Clinical Evaluation of Intraperitoneal Pseudomonas Exotoxin
`Immunoconjugate OVB3-PE in Patients With Ovarian Cancer
`
`By Lee H. Pai, Michael A. Bookman, Robert F. Ozols, Robert C. Young, John W. Smith II, Dan L. Longo,
`Bruce Gould, Arthur Frankel, Edward F. McClay, Stephen Howell, Eddie Reed, Mark C. Willingham,
`David J. FitzGerald, and Ira Pastan
`
`OVB3-PE is an immunotoxin composed of a murine
`monoclonal antibody reactive with human ovarian
`cancer and conjugated to Pseudomonas exotoxin (PE).
`Twenty-three patients with refractory ovarian cancer
`were treated intraperitoneally (IP) with escalating doses
`of OVB3-PE to study toxicity, pharmacokinetics, antiimmu-
`notoxin antibody formation, and antitumor response.
`Dose-limiting CNS toxicity occurred after repeated doses
`at 5 and 10 pg/kg. Other non-dose-limiting toxicities
`included transient elevation of liver enzymes, fever,
`and gastrointestinal toxicity. Pharmacokinetics of IP
`and serum OVB3-PE were determined in 16 patients.
`Peak peritoneal fluid levels exceeded the in vitro
`median effective dose at all doses tested. At doses of 1
`Ig/kg, the immunotoxin concentration in the
`to 2
`peritoneal fluid remained constant for up to 8 hours
`and dropped to negligible levels after 12 hours. At the
`5 and 10 pg/kg doses, levels remained high for up to
`24 hours (> 100 ng/mL) and then gradually de-
`creased and became undetectable (< 4 ng/mL) after
`72 hours. Serum levels of OVB3-PE were also analyzed
`
`O VARIAN CANCER is a common gyneco-
`
`logic malignancy that accounts for the death
`of 11,600 women per year in the United States.'
`Disseminated peritoneal disease is not amenable
`to surgical cure, and aggressive chemotherapy has
`been used as primary treatment. For those pa-
`tients who fail or relapse after initial cisplatin-
`based chemotherapy, the overall prognosis re-
`mains poor. It is clear that the development of new
`therapeutic approaches for this disease is needed.
`In the past decade, several clinical studies have
`examined the potential therapeutic value of vari-
`ous monoclonal antibody conjugates with plant
`and bacterial toxins, radionuclides, or chemother-
`apeutic agents.2 5 These efforts have been aimed at
`testing the concept that monoclonal antibodies
`could serve as targeting agents to human tumors.
`We have been investigating the utility of immuno-
`toxins composed of antibodies linked to Pseudo-
`monas exotoxin (PE), a 66-kd protein produced by
`P aeruginosa. This protein has been crystallized,
`and x-ray diffraction analysis has revealed three
`prominent domains.6 The amino terminal domain
`
`in 16 patients. At doses of 1 j1g/kg and 2 pg/kg,
`serum levels were not detectable (< 5 ng/mL). How-
`ever, after doses of 5 or 10 ipg/kg, peak serum level
`occurred at 24 hours after each dose and dropped to
`negligible levels by 72 hours. Sera from 12 patients
`were analyzed for anti-PE antibodies and antibodies to
`mouse immunoglobulin (HAMA). All patients devel-
`oped antibodies against PE within 14 days of therapy.
`Domain II of PE appeared to be the most immunogenic
`portion of the PE molecule. HAMA was detected on day
`14 of therapy in nine patients, on day 21 in two, and on
`day 28 in one patient. No clinical antitumor responses
`were observed. We conclude that IP OVB3-PE at dose
`levels of 5 pg/kg (x 3) and 10 jIg/kg (x 2) is accom-
`panied by dose-limiting toxic encephalopathy. Neuro-
`logic toxicity is likely to be due to crossreactivity of
`OVB3 to normal human brain tissue, which was not
`appreciated during preclinical screening.
`J Clin Oncol 9:2095-2103, 1991. This is a US govern-
`ment work. There are no restrictions on its use.
`
`mediates receptor binding (domain Ia); the mid-
`dle one, translocation (domain II); and the car-
`boxyl terminal domain (domain III), the adenosine
`diphosphate (ADP) ribosylation and inactivation of
`
`From the Laboratory of Molecular Biology. Division of
`Cancer Biology, Diagnosis and Center, Bethesda; Medicine
`Branch, Division of Cancer Treatment, National Cancer
`Institute, National Institutes of Health, Bethesda; Biological
`Response Modifiers Program, Fredenck CancerResearch Facil-
`ity, Fredenck, MD; Hematology-Oncology Division, Depart-
`ment of Medicine, Duke University Medical Center, Durham,
`NC; and Cancer Center, University of California, San Diego,
`La Jolla, CA.
`Submitted March 19, 1991; accepted June 11, 1991.
`M.A.B., R.F.O., R.C.Y, and E.R. are currently at the Fox
`Chase Cancer Center, Philadelphia, PA. A.F. is currently at the
`Florida Hospital Cancer and Leukemia Research Center,
`Orlando, FL.
`Address reprint requests to Ira Pastan, MD, Laboratory of
`Molecular Biology, National Cancer Institute, National Insti-
`tutes of Health, 9000 Rockville Pike, 37/4E16, Bethesda, MD
`20892.
`This is a US government work. There are no restrictions on
`its use.
`0732-183X191/0912-0014$0.00/0
`
`Journal of Clinical Oncology, Vol 9, No 12 (December), 1991: pp 2095-2103
`
`2095
`
`Downloaded from jco.ascopubs.org on October 23, 2014. For personal use only. No other uses without permission.
`Copyright © 1991 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2030, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`2096
`
`PAI ET AL
`
`elongation factor 2 (EF-2), which inhibits protein
`synthesis and leads to cell death.7 PE was coupled
`with OVB3, a murine immunoglobulin G2b (IgG2b,)
`antibody that reacts with all human ovarian can-
`cers tested. The resulting immunotoxin, OVB3-
`PE, was shown to kill human ovarian cancer cells
`in cell culture and to prolong the life of nude mice
`bearing human ovarian tumor xenografts.'" Toxin-
`conjugates and conventional chemotherapeutic
`drugs have different mechanisms of action. There-
`fore, those cancer cells with natural or acquired
`resistance to chemotherapeutic drugs cannot be
`crossresistant to toxin-based therapies.
`Ovarian cancer patients with small-volume resid-
`ual disease are good candidates for therapeutic
`evaluation of reagents administered directly into
`the peritoneal cavity. In addition to our own
`studies on ovarian cancer,8 "(cid:127) 0 Griffin et al" have
`shown that immunotoxins constructed with ricin A
`chain and given intraperitoneally (IP) have signifi-
`cant tumoricidal activity in nude mice bearing IP
`human malignant mesothelioma. Pharmacologic
`modeling12 suggests that prolonged IP administra-
`tion of large molecules, such as proteins, results in
`an increased depth of penetration into normal
`parietal tissues such as the diaphragm and abdom-
`inal wall musculature'3 compared with smaller
`molecules. Penetration into tumors has been less
`well characterized but may be no better than that
`observed after intravenous administration. Al-
`though conventional drugs can be cleared from the
`peritoneal space by transmembrane diffusion into
`capillaries, macromolecules are limited to clear-
`ance via tissue lymphatics. Dissemination of ovar-
`ian cancer results in lymphatic obstruction with
`further impairment of IP clearance. Therefore, IP
`immunotoxin should have a particularly prolonged
`half-life in this group of patients. We undertook a
`phase I study of IP OVB3-PE in patients with
`ovarian cancer limited to the peritoneal cavity.
`
`PATIENTS AND METHODS
`Patient Selection
`
`All patients had histologic confirmation of refractory
`invasive epithelial cancer of the ovary limited to the perito-
`neal cavity after platinum-based chemotherapy that could
`be serially evaluated by noninvasive or invasive techniques.
`Other criteria for eligibility included Karnofsky perfor-
`mance status greater than 70 and minimum life expectancy
`of 3 months. Chemotherapy, immunotherapy, or radiation
`therapy was not allowed within 3 weeks of study entry (6
`weeks for nitrosourea or mitomycin). Laboratory criteria
`
`included serum creatinine level less than 2 mg/dL, 24-hour
`creatinine clearance greater than 70 cc/min, serum bilirubin
`level less than 1.5 mg/dL, serum SGOT level less than 50
`IU/L, and prothrombin time less than 14.0 seconds. Pa-
`tients were required to tolerate Tenckhoff catheter inser-
`tion, maintain catheter integrity, and have an adequate
`peritoneal space to permit IP therapy. Patients with disease
`outside the peritoneal cavity, including chest masses or
`effusion, intraparenchymal liver disease, subcutaneous nod-
`ules or CNS metastasis, were excluded. Patients were
`excluded if they had previously received any murine mono-
`clonal antibody or if significant neutralizing antibody to PE
`was demonstrated before treatment. All patients gave
`informed consent before study entry. The study was ap-
`proved by the Clinical Research Subpanel of the National
`Cancer Institute (NCI), the investigational review board of
`the NCI-Frederick Cancer Research Development Center
`(FCRDC), and the Cancer Therapy Evaluation Program of
`the Division of Cancer Treatment.
`
`OVB3-PE
`
`OVB3-PE is an immunotoxin consisting of the murine
`IgG2b monoclonal antibody OVB3 coupled with PE. Preclin-
`ical activity against human ovarian cancer has been de-
`scribed by Willingham et al' and FitzGerald et al. 9 For the
`production of OVB3, mice were immunized with mem-
`branes from the human ovarian cancer cell line OVCAR-3.
`Immune spleen cells were fused with NS-1 cells, and the
`resulting hybridoma was selected on the basis of antibody
`binding to OVCAR cells. OVB3 reacts with a variety of
`ovarian cancer cell lines, fresh ovarian cancer tissue, and
`ascites specimens with minimal reactivity against normal
`tissues. PE was purified from the culture medium of P
`aeruginosa by Swiss Serum and Vaccine Institute, Berne,
`Switzerland. OVB3 was coupled with PE by a thioether
`bond. Clinical lots of OVB3-PE were prepared by the
`Laboratory of Molecular Biology, NCI/National Institutes
`of Health and by Hybritech Inc, LaJolla, CA. Each lot met
`the specifications of United States Pharmacopeia sterility,
`rabbit pyrogenicity, general safety, and murine virus testing.
`The median effective dose of OVB3-PE for inhibition of
`protein synthesis against the ovarian carcinoma cell line
`OVCAR-3 in vitro was 0.5 ng/mL.
`
`Study Design
`
`This was a nonrandomized, fixed, multiple dose-escala-
`tion study. The first group of 19 patients received a single
`cycle of therapy, consisting of a fixed dose of OVB3-PE
`administered IP via a Tenckhoff catheter on days 1 and 4
`(Table 1). Because of two episodes of CNS encephalopathy
`at the 10 pg/kg dose level, the protocol was amended to
`reduce the dose of OVB3-PE to 5 Ig/kg and to administer
`
`Table 1. Study Design of OVB3-PE IP Therapy
`
`Escalating Dose, Fixed Schedule
`
`Fixed Dose, Escalating Schedule
`
`I. 1 pg/kg, days 1,4
`11. 2 gg/kg, days 1,4
`III-A. 5 gg/kg, days 1,4
`IV. 10 pg/kg, days 1,4
`
`III-B. 5 gg/kg, days 1,4
`V. 5 ig/kg, days 1,4, 7
`VI. 5 pig/kg, days 1,3, 5, 7
`
`Downloaded from jco.ascopubs.org on October 23, 2014. For personal use only. No other uses without permission.
`Copyright © 1991 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2030, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`IMMUNOTOXIN AGAINST OVARIAN CANCER
`
`an escalating number of doses (Table 1). Spontaneous
`ascites was drained before each infusion. Before each
`treatment dose, patients received an IP test dose of
`iLg total) over 10 minutes, followed by
`OVB3-PE (20
`observation over 2 hours. In the absence of systemic
`reaction to the test dose, treatment doses were given. For
`each treatment dose, patients received 1 L of normal saline
`followed by immunotoxin dissolved in 50 cc normal saline
`with 5% human serum albumin, followed by an additional 1
`L of normal saline. Patients were monitored closely for vital
`signs and fluid status, with support for anaphylaxis available
`at the bedside. All patients were hospitalized during treat-
`ment and for a minimum of 24 hours after the last dose of
`immunotoxin.
`
`Patient Evaluation
`
`Patients were restaged with noninvasive techniques at the
`end of the first cycle, and those with progressive disease
`were removed from study. Responding patients were eligi-
`ble for additional cycles of therapy at 28-day intervals,
`provided they had not developed anti-OVB3-PE antibodies
`or experienced unacceptable toxicity.
`
`Pharmacology Studies
`
`Ascitic fluid was collected at 5 and 30 minutes and 1, 2, 4,
`8, 12, 24, and 72 hours after each dose. Blood was collected
`at 4, 8, 24, 48, and 72 hours and immediately before each
`dose, then every 24 hours until patient's discharge from
`hospital.
`OVB3-PE was measured in serial samples of ascitic fluid
`and serum by an enzyme-linked
`immunosorbent assay
`(ELISA). Ninety-six-well microtiter plates were coated
`with affinity-purified goat antimouse F(ab') 2 (Jackson Immu-
`noresearch Laboratories, West Grove, PA), and nonspecific
`binding was blocked with 3% gelatin. Test samples were
`diluted 1:10 to 1:1,000 in phosphate-buffered saline (PBS)
`containing 1% bovine serum albumin (BSA) and incubated
`for 1 hour at 37 0C. Rabbit anti-PE (1:2,000 dilution) was
`added for 1 hour and then washed off. Peroxidase-
`conjugated, affinity-purified goat antirabbit antibody (Jack-
`son Laboratories) was added for 30 minutes. The reaction
`was developed using 2.2-azino-di-(3-athyl-benzthiazolinsul-
`fonate 6) (ABTS; Boehringer Mannheim, Indianapolis, IN).
`Optical densities were read at 405 nm in a MicroElisa
`autoreader (Dynatech, Alexandria, VA). For each plate, a
`standard curve of absorbance versus concentration of
`OVB3-PE was generated. The threshold for the peritoneal
`fluid assay was 5 ng/mL and for the serum assay, 4 ng/mL.
`
`Human Anti-PE Antibody
`
`Anti-PE antibody was also measured in serum by ELISA.
`Ninety-six-well microtiter plates were coated with whole
`PE, domain I of PE, domain II plus III (PE-40), or domain
`III of PE, incubated for 90 minutes at 37°C and then
`blocked with PBS-containing 1% BSA. Serum samples were
`added in dilutions beginning at 1:10, incubated, washed,
`incubated with peroxidase conjugates of goat antihuman
`IgG (Jackson Laboratories), and developed using ABTS.
`Results are reported as optical density values of serum
`specimens diluted 1:100.
`
`2097
`
`Human Antimouse Antibody
`Human antimouse antibody (HAMA) was also measured
`in serum by ELISA. Plates were coated with OVB3 and
`blocked with 3% gelatin. Serum samples were added in
`dilutions beginning at 1:10 and incubated for 60 minutes at
`370 C followed by alkaline phosphatase conjugates of goat
`antihuman IgG (Jackson Laboratories), and finally devel-
`oped with ABTS. The results were read at 405 nm. Back-
`ground optical density was approximately 5% of the maxi-
`mum. Positive wells were scored when the optical density
`was at least twice the background.
`
`RESULTS
`
`Patient Characteristics
`
`Twenty-three patients were entered onto the
`trial between November 1987 and November 1989.
`All patients had a history of refractory invasive
`epithelial cancer of the ovary and had received
`and failed prior treatment with a platinum-based
`chemotherapy regimen. Their ages ranged from 39
`to 68 years; the mean age was 53 years. Karnofsky
`performance status ranged from 70 to 90 (median,
`90). All patients had disease limited to the perito-
`neal cavity, except one patient, who had liver
`metastases at the time of therapy. All patients
`were screened and found to be negative for anti-PE
`antibodies before entering the trial. Two patients
`were initially treated despite low levels of neutral-
`izing activity against PE. In these patients
`OVB3-PE was immediately neutralized in vivo,
`and neutralizing activity could not be eliminated
`by peritoneal lavage. Therefore, additional pa-
`tients were only treated in the absence of neutral-
`izing activity. Before treatment, patients under-
`went peritoneoscopy and were found to have an
`adequate peritoneal space to permit IP therapy.
`At the time of the procedure, a Tenckhoff catheter
`(if not already present) was placed for immuno-
`toxin delivery.
`
`Toxicity
`
`All patients entered on the protocol were evalu-
`ated for toxicity (Table 2). Nineteen patients
`developed peritoneal or abdominal pain after
`therapy (83%), seven of which required parenteral
`narcotics for pain control. There was no evidence
`of inflammatory or hemorrhagic peritonitis on the
`basis of peritoneal fluid cell counts (data not
`shown). One patient was found to have abdominal
`infection after the first dose, and therapy was
`discontinued. Twelve patients (52%) developed
`mild nausea and vomiting (grade 1 to 2) easily
`
`Downloaded from jco.ascopubs.org on October 23, 2014. For personal use only. No other uses without permission.
`Copyright © 1991 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2030, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`2098
`
`Table 2. Toxicity
`
`Toxicity
`
`No. of Patients
`
`Abdominal pain
`1
`2
`Nausea and vomiting
`1
`2
`Fever
`2
`Chemical hepatitis
`Elevated transaminases
`1
`2
`Elevated LDH
`1
`2
`Elevated Alk phosp
`1
`2
`Elevated bilirubin
`1
`Neurocortical toxicity
`3
`4
`
`19
`12
`7
`12
`4
`8
`5
`5
`12
`8
`4
`4
`4
`2
`2
`9
`8
`1
`2
`2
`3
`2
`1
`
`%
`
`83
`63
`36
`52
`33
`67
`22
`100
`52
`34
`50
`50
`17
`50
`50
`59
`89
`11
`9
`100
`13*
`66
`33
`
`Abbreviations: LDH, lactate dehydrogenase; Alk phos, alkaline
`phosphatase.
`*Neurocortical toxicity occurred after 10 pIg/kg x 2 (two
`patients) and 5 ig/kg x 3 doses (one patient).
`
`controlled with antiemetics. Eight patients (35%)
`had transient mild elevations of SGOT, SGPT, or
`alkaline phosphatase. Five patients had low-grade
`fever with no infection source.
`Dose-limiting central neurologic toxicity was
`documented in three patients. Two incidents of
`encephalopathy occurred among five patients
`treated at the 10 jig/kg dose level and were
`characterized by confusion, apraxia, and dysar-
`thria beginning several hours after the second
`dose. Both patients eventually recovered after
`several months; one patient has no residual neuro-
`logic deficit, and one has minimal residual apraxia
`and dysarthria. Cranial computed tomographic
`(CT) scans were normal in two patients, whereas
`gadolinium-enhanced magnetic resonance imag-
`ing (MRI)
`in the third patient showed focal
`inflammatory abnormalities in the pons and mid-
`brain (Fig 1). There was no evidence of metastatic
`tumor or carcinomatous meningitis in any of the
`three patients. No neurologic toxicity occurred
`among six patients who received two doses, two
`patients who received three doses, and one patient
`who received four doses, at 5 jig/kg. Fatal neuro-
`toxicity was seen, however, in one patient (no. 16)
`
`PAI ET AL
`
`after a third dose of OVB3-PE at the 5 jig/kg dose
`level. The patient became progressively confused
`and, within 6 hours of treatment, developed a
`severe acute encephalopathy characterized by my-
`oclonus, disorientation, apraxia, and dysarthria.
`By 12 hours after treatment, she developed petit
`mal seizures and an upper gastrointestinal bleed.
`This was followed by coma and a grand mal
`seizure requiring both diphenylhydantoin and phe-
`nobarbital for control. She was transferred to the
`intensive care unit and intubated. Lumbar punc-
`ture revealed a CSF protein of 700 to 800 mg/dL
`and WBC count of 2/iLL to 3/jIL; no OVB3-PE
`was detected
`in the CSF by ELISA. CT scan
`without contrast was negative; MRI revealed in-
`flammatory abnormalities in the brain stem, cere-
`bellum, deep nuclei, and deep white matter. The
`patient remained comatose. Five days after treat-
`ment, CT scan showed increased edema with
`sulcal and cisternal effacement. Electroencephalo-
`gram showed persistent seizure activity. Aggres-
`sive life support was discontinued, and the patient
`died 12 days posttherapy of respiratory failure. An
`autopsy was not performed. This toxicity necessi-
`tated immediate termination of the study.
`
`Response
`No objective partial or complete antitumor
`responses were noted
`in any of the patients.
`Peritoneal fluid cytology remained positive in all
`
`Fig 1. Gadolinium-enhanced MRI of patient no. 12, show-
`ing increased signal intensity in the pans and midbrain.
`
`Downloaded from jco.ascopubs.org on October 23, 2014. For personal use only. No other uses without permission.
`Copyright © 1991 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2030, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`2099
`
`E
`
`300
`
`250
`
`200-
`
`150'
`
`100.
`
`50-
`
`9
`
`0.
`
`! I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`0
`-12 12 36 60 84 108-132156180
`time
`
`(hours)
`
`Fig 3. Ascitic fluid (0) and serum levels (0) of OVB3-PE of
`patient no. 16, who received immunotoxin 5 pg/kg on days 1,
`3, and 5. Levels in CSF were undetectable. Arrow indicates the
`onset of neurotoxicity after the third dose of immunotoxin.
`
`immunotoxin present in the peritoneal cavity.
`Table 3 shows the peak immunotoxin levels in the
`peritoneal fluid of these patients after each dose.
`Serum concentration of OVB3-PE. Concentra-
`tions of OVB3-PE in the serum were determined
`in 16 patients. At the dose level of 1 and 2 t±g/kg,
`no significant amounts of intact OVB3-PE (ie, < 4
`ng/mL) were detected. At the dose level of 5
`pCg/kg, six of six patients who received IP infusions
`
`Table 3. Peak OVB3-PE Level in Peritoneal Fluid
`
`Schedule/Patient No.
`1 pg/kg days 1 and 4
`1
`2
`3
`2 ýg/kg days 1 and 4
`4
`5
`6
`5 ig/kg, days 1 and 4
`7
`8
`9
`10 ig/kg, days 1 and 4
`10
`11
`12
`13
`5 p.g/kg, days 1,4, 7
`14
`15
`5 Rg/kg, days 1,3, 5
`16
`
`First Dose
`
`Second Dose
`
`Third Dose
`
`45
`35
`28
`
`90
`60
`90
`
`120
`210
`400
`
`430
`346
`500
`342
`
`400
`250
`
`182
`
`35
`25
`25
`
`92
`92
`110
`
`410
`560
`550
`
`310
`324
`380
`338
`
`400
`360
`
`265
`
`-
`-
`-
`
`-
`-
`-
`
`-
`-
`-
`
`-
`-
`-
`-
`
`500
`400
`
`240
`
`IMMUNOTOXIN AGAINST OVARIAN CANCER
`
`patients in whom positive cytology was present
`before therapy.
`
`Pharmacokinetics
`Peritoneal fluid. Peritoneal fluid from 16 pa-
`tients was analyzed for the concentration of intact
`OVB3-PE using a sandwich ELISA. At the dose
`level of 1 I.g/kg, the concentration remained
`stable up to 4 hours after infusion, then gradually
`decreased to undetectable levels (< 5 ng/mL) 12
`to 24 hours after each dose (days 1 and 4). At the 2
`pig/kg dose level, immunotoxin concentration re-
`mained high for up to 8 hours and became unde-
`tectable 12 to 24 hours after the first and second
`doses. At the dose level of 5 jig/kg, four of six
`patients had high concentrations of OVB3-PE
`(> 50 ng/mL) in the peritoneal fluid 24 hours
`after the first dose; it became undetectable after
`72 hours. Figure 2 shows a pharmacokinetic curve
`of patient no. 7, who received 5 RIg/kg of immuno-
`toxin on days 1 and 4. Two patients received a
`third dose of OVB3-PE on day 7, and the pattern
`of clearance did not differ greatly from the first
`two doses. Patient no. 16 received three doses of
`OVB3-PE at 5 Rig/kg/dose on days 1, 3, and 5. The
`concentration of OVB3-PE remained greater than
`100 ng/mL 24 hours after each dose and greater
`than 50 ng/mL before each dose. It became
`undetectable only 96 hours after the last dose (Fig
`3). At the 10 jig/kg dose level, concentrations of
`OVB3-PE remained greater than 100 ng/mL up to
`12 hours after each infusion, but gradually de-
`creased to insignificant levels after 72 hours in all
`cases. Before each dose, there was no detectable
`
`--
`7'
`
`61
`
`51
`
`4(
`
`31
`
`21
`
`14
`
`time
`
`(hours)
`
`E a
`
`t
`
`c
`
`Fig 2. Concentration of OVB3-PE in ascitic fluid of patient
`no. 7, who received OVB3-PE 5 ig/kg IP on days 1 and 4.
`
`NOTE. Values indicate ng/mL at 2 to 4 hours after IP adminis-
`tration.
`
`Downloaded from jco.ascopubs.org on October 23, 2014. For personal use only. No other uses without permission.
`Copyright © 1991 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2030, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`2100
`
`on day 1, day 4, or days 1, 4, and 7 had very low
`serum levels of OVB3-PE 24 hours after each IP
`infusion (range, 4 to 13.6 ng/mL). Serum levels
`remained constant and were still detected 72
`hours after the first IP dose but were negligible
`before the second dose at 96 hours. Patient no. 16
`received three doses of OVB3-PE 5 pýg/kg on days
`1, 3, and 5. The immunotoxin was detected 8 hours
`after IP infusion (5 ng/mL), remained detectable
`(6 ng/mL) before the second dose, and increased
`in a cumulative fashion after the second and third
`doses (Fig 3). At the dose level of 10 (xg/kg, serum
`levels were detected at 24 hours after infusion,
`with a mean level of 19 ng/mL (range, 7 to 40
`ng/mL). It was no longer detectable before the
`second dose (96 hours after infusion). One patient
`did not receive a second dose due to abdominal
`pain. The mean level at 24 hours after the second
`dose was 13 ng/mL (range, 7 to 23 ng/mL). One
`patient had a peak serum level of 40 ng/mL 48
`hours after the second IP infusion, which gradu-
`ally decreased to insignificant levels after 96 hours.
`
`Anti-PE Antibodies
`Serum samples from 12 patients were analyzed
`for antibodies against PE. This toxin is composed
`of three structural domains, thus, analysis was
`performed against both whole PE and the individ-
`ual domains. All patients developed antibodies
`
`PAl ET AL
`
`against PE within 14 days of therapy. No major
`difference in the magnitude of antibody response
`was noted among the different treatment groups.
`Results are shown in Fig 4 and reported as optical
`density values of serum specimens on day 14,
`diluted 1:100. In all treatment groups, antibody
`responses to isolated domains I (amino acids 1 to
`252) and III (amino acids 400 to 613) were low as
`compared with PE40 (amino acids 253 to 613). We
`were unable to prepare sufficient quantities of
`domain II to measure reactivity directly, but we
`interpret our results to conclude that domain II is
`the most immunogenic portion of the PE. Due to
`formation of anti-PE antibodies, none of the
`patients received a second cycle.
`
`HAMA
`Serum samples from 12 patients were analyzed
`for the appearance of HAMA IgG as long as 4
`weeks after therapy (Table 4). HAMA became
`detectable within 14 days after the first dose of
`OVB3-PE in nine patients, within 21 days in two
`patients, and on day 28 in one patient. No major
`difference in the magnitude of antibody response
`was noted in different treatment groups.
`
`DISCUSSION
`When OVB3 is chemically coupled with PE, an
`immunotoxin is produced that, when administered
`
`--
`Z.U
`
`1.5
`
`ErS
`
`1.0
`
`3
`
`1
`
`18
`
`8
`
`7
`
`19
`
`17
`
`0.5
`
`0.0
`
`B
`
`2.0
`
`'10
`
`E
`LO
`0 1.0
`6 0.
`0 0.5
`
`0.0
`
`D
`
`5
`
`4
`
`11
`
`10
`11 10
`
`12
`12
`
`Fig 4. Histogram of human
`antibody response (by patient
`UdfU* *
`* U,* L UII U,,,fl fl
`Ito
`an Ulrentll
`oll(cid:127)ans
`no.j
`of PE 14 days after therapy with
`escalating doses of OVB3-PE IP:
`(A) 1 pig/kg, days 1 and 4; (B) 2
`iLg/kg, days 1 and 4; (C) 5 aIg/
`kg, days 1 and 4; (D) 10 Rg/kg,
`days 1 and 4. Antibody response
`was measured by ELISA, and re-
`sults are reported as absorbance
`(O.D.) values of serum speci-
`mens diluted 1:100. PE (0), do-
`main I of PE (tB. domain III of PE
`(
`.
`(m), and PE40 ([]).
`,
`.
`(...
`andPE40
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`E
`OU,
`
`6
`
`A
`
`E oI
`
`, 0 6
`
`A
`C
`
`Downloaded from jco.ascopubs.org on October 23, 2014. For personal use only. No other uses without permission.
`Copyright © 1991 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2030, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`IMMUNOTOXIN AGAINST OVARIAN CANCER
`
`Table 4. Antibody Against Mouse IgG After IP Therapy
`With OVB3-PE
`
`Patient No.
`
`Dose (Lg/kg)
`
`4
`
`2
`3
`I
`5
`4
`8
`17
`18
`11
`10
`12
`
`1
`1
`I
`2
`2
`5
`5
`5
`10
`10
`10
`
`-
`-
`
`-
`-
`
`-
`-
`
`-
`
`Days Posttherapy
`
`7
`
`+
`-
`
`+
`+
`
`-
`
`14
`
`+
`+
`
`+
`
`+
`
`+
`+
`
`28
`
`21
`
`+
`
`+
`
`+
`+
`
`+
`+
`
`+
`
`intraperitoneally at a dose of 50 to 100 KIg/kg,
`prolongs the survival of mice bearing IP human
`ovarian cancer xenografts. 8-9 It also prolongs the
`survival of mice with an IP human colon tumor
`(HT-29) when given IP either alone or with
`cyclophosphamide. 14 Doses greater than 100 lg/kg
`could not be administered to mice because of liver
`toxicity. OVB3 reacts with an unknown antigen on
`the surface of several human adenocarcinomas. It
`also reacts with a small number of normal human
`tissues. However, it does not react with any mon-
`key or rodent tissues. A clinical trial with OVB3-PE
`was initiated to determine dose-limiting toxicities
`and the pharmacokinetics of this immunotoxin.
`Based on antibody localization studies using fro-
`zen sections of normal human tissue, pancreatic
`and thyroid toxicity was anticipated. However,
`toxicities to these organs were not observed; in-
`stead, neurocortical toxicity proved to be dose-
`limiting. This was manifested by a severe acute
`encephalopathy characterized by confusion,
`apraxia, and dysarthria in three patients. One
`patient progressed with seizures and coma, which
`eventually lead to her death. MRI in this patient
`showed inflammatory abnormalities in the brain
`stem, cerebellum, deep nuclei, and deep white
`matter.
`We subsequently obtained fresh samples from
`various portions of normal human brain and per-
`formed immunohistochemical studies. In one sam-
`ple we were able to detect a weak reactivity of
`OVB3-PE with the molecular layer of the cerebel-
`lum; it was negative for cerebellar granular layer,
`white matter, capillaries, and cortical gray matter.
`Similar studies were performed in monkey brain
`tissue, which showed no reactivity to OVB3-PE.
`
`2101
`
`This probably explains the lack of neurologic
`symptoms in preclinical experiments performed in
`these primates. Although we were not able to
`detect the presence of OVB3-PE in the CSF, it is
`possible that small amounts of the immunotoxin
`(not detectable by our assay, ie, < 4 ng/mL) may
`have entered the cerebrospinal space and dam-
`aged critical cells in the brain due to crossreactiv-
`ity with the monoclonal antibody OVB3. It is
`unlikely that the neurotoxicity seen in these pa-
`tients was due to free PE, since the thioether bond
`used for conjugation is known to be very stable.
`Furthermore, in experiments performed in mon-
`keys, we administered OVB3-PE intravenously at
`doses of 50 I±g/kg on days 1 and 4 and achieved
`serum blood levels of several hundreds of nano-
`grams per milliliter, which fell to 44 ng/mL at 24
`hours, yet no signs of neurotoxicity were observed.
`Two other PE-containing immunotoxins, anti-
`Tac-PE and B3-PE, also have been administered
`to monkeys in preclinical studies at doses up to 66
`pIg/kg with no signs of neurotoxicity (Pai et al,
`manuscript submitted, September 1991). There-
`fore, we conclude that the neurotoxicity observed
`in this trial was most likely due to the specific
`reactivity of OVB3 with brain cells-reactivity that
`was not detected in the preclinical screening-
`rather than due to free PE. Other side effects in
`this trial include transient grade 1 to 2 elevation of
`liver enzymes attributed to PE. In a phase I trial
`using monoclonal antibody coupled with ricin A
`chain for metastatic colon carcinoma,15 the nonspe-
`cific side effects included proteinuria, decreased
`serum albumin, and flu-like symptoms; however,
`these side effects were not observed in our study.
`Analysis of the levels of OVB3-PE in the perito-
`neal fluid and blood showed that intact immuno-
`toxin was present at concentrations that were in
`excess of that necessary to kill many different types
`of ovarian cancer cell lines. The immunotoxin
`remained at high levels within the peritoneal
`cavity for prolonged periods, as shown by our
`pharmacokinetic data. Thus, the OVB3-PE was
`not rapidly cleared by binding to antigen, by
`proteolytic degradation, or by rapid entry into the
`circulation or lymphatics. Also, once the immuno-
`toxin did reach the blood, it remained detectable
`for several days. Despite the high IP levels of
`immunotoxin, no clear clinical response was ob-
`served in this trial. Immunotoxin delivered by the
`IP route may not have had adequate distribution.
`
`Downloaded from jco.ascopubs.org on October 23, 2014. For personal use only. No other uses without permission.
`Copyright © 1991 American Society of Clinical Oncology. All rights reserved.
`
`IMMUNOGEN 2030, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`2102
`
`PAI ET AL
`
`It is also possible that the cytotoxic activity of the
`immunotoxin was limited to cells that are in the
`periphery of the tumor mass and cells present in
`the ascitic fluid, and because of poor penetration
`to bulk disease, no clear clinical response was
`detected. Unfortunately, in the few patients with
`ascites that contained tumor cells, we were unable
`to obtain samples necessary to perform immunohis-
`tochemical analysis. In preclinical studies per-
`formed in mice, 50 to 100 kg/kg of OVB3-PE was
`required to demonstrate activity in nude mice
`bearing ovarian cancer xenografts, and we were
`not able to deliver that amount to patients due to
`CNS toxicity.
`Antibodies to PE developed 10 to 14 days after
`initial administration. PE is a highly immunogenic
`molecule, and this was anticipated in these nonim-
`munosuppressed patients. Major reactivity oc-
`curred with domain II of PE (amino acids 253 to
`364). This region also appears to be highly immu-
`nogenic in mice.16 To permit therapy for longer
`than 10