    | 16   | 68    | 8    | 8   | 191 | 3    | 39    |
| 53                                         | 9     | 26   | 2     | 2    | 82  | 6   | 180  |       |
| Pruntus                                    |       |      |       |      |     |     |      |       |
| Necrosis                                   | 3     | l    | 2     | i    | l   | .   | l    | S     |
| Anaphylaxis                                | i     | l    | l     | l    | l   | l   | i    | 1     |
| Mucositis (requiring liquid diet)          | 6     | I    | 7     | l    | 2   | 12  | 2    | 30    |
| Alimentation not possible                  | i     | l    | .     | J    | 4   |     |      |       |
| A                                          | .     |      |       |      |     |     |      |       |
| Nausea and vomiting                        | 162   | 42   | 17    | 14   | 20  | 263 | 48   | 666   |
| 144                                        | 38    | 98   | 15    | 13   | 250 | 38  | 596  |       |
| Diarrhea                                   |       |      |       |      |     |     |      |       |
| Hyperbilirubinemia (maximum/mg %)          |       |      |       |      |     |     |      |       |
| 2.1-6.0                                    | 126   | 49   | 97    | 21   | 18  | 190 | 46   | 547   |
| 6.1-10.0                                   | 49    | 3    | 12    | 8    | 9   | 72  | 26   | 179   |
| 10.1+                                      | 26    | l    | 4     | 3    | l   | 40  | 8    | 83    |
| Oliguria                                   |       |      |       |      |     |     |      |       |
| <80 ml/8 hours                             | 81    | 37   | 67    | 14   | 9   | 14  | 25   | 347   |
| <240 ml/24 hours                           | 19    | 2    | 3     | l    | 12  | S   | 42   |       |
| Weight gain (% body weight)                |       |      |       |      |     |     |      |       |
| 0.0-5.0                                    | 106   | 23   | 65    | 8    | 9   | 17  | 49   | 37    |
| 22                                         | 10    | .436 |       |      |     |     |      |       |
| 5.1-10.0                                   | 178   | 41   | 11    | 148  | 26  |     |      |       |
| 43                                         | 17    | 175  |       |      |     |     |      |       |
| 10.1-15.0                                  | 26    | 3    | 9     | 62   | 15  |     |      |       |
| 15.1-20.0                                  | 17    | 3    | 8     | l    | l   | 15  | 3    | 38    |
| 20.1+                                      | 2     | I    | l     | I    | 6   | 2   | 13   |       |
| Elevated creatinine (maximum/mg %)         |       |      |       |      |     |     |      |       |
| 2.1-6.0                                    | 148   | 43   | 121   | 20   | 14  | 237 | 54   | 637   |
| 6.1-10.0                                   | 21    | I    | 14    | 3    | l   | 34  | 12   | 85    |
| 10.1+                                      | S     | l    | l     | i    | 2   | l   | 10   |       |
| l                                          |       |      |       |      |     |     |      |       |
| Hematuria (gross)                          | 2     | 2    |       |      |     |     |      |       |
| l                                          | l     | l    | l     | l    |     |     |      |       |
| Edema (symptomatic nerve or vessel         |       |      |       |      |     |     |      |       |
| compression)                               | 4     | l    | 6     | l    | 7   | l   | 17   |       |
| Tissue ischemia                            | l     | l    | i     | I    | l   | 2   |      |       |
| l                                          |       |      |       |      |     |     |      |       |
| Resp. distress:                            | 17    | l    | 19    | 4    | 28  | 7   | 67   |       |
| not intubated                              | l     |      |       |      |     |     |      |       |
| intubated                                  | 15    | l    | 6     | 3    | l   | 12  | 5    | 41    |
| Bronchospasm                               | 2     | l    | 2     | l    | 4   | 9   |      |       |
| Pleural effusion (requiring                |       |      |       |      |     |     |      |       |
| thoracentesis)                             | 4     | l    | l     | 2    | 8   | l   | 17   |       |
| Somnolence                                 | 29    | 2    | 22    | 6    | 2   | 45  | 8    | 14    |
| Coma                                       | 9     | l    | 8     | l    | 2   | 8   | s    | 3     |
| Disorientation                             | 52    | 3    | 50    | 7    | 4   | 89  | 10   | 215   |
| Hypotension (requiring pressors)           | 19    | 16   | 40    | 17   | 12  | 259 | 45   | 508   |
| s                                          | 8     | 2    |       |      |     |     |      |       |
| Angina                                     | i     |      | 8     |      |     |     |      |       |
| Myocardial infarction                      | 4     | i    | l     | l    | .1  | l   | 66   |       |
| Arrythmias                                 | 15.   | 2    | 13    | 3    | i   | 39. | 6    | 78    |
| Anemia requiring transfusion (number       |       |      |       |      |     |     |      |       |
| units transfused)                          | 77    | 53   | 9     | 37   |     |     |      |       |
| 1-15                                       | 16    | 6    | 176   | 40   |     |     |      |       |
| 6-10                                       | 2     | l    | s     | 3    | 2   | 53  | 9    | 95    |
| 1-15                                       | 4     | l    | i     | 15   | 4   | 24  |      |       |
| l                                          | l     |      |       |      |     |     |      |       |
| 16+                                        | l     | l    | i     | i    | l   | l   | I    | 14    |
| Thrombocytopenia (minimum/mm 3 )           |       |      |       |      |     |     |      |       |
| <20,000                                    | 28    | i    | 2     | 4    | 6   | 71  | 19   | 131   |
| 20,001-60,000                              | 82    | 1    | 62    | 14   | 12  | 150 | 30   | 361   |
| 60,001-100,000                             | 53    | 36   | 76    | 11   | 8   | 79  | 2222 | 285   |
| 7                                          |       |      |       |      |     |     |      |       |
| Central line sepsis                        | 13    | l    | l     | 4    | 36  | NRN | .63  |       |
| Death                                      | 4     | 1    | .3    | 10   |     |     |      |       |
| * Eleven patients are in two protocols.    |       |      |       |      |     |     |      |       |  
| Expected IL-2 Toxicities and their Management                                                               |               |                                                                                                                                                |                                                                  |                   |
|-------------------------------------------------------------------------------------------------------------|---------------|------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------|-------------------|
| Expected                                                                                                    | Expected      | Supportive                                                                                                                                     | Stop Cycle*                                                      | Stop Treatment ** |
| toxicity                                                                                                    | grade         | Measures                                                                                                                                       |                                                                  |                   |
| Chills                                                                                                      | 3             | IV Meperidine 25-50                                                                                                                            | No                                                               | No                |
| mg, IV q1h, prn,                                                                                            |               |                                                                                                                                                |                                                                  |                   |
| Fever                                                                                                       | 3             | Acetaminophen 650  mg, po, q4h;  Indomethicin 50-75 mg, po, q8h                                                                                | No                                                               | No                |
| Pruritis                                                                                                    | 3             | Hydroxyzine HCL  10-20 mg po q6h,  prn;  Diphenhydramine  HCL25-50 mg, po,  q4h, prn                                                           | No                                                               | No                |
| Nausea/ Vomiting/                                                                                           | 3             | Ondansetron 10 mg,                                                                                                                             |                                                                  |                   |
| Anorexia                                                                                                    | IV, q8h, prn; |                                                                                                                                                |                                                                  |                   |
| Granisetron 0.01                                                                                            |               |                                                                                                                                                |                                                                  |                   |
| mg/kg IV daily prn;  Droperidol 1 mg, IV  q4-6h, prn;  Prochlorperazine 25  mg pr, prn or 10 mg  IV q6h prn | No            | No                                                                                                                                             |                                                                  |                   |
| Diarrhea                                                                                                    | 3             | Loperamide 2mg, po, q3h, prn;  Diphenoxylate HCl  2.5 mg and atropine  sulfate 25 mcg, po,  q3h, prn; codeine  sulfate 30-60 mg, po,  q4h, prn | If uncontrolled after  24 hours despite all  supportive measures | No                |
| Malaise                                                                                                     | 3 or 4        | Bedrest                                                                                                                                        | If other toxicities  occur  simultaneously                       | No                |
| Hyperbilirubinemia                                                                                          | 3 or 4        | Observation                                                                                                                                    | If other toxicities  occur  simultaneously                       | No                |
| Anemia                                                                                                      | 3 or 4        | Transfusion with                                                                                                                               | If uncontrolled                                                  |                   |
| PRBCs                                                                                                       | despite all   |                                                                                                                                                |                                                                  |                   |
| supportive measures                                                                                         | No            |                                                                                                                                                |                                                                  |                   |
| Thrombocytopenia                                                                                            | 3 or 4        | Transfusion with                                                                                                                               | If uncontrolled                                                  |                   |
| platelets                                                                                                   | despite all   |                                                                                                                                                |                                                                  |                   |
| supportive measures                                                                                         | No            |                                                                                                                                                |                                                                  |                   |
| Edema/Weight gain                                                                                           | 3             | Diuretics prn                                                                                                                                  | No                                                               | No                |
| Hypotension                                                                                                 | 3             | Fluid resuscitation                                                                                                                            | If uncontrolled                                                  |                   |
| Vasopressor support                                                                                         | despite all   |                                                                                                                                                |                                                                  |                   |
| supportive measures                                                                                         | No            |                                                                                                                                                |                                                                  |                   |
| 08-C-0121                                                                                                   | 80            |                                                                                                                                                |                                                                  |                   |  
| Dyspnea                                                                                                                           | 3 or 4                                      | Oxygen or ventilatory                      | If requires                  | No                        |
|-----------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------|--------------------------------------------|------------------------------|---------------------------|
| support                                                                                                                           | ventilatory support                         |                                            |                              |                           |
| Oliguria                                                                                                                          | 3 or 4                                      | Fluid boluses or  dopamine at renal  doses | If uncontrolled  despite all |                           |
| supportive measures                                                                                                               | No                                          |                                            |                              |                           |
| Increased creatinine                                                                                                              | 3 or 4                                      | Observation                                | Yes (grade 4)                | No                        |
| Renal failure                                                                                                                     | 3 or 4                                      | Dialysis                                   | Yes                          | Yes                       |
| Pleural effusion                                                                                                                  | 3                                           | Thoracentesis                              | If uncontrolled  despite all |                           |
| supportive measures                                                                                                               | No                                          |                                            |                              |                           |
| Bowel perforation                                                                                                                 | 3                                           | Surgical intervention                      | Yes                          | Yes                       |
| Confusion                                                                                                                         | 3                                           | Observation                                | Yes                          | No                        |
| Somnolence                                                                                                                        | 3 or 4                                      | Intubation for airway                      | Yes                          | Yes                       |
| protection                                                                                                                        |                                             |                                            |                              |                           |
| Arrhythmia                                                                                                                        | 3                                           | Correction of fluid  and electrolyte       |                              |                           |
| imbalances; chemical  conversion or  electrical conversion  therapy                                                               | If uncontrolled  despite all                |                                            |                              |                           |
| supportive measures                                                                                                               | No                                          |                                            |                              |                           |
| Elevated Troponin                                                                                                                 | 3 or 4                                      | Observation                                | Yes                          | If changes in LV function |
| levels                                                                                                                            | have not improved to  baseline by next dose |                                            |                              |                           |
| Myocardial                                                                                                                        | 4                                           | Supportive care                            | Yes                          | Yes                       |
| Infarction Elevated                                                                                                               | 3 or 4                                      | Observation                                | For grade 4 without          | If changes have not       |
| transaminases                                                                                                                     | liver metastases                            | improved to baseline by  next dose         |                              |                           |
| Hyperbilirubinemia                                                                                                                | 3 or 4                                      | Observation                                | For grade 4 without          | If changes have not       |
| liver metastases                                                                                                                  | improved to baseline by  next dose          |                                            |                              |                           |
| Electrolyte                                                                                                                       | 3 or 4                                      | Electrolyte                                | If uncontrolled              |                           |
| imbalances                                                                                                                        | replacement                                 | despite all                                |                              |                           |
| supportive measures                                                                                                               | No                                          |                                            |                              |                           |
| Neutropenia                                                                                                                       | 4                                           | Observation                                | No                           | No                        |
| *Unless the toxicity is not reversed within 12 hours ** Unless the toxicity is not reversed to grade 2 or less by next treatment. |                                             |                                            |                              |                           |  
| TABLE 8. Toxicity of Treatment with Interleukin-2   |       |         |       |       |      |     |      |       |
|-----------------------------------------------------|-------|---------|-------|-------|------|-----|------|-------|
| Interleukin-2 Plus                                  | Alone | TNF     | a-IFN | MoAB  | CYT  | LAK | TIL  | Total |
| 155                                                 | 652*  |         |       |       |      |     |      |       |
| Number of Patients                                  | 38    | 128     | 32    | s     | 19   | 214 | 666  |       |
| Number of Courses                                   | 236   | 85      | 210   | 35    | 30   | 348 | 95   | 1039  |
| Chills                                              | 75    | 16      | 68    | 8     | 8    | 191 | 33   | 399   |
| Pruntus                                             | 53    | 9       | 26    | 2     | 2    | 82  | 6    | 180   |
| Necrosis                                            | 3     | l       | 2     | l     | S    |     |      |       |
| Anaphylaxis                                         | i     | l       | l     |       |      |     |      |       |
| Mucositis (requiring liquid diet)                   | 6     | I       | 7     | 2     | 12   | 2   | 30   |       |
| l                                                   |       |         |       |       |      |     |      |       |
| Alimentation not possible                           | l     | 4       |       |       |      |     |      |       |
|                                                     |       |         |       |       |      |     |      |       |
| Nausea and vomiting                                 | 162   | 42      | 17    | 14    | 20   | 263 | 48   | 666   |
| 38                                                  | 250   | 38      | 596   |       |      |     |      |       |
| Diarrhea                                            | 144   | 98      | 15    | 13    |      |     |      |       |
| Hyperbilirubinemia (maximum/mg %)                   | 126   | 49      | 97    | 21    | 18   | 190 | 46   | 547   |
| 2.1-6.0                                             |       |         |       |       |      |     |      |       |
| 6.1-10.0                                            | 49    | 3       | 12    | 8     | 9    | 72  | 26   | 179   |
| 10.1+                                               | 26    | i       | 4     | 3     | i    | 40  | 8    | 83    |
| Oliguria                                            |       |         |       |       |      |     |      |       |
| <80 ml/8 hours                                      | 81    | 37      | 67    | 14    | 9    | 25  | 347  |       |
| 14                                                  |       |         |       |       |      |     |      |       |
| <240 ml/24 hours                                    | 19    | 2       | 3     | i     | 12   | s   | 42   |       |
| Weight gain (% body weight)                         | 106   | 23      | 65    | 8     | 9    | 17  | 49   | 377   |
| 0.0-5.0                                             |       |         |       |       |      |     |      |       |
| 5.1-10.0                                            | 78    | 41      | 111   | 22222 | 10   | 148 | 26   | 436   |
| 10.1-15.0                                           | 43    | 17      | 26    | 3     | 9    | 62  | 15   | 175   |
| 15.1-20.0                                           | 7     | 3       | 8     | l     | l    | 15  | 3    | 38    |
| 20.1+                                               | 2     | l       | l     | l     | 6    | 2   | 13   |       |
| Elevated creatinine (maximum/mg %)                  |       |         |       |       |      |     |      |       |
| 2.1-6.0                                             | 148   | 43      | 121   | 20    | 14   | 237 | 54   | 637   |
| 6.1-10.0                                            | 12    | 85      |       |       |      |     |      |       |
| 21                                                  | 1     | 14      | 3     | 34    |      |     |      |       |
| 10.1+                                               | s     | l       | l     | l     | 2    | l   | 10   |       |
| Hematuria (gross)                                   | 2     | 2       |       |       |      |     |      |       |
| l                                                   | l     | l       |       |       |      |     |      |       |
| Edema (symptomatic nerve or vessel                  | 17    |         |       |       |      |     |      |       |
| compression)                                        | 4     | l       | 6     | l     |      | 7   | l    |       |
| Tissue ischemia                                     | l     | l       | l     | i     | l    | 2   |      |       |
| Resp. distress:                                     |       |         |       |       |      |     |      |       |
| not intubated                                       | 17    | l       | 9     | 4     | l    | 28  | 7    | 67    |
| intubated                                           | 15    | 6       | 3     | 12    | 5    | 41  |      |       |
| i                                                   |       |         |       |       |      |     |      |       |
| Bronchospasm                                        | 2     | 2       | l     |       |      |     |      |       |
| l                                                   |       | 4       | 1.9   |       |      |     |      |       |
| Pleural effusion (requiring                         |       |         |       |       |      |     |      |       |
| thoracentesis)                                      | 4     | i       | l     | l     | 2    | 8   | i    | 17    |
| 29                                                  | 2     | 22      | 6     | 2     | 45   | 8   | 14   |       |
| Somnolence                                          |       |         |       |       |      |     |      |       |
| Coma                                                | 9     | l       | 8     | 2     | 8    | s   | 33   |       |
| Disorientation                                      | 52    | 3       | 50    | 7     | 4    | 89  | 10   | 215   |
| Hypotension (requiring pressors)                    | 19    | 16      | 40    | 17.   | 12   | 259 | 45   | 508   |
| Angina                                              | 5     | I       | 8     | 8     | 22   |     |      |       |
| l                                                   | l     | l       |       |       |      |     |      |       |
| Myocardial infarction                               | 4     | l       | l     |       |      |     |      |       |
| l                                                   | l     |         | 16    |       |      |     |      |       |
| Arrythmias                                          | 15    | 2       | 13    | 3     | 39   | 78  |      |       |
| l                                                   | 6     |         |       |       |      |     |      |       |
| Anemia requiring transfusion (number                |       |         |       |       |      |     |      |       |
| units transfused)                                   | 77    | 53      |       |       |      |     |      |       |
| 1-15                                                | 16    | 9       | 6     | 176   | 40   | 37  |      |       |
| 6-10                                                | 22    | l       | s     | 3     | 2    | 53  | 9    | 95    |
| 1-15                                                | 4     | l       | i     | 15    | 4    | 24  |      |       |
| l                                                   | l     |         |       |       |      |     |      |       |
| 16+                                                 | l     | l       | l     | 11    | I    | 14  |      |       |
| l                                                   | l     |         |       |       |      |     |      |       |
| Thrombocytopenia (minimum/mm')                      |       |         |       |       |      |     |      |       |
| <20,000                                             | 28    | l       | 2     | 4     | 1: 6 | 71  | 19   | 131   |
| 20.001-60.000                                       | 82    | 1111111 | 62    | 14    | 12   | 150 | 30   | .361  |
| 60.001-100.000                                      | 53    | 36      | 76    | 11    | 8    | 79  | 22   | 285   |
| Central line sepsis                                 | 13    | l a     | 7     | l     | 4    | 36  | 12 N | 63    |
| Death                                               | 4     | al      | l     | 3     | 10   |     |      |       |
| * Eleven patients are in two protocols.             |       |         |       |       |      |     |      |       |
certificate of analysis anti-eso-1 tcr
appendix 1

Date of preparation of final product:
Patient:
Allogeneic PBMC Donor Name: Pheresis date:
Tests performed on final product:  
| Test                                                                                                                     | Method                    | Limits     | Result   | Initials/ Date   |
|--------------------------------------------------------------------------------------------------------------------------|---------------------------|------------|----------|------------------|
| Cell viability1                                                                                                          | trypan blue exclusion     | >70%       |          |                  |
| Total viable cell                                                                                                        | visual microscopic count  | >1 x108    |          |                  |
| number1 Tumor reactivity2                                                                                                | J-IFN release vs. peptide | >200 pg/ml |          |                  |
| pulsed T2 cells                                                                                                          |                           |            |          |                  |
| TCR expression2                                                                                                          | FACS analysis of the      | PBL, >10%  |          |                  |
| transduced cells gram stain1,3,                                                                                          | no microorganisms seen                           |            |          |                  |
| Microbiological  studies                                                                                                 | aerobic culture3,4        | no growth  |          |                  |
| fungal culture3,4                                                                                                        | no growth                 |            |          |                  |
| anaerobic culture3,4                                                                                                     | no growth                 |            |          |                  |
| mycoplasma test5                                                                                                         | negative                  |            |          |                  |
| Endotoxin                                                                                                                | limulus assay1            | <5 E.U./kg |          |                  |
| RCR                                                                                                                      | S+L- Assay4               | negative   |          |                  |
| RCR-PCR6                                                                                                                 |                           |            |          |                  |
| 1 Performed on sample of the final product immediately prior to infusion. Results are available at the time of infusion. |                           |            |          |                  |  
1 Performed on sample of the final product immediately prior to infusion. Results are available at the time of infusion.  
2 Performed 2-10 post transduction. Results are available at the time of infusion.  
3 Performed 2-4 days prior to infusion. Results are available at the time of infusion but may not be definitive.  
4 Sample collected from the final product prior to infusion. Results will not be available before cells are infused into the patient. 5 Performed 2-10 days prior to infusion. Results are available at the time of infusion.  
6 Performed on sample approximately 1-4 days prior to infusion. Results are available at the time of infusion.  
Prepared by: _______________________________________ Date: ____________________  
  
  
 QC sign-off: _______________________________________ Date: ____________________  
  
  
  
  
James C. Yang, M.D. or designee