`NEUROSURGERY
`
`OFFICIAL JOURNAL OF THE CONGRESS OF NEUROLOGICAL SURGEONS
` 7005
` Radical Resection of Meningiomas and Arteriovenous Fistulas
`':CI.INICA " teams 5.
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`LINEAR ACCELERATOR
`RADIOSURGERY FOR CRANIAL
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`BASE MENINGIOMAS
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`Frags: Hi 3 “3"
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`;
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`l
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`Involving Critical Dural Sinus Segments: Experience with lntraoperative
`Sinus Pressure Monitoring and Elective Sinus Reconstruction in 10 Patients:
`
`Robert Sclimiclflsaesser, HansJakob Steiger, Tarek Yousry, Klaus Christian See/Os, HansJiirgen Reulen
`Treatment of Cranial Base Meningiomas with Linear Accelerator Radiosurgery:
`Steven D. Chang, john R. Adler, Ir.
`
`Quantitative Imaging Study of Extent of Surgical Resection and Prognosis of Mali mutt Astroe ’tolpas:
`Andrew Kowalczuk, R, Loch Macdonald, Chris Amidei, George Dohrmann III,
`I WEb Site Feallljil
`Robert K, Erickson, javad Hekmatpanah, Stuart Krauss, Siva/Ira Krislmasamy, Gregory Masters,
`Sean Ft Mullah, Arno j,Mund/, Patrick Sweeney, Everett E. Vokes, Bryce K.A Weir, Robert L Wollman
`Intraventricular Immunotoxin Therapy for Leptomeningeal Neoplasia:
`Douglas W. Laske, Karin M. Muraszko, Edward H. Oldfield, Hetty L. DeVroom, Cynthia Sung,
`Robert L. Dedrick, Theodore R. Simon, jean Colandrea, Christie Copeland, David Katz, Larry Greenfield,
`Eric 5, Groves, LL Houston, Richard j. You/e
`
`Tenascin-C Expression in the Cyst Wall and Fluid of Human Brain Tumors Correlates with Angiogenesis:
`George I. jal/o, David R. Fried/antler, Patrick 1. Kelly, jeffrey H: Wisot'f, Martin Grumet, David Zagzag
`Mass Effect Caused by Clinically Unruptured Cerebral Arteriovenous Malformations:
`Yoshio MiyasakaiAkira Kurata, Ryusui Tanaka, Shigeki Nagai, Masaru Yamada, Katsumi Irikura, Kiyotaka Fu/ii
`Evaluating the Effect of Superficial Telnporal Artery to Middle Cerebral Artery Bypass on Pure Motor
`Function Using Motor Activation Single Photon Emission Computed Tomography:
`Shoichiro Kawaguchi, Hiroshi Noguchi, Toshisuke Sakaki, Tetsuya Morimoto,
`Toru Hoshida, Taiji Yonezawa,Te/uhiko lmai, Haj/me Ohishi
`Risk Factors for Neurosurgical Site Infections after Craniotomy: A Prospective Multicenter Study of 2944 Patients:
`WCb Site Feature]
`AnneiMarie Korinek, the French Study Group oiNeuI'oiur‘gica/ infections, the SEHP, and the C—CL/N ParisNorrl
`Continuous Monitoring of Cerebral Substrate Delivery and Clearance:
`Initial Experience in 24 Patients with Severe Acute Brain Injuries:
`Alois Zauner, Egon M.R. Doppenberg, john j. Woodward, Sung C. Choi, Harold F. Young, Ross Bullock
`Surgical Resection of Intramedullary Spinal Cord cavernous Malformations: Delayed Complications, Long-term Outcomes, and Association with
`Cryptic Venous Malformations:
`A. Giancarlo Vishteh, Suresh San/(hla, john A. Anson, joseph M. Zabramski, Robert F. Spetzlei
` INTRAVENTRICULAR
`
`IMMUNOTOXIN FOR
`lntramedullary Pressure in Syringomyelia: Clinical and Pathophysiological Correlates of Syrinx Distension:
`
`
`N
`LEPTOMENENGEAL
`Thomas H‘ Mil/iorat, Anthony L. Capocelli, jr., Rene M. Kolzen, Paolo Bolognese, Ian M. Heger, james E. Cottrell
`Outcome For Preterm Infants with Germinal Matrix Hemorrhage and
`Progressive Hydrocephalus:
`Michael L. Levy, Lena S. Masri, 1. Gordon McComb
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`7079
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`7020
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`7039
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`7052
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`7060
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`7065
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`7073
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`7082
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`7094
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`7702
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`7777
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`suaqucatg-Maromv AND TECHNIQUE _
`7779
`Direct Microsurgery of Dural Arteriovenous Malformation Type
`Carotid-Cavernous Sinus Fistulas:
`Indications, Technique, and Results:
`1. Diaz Day, Takanori Fukushima
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`TENASCIN-C EXPRESSION
`AND TUMOR ANGIOGENESIS
`Page ESE»???
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`7727
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`Occipitotranstentorial Approach for Lesions of the Superior Cerebellar Hemisphere:
`Technical Report:
`Ronald H.M.A. Bane/5, joost cle Vries, jacobus 1. Van Overbeeke, j. André Grotenhuis
`Experimental Study for Identification of the Facial Colliculus Using Electromyography
`and Antidromic Evoked Potentials:
`
`7130
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`Kyouichi Suzuki, Masato Matsumoto, Mamoru Ohta, Tatsuya Sasaki,‘ Narnia Kodama
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`IMMUNOGEN 2300, pg. 1
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`IMMUNOGEN 2300, pg. 1
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`lntraventricular immunotoxin Therapy for
`Leptomeningeal Neoplasia
`
`
`Douglas W. Laske, M.D., Karin M. Muraszko, M.D.,
`Edward H. Oldfield, M.D., Hetty L. DeVroom, R.N.,
`Cynthia Sung, Ph.D., Robert L. Dedrick, Ph.D.,
`Theodore R. SimOn, M.D., Jean Colandrea, M.D.,
`Christie Copeland, C.T., David Katz, M.D.,
`Larry Greenfield, M.D., Eric S. Groves, M.D.,
`LL. Houston, Ph.D., Richard J. Youle, Ph.D.
`
`Surgical Neurology Branch (DWL, KMM, EHO, HLD, RJY), National Institute ot
`Neurological Disorders and Stroke, Biomedical Engineering and Instrumentation
`Program (CS, RLD), Division of Intramural Research Resources, National Center for
`Research Resources, Nuclear Medicine Department (TRS), Clinical Center, Division
`of Cancer Biology and Diagnosis (JC, CC, DK), Laboratory of Pathology, National
`Cancer Institute, and Office of the Clinical Director (DK), National Institute of
`Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland,
`and Cetus Corporation (LG, ESQ, LLH), Emeryville, California
`
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`l OBJECTIVE: The goals of this clinical trial of intraventricular 454A12-rRA therapy were to identify dose-limiting
`toxicities, to evaluate the pharmacokinetics of single—dose intraventricular 454A12-rRA, and to detect antitumor
`activity.
`METHODS: We performed a pilot study of intraventricular therapy with the immunotoxin 454A12-rRA in eight
`patients with leptomeningeal spread of systemic neoplasia. The immunotoxin 454A12—rRA is a conjugate of a
`monoclonal antibody against the human transferrin receptor and recombinant ricin A chain, the enzymatically
`active subunit of the protein toxin ricin. Patients were treated with single doses of 454A12-rRA ranging from
`1.2 to 1200 pg.
`RESULTS: The early phase half-life of 454A1 2-rRA in ventricular cerebrospinal fluid (CSF) averaged 44 i 21 minutes,
`and the late phase half-life averaged 237 i 86 minutes. The clearance of the immunotoxin was faster than the
`Clearance of coinjected technetium-99m-diethylenetriamine penta-acetic acid, averaging approximately 2.4-fold
`greater. No 454A12-rRA degradation was detected by Western blot analysis of ventricular CSF for a period of
`24 hours, and bioactivity was retained in CSF paralleling the concentration of immunotoxin. No acute or chronic
`drug toxicity was identified in patients who received less than or equal to 38 Mg of 454A12-rRA by intraven-
`tricular injection. Doses more than or equal to 120 pig caused a CSF inflammatory response that was associated
`with transient headache, vomiting, and altered mental status. This acute syndrome was responsive to steroids and
`CSF drainage. No systemic toxicity was detected. In four of the eight patients, a greater than 50% reduction of
`tumor cell counts in the lumbar CSF occurred within 5 to 7 days after the intraventricular dose of 454A12-rRA;
`however, no patient had their CSF cleared of tumor, and clinical or magnetic resonance imaging evidence of
`tumor progression was demonstrated in seven of the eight patients after treatment.
`CONCLUSION: Tumoricidal concentrations of the immunotoxin 454A12-rRA can be attained safely in the CSF of
`patients with leptomeningeal tumor spread. (Neurosurgery 41 :10394051, 1997)
`KEV words: Antibody, Cancer, Cerebrospinal fluid, Pharmacokinetics, Ricin toxin
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`Neurosurgery, Vol. 4/, N0. 5, November 1997
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`1039
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`IMMUNOGEN 2300, pg. 2
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`IMMUNOGEN 2300, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
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`complexes into cells and are overexpressed on rapidly divid.
`ing cells, including cancer cells, reflecting increased iron re.
`quirements (15, 17, 18, 32, 51, 57759). Within the normal adult
`CNS,
`transferrin receptors are sparse and are largely re-
`stricted to the luminal surface of brain capillaries (3, 12, 26,
`28). Ricin, a potent protein toxin consisting of two subunits,
`binds cells and, when internalized, kills them by enzymati—
`cally inactivating protein synthesis (16, 40, 69). Recombinant
`ricin A chain, the catalytic subunit, lacks the 13 chain that
`mediates nonspecific binding and internalization. By linking
`ricin A chain to certain monoclonal antibodies, potent cyto—
`toxic drugs are produced that are specific for cells bearing the
`antigen recognized by the antibody (20, 31, 68, 72). The im—
`munotoxin 454A12—rRA demonstrates
`cytotoxic
`activity
`against many tumor cell lines, including breast carcinomas,
`leukemia, glioblastomas, and medulloblastomas (29, 72).
`This study initiates the evaluation of immunotoxin therapy
`for human CNS tumors. The objectives of this study were to
`identify dose-limiting toxicities for intraventricular 454A12-
`rRA therapy, to evaluate the pharmacokinetics of immuno—
`toxins in CSF, and to detect in vivo antitumor activity against
`human tumors.
`
`PATIENTS AN D METHODS
`
`Patients
`
`Patients who were more than 18 years of age and not
`pregnant were eligible for this study if their CSF prepared by
`cytocentrifugation contained malignant cells, they had a con—
`firmed tissue diagnosis of cancer, and their Karnofsky perfor-
`mance score was greater than or equal to 30 (30). Patients
`were excluded if they suffered obstructive hydrocephalus
`requiring a shunt or if they received previous therapy with
`immunotoxin conjugates, murine monoclonal antibodies, or
`ricin. The protocol was approved by the institutional review
`board of the National Institutes of Health (National Institute
`of Neurological Disorders and Stroke protocol No. 90—N436).
`Written informed consent was obtained from all patients or
`their responsible next—of—kin.
`
`Drug preparation
`
`lmmunotoxin 454A12—rRA was supplied by Cetus Corpo—
`ration (Emeryville, CA). This immunotoxin is composed of a
`murine immunoglobulin (1g)G1 subclass monoclonal anti—
`body, 454A12, covalently coupled to recombinant ricin A chain
`via a disulfide linkage. Monoclonal antibody 454A12 binds to
`the human transferrin receptor (7). The immunotoxin was a
`mixture of antibody coupled to recombinant ricin A chain in
`a 1:1 molar ratio, a 1:2 molar ratio, and a 1:3 and higher molar
`ratios. The percent composition of the immunotoxin was 27%
`(1:1), 32% (1:2), 22% (1:3), and 18% greater than 1:3. There was
`no detectable free antibody or recombinant ricin A chain. The
`composition, purity, and reproducibility of the immunotoxin
`were approved by the Food and Drug Administration in
`an investigational new drug application. The immunotoxin
`454A12—rRA was supplied in vials containing 4.4 mg of ly—
`ophilized drug formulated with human serum albumin and
`
`median survival of only a few months (10, 19, 21, 22, 41,
`52, 55, 66). In adults, breast and lung carcinoma cause the
`majority of cases of leptomeningeal neoplasia (2, 39, 66). Stud—
`ies indicate that 35 to 40% of all breast cancer patients expe-
`rience central nervous system (CNS) involvement at some
`point in their disease and that 5% experienceAleptomeningeal
`involvement (54, 61, 66). In addition, primary CNS tumors,
`including medulloblastomas, ependymomas, pineal tumors, and
`gliomas, can spread diffusely to the leptomeninges, thwarting
`efforts at treatment.
`
`Leptomeningeal neoplasia produces a clinical course that is
`characterized by progressive neurological deficits (21, 41, 62,
`66). Cerebral symptoms, which include headache, changes in
`mental status, and seizures, occur in 50% of patients, cranial
`nerve symptoms arise in 38%, and spinal nerve root symp—
`toms affect 70%. With leptomeningeal spread of systemic
`cancers, the median survival in untreated patients and pa—
`tients unresponsive to treatment is less than 2 months (41, 55).
`With aggressive treatment, including radiation therapy and
`intrathecal methotrexate,
`the average survival is only pro—
`longed to 6 to 7 months (19, 41, 66). However, even with
`aggressive treatment, 40 to 50% of patients fail to stabilize.
`Nonetheless, meningeal involvement with cancer is often not
`a premorbid phenomenon and in two—thirds t0 three—quarters
`of patients, it occurs when systemic disease is stable or in
`complete remission (19, 66). Effective therapy in these patients
`would significantly extend survival.
`Chemotherapy is commonly used for leptomeningeal neo-
`plasia; however, it can have serious side effects. The most
`well-known and studied chemotherapeutic agents used intra—
`thecally are methotrexate, cytosine arabinoside, and thiotepa
`(9, 19, 24, 42, 62, 66). Intraventricular methotrexate is the most
`effective current treatment for meningeal spread of various
`tumors, including breast cancer (42). However, long—term use
`of methotrexate, particularly in high doses and in patients who
`have received radiation therapy, may cause significant CNS
`toxicity, including necrotizing leukoencephalopathy (62).
`The development of monoclonal antibodies that recognize
`specific tumor—associated antigens provides the possibility of
`creating more specific therapeutic reagents. Antibodies have
`been conjugated to radionuclides and various peptide toxins
`to create new drugs with high tumor selectivity in vitro (7, 20,
`25, 31, 33, 57, 60, 67, 68). However, attempts to develop these
`compounds for clinical use indicate that transcapillary and
`interstitial barriers limit the delivery of antibodies or antibody
`conjugates into solid tumors. In leptomeningeal neoplasia, the
`malignant cells often grow as thin sheets bathed in cerebro—
`spinal fluid (CSF) (41), which reduces the problems of drug
`delivery and tissue penetration;
`therefore,
`leptomeningeal
`neoplasia is an attractive candidate for intrathecal therapy
`with antibody—toxin conjugates (immunotoxins).
`We conducted a pilot study of intraventricular therapy with
`the immunotoxin 454A12—rRA, a chemically linked conjugate
`of a monoclonal antibody against the human transferrin re—
`ceptor (454A12) and a protein toxin (recombinant ricin A
`chain [rRADQ Transferrin receptors transport iron—transferrin
`
`Neurosurgery, Vol. 47, N0. 5, November 7997
`
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`IMMUNOGEN 2300, pg. 3
`Phigenix v. lmmunogen
`|PR2014-00676
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` patients and results in a very poor prognosis, with a
`
`I eptomeningeal neoplasia occurs in 5 to 20% of all cancer
`
`1040
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`Laske et al.
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`IMMUNOGEN 2300, pg. 3
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`was reconstituted with 2.2 ml of 0.9% sodium chloride,
`
`preservative—free. Subsequent dilutions Were prepared with
`0.1% human serum albumin in normal saline.
`
`Trial design
`Pretreatment evaluation included baseline physical exami—
`nation, Karnofsky performance score determination, and
`gadolinium-enhanced magnetic resonance imaging (MRI) of
`the head and complete spine. Patients were required to have
`liver and renal function values of less than two times normal
`values, normal metabolic parameters, and a normal coagula~
`tion profile.
`A right frontal reservoir (Ommaya type) was attached to a
`catheter with the tip in the right frontal horn of the lateral
`ventricle, if not already present. Ventricular and lumbar CSF
`samples were obtained before therapy for cell count with
`differential, total protein, glucose, lactate dehydrogenase (LDH)
`isoenzymes, and cytology (5, 6, 22, 41, 44, 61, 62, 64, 70). An
`estimate of the number of malignant cells in the lumbar
`CSF was determined. Before treatment, all patients under—
`went teChnetium—99m—diethy1enetriamine penta—acetic acid
`(99”‘Tc—DTPA) ventriculocisternography to evaluate flow
`within the CSF pathways (see Fig. 1) and, 12 to 18 hours before
`intraventricular injection of 454A12—1‘RA, a lumbar subarach—
`noid drain was placed for frequent lumbar CSF sampling.
`Treatment consisted of a single dose of 454A12—1‘RA delivered
`through the Ommaya reservoir in a constant injection volume
`of 10 ml, and then barbotage of the reservoir was performed
`four times.
`
`The initial dose of 454A12—rRA was 1.2 [.Lg (to yield an
`estimated CSF concentration of 5.6 X 10“11 mol/L if uni—
`formly distributed over the predicted CSF volume of approx-
`imately 120 ml). This dose was chosen because it was calcu—
`lated to result in an expected CSF concentration 2000—fold
`lower than that which produced toxicity in preclinical animal
`studies and because the calculated CSF concentration was
`
`within the lower range in which human tumor cells were
`killed in vitro (38, 72). The first patient received a total of three
`
`FIGURE 1. 99mTc-DTPA
`
`ventriculocisternography
`(left lateral planar gamma
`camera image). Image
`obtained in Patient 1
`30 minutes after
`intraventricular injection of
`265 ”Ci of 99'"Tc-DTPA
`(injection was administered
`into an Ommaya reservoir
`and barbotage was
`performed four times), which
`demonstrates satisfactory
`distribution through the
`foramen magnum (arrow)
`into the spinal subarachnoid
`space (arrowheads).
`
`
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`4
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`lntraventricular Immunotoxin Therapy
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`1041
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`doses that were increased by 1/2 log increments; subsequent
`patients (two patients per dose level) received single doses
`that were increased by 1/2 log increments. A very low initial
`dose and extensive preclinical pharmacokinetic and toxicity
`data (38) supported the use of
`the rapid dose—escalation
`scheme.
`
`Pharmacokinetics
`
`To compare the CSF pharmacokinetics of 454A12-rRA to
`bulk CSF flow, 200 to 550 qui of 99mTc—DTPA was coinjected
`with the immunotoxin through the Ommaya reservoir (4, 11,
`25, 36, 38). Using an aseptic technique, CSF samples of 2.0 ml
`were obtained from the Ommaya reservoir and the lumbar
`drain 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours after injection. The
`454A12-rRA concentration was determined by an enzyme—
`linked immunosorbent sandwich assay (36), and measure—
`ment of 99mTc—DTPA was performed by gamma counting
`(Packard B5003; Hewlett Packard, Avondale, PA). The amount
`of 99mTc—DTPA in each sample was determined after correction
`for the rapid isotope decay (half-life of 6 h). Additional informa-
`tion on the clearance of the labeled tracer was obtained by
`sequential gamma camera imaging. Gamma camera images
`(Siemens Orbiter Camera,
`low energy all purpose colli—
`mator; Siemens Medical Systems, Hoffman Estates, IL; and
`Elscint Apex Computer; Elscint, Inc., Hackensack, Nj) were
`obtained 0, 0.5, 4, and 24 hours after injection of 99mTc-DTPA.
`Biexponential decay curves of the form Concentration = a
`exp(—)\1t) + B exp(*)\2t) were fit to the ventricular (99”‘Tc—
`DTPA and 454A12—rRA) data of each patient using IMSL
`subroutines (lMSL lnc., Houston, TX). The IMSL subroutines
`perform nonlinear regressions by means of a finite difference
`Levenberg-Marquardt algorithm. The ratio R, equal to a/(a+B),
`represents the fractional contribution of the early or u-phase.
`The half—lives of the two phases, t, H” and t-J p43, are equal to
`(ln 2)/)\l and (ln 2)/)\2, respectively. Clearances were calcu-
`vent! ides
`lated as dose/AUCVCMIMCS, where AUC
`is the area
`under the curve (AUC) for the best—fit curve to the ventricular
`data. AUClumbm. was calculated by the linear
`trapezoidal
`method. The AUC beyond the last sampling point was esti—
`mated by exponential extrapolation. The apparent volume of
`distribution, Vd, was calculated by dividing the dose by the
`concentration—intercept of the ventricular curve.
`
`In vitro bioassays
`
`In vitro testing was used to assess the bioactivity of each
`patient’s CSF after intraventricular injection of 454A12—1‘RA
`and to assess the sensitivity of each patient’s tumor cells
`(harvested from the CSF) to the immunotoxin.
`
`Bioactivity of 454A72—rRA in CSF of patients
`
`Samples of CSF obtained from the Ommaya reservoir and
`the lumbar drain at
`timed intervals after treatment were
`
`tested at various dilutions in protein synthesis assays with the
`K562 erythroleukemia cell line, as described previously (29, 72).
`The results are expressed as a percentage of l4C—leucine incor-
`poration in mock—treated control cultures.
`
`Neurosurgery, Vol. 41, No. 5, November 7997
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`if
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`Detection of anticonjugate antibody formation
`Patient serum and CSF before treatment and 1, 2, 3, 6, and
`16 weeks after treatment were assayed for antibodies to
`454A12—rRA or either of its components (454A12 or rRA)
`using a secondary radiolabeled antibody in an immunosor—
`bent assay (63).
`‘
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`1042
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`Laske et al.
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`Sensitivity of tumor ce/ls in CSF of patients
`
`In three patients, CSF tumor cells were harvested from 25 to
`35 ml of lumbar CSF by centrifugation 3 to 5 days before
`treatment and 2 to 3 weeks after treatment. The cytotoxicity of
`serial dilutions of 454A12—rRA was then tested against these
`tumor cells based on the protein synthesis assay described
`previously (29, 72).
`
`
`
`
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`Western blot analysis of CSF of patients for
`454A12-rRA
`
`Ventricular CSF samples were obtained from Patients 6 and
`8 at O, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after 454/412-rRA
`injection and run on 6.5% nonreducing sodium dodecyl sul-
`fate polyacrylamide gels. Each lane was loaded with 20 ul of
`CSF or conjugate control and 10 ul of loading buffer. Gels
`were transferred to nitrocellulose filters,
`incubated with
`avidin—labeled goat antimouse antibodies to lgG, IgA, and
`IgM (heavy and light chains) (Zyrned Laboratories, South San
`Francisco, CA), and then detected with biotin—horseradish
`peroxidase.
`
`Measurement of antitumor efficacy
`Tumor response was assessed directly by the determination
`of CSF tumor cell counts. CSF tumor cell counts were ob—
`
`tained by averaging the tumor cell counts of three high-power
`fields after a fixed volume (4 ml) of lumbar CSF was filtered
`through a 5—um small Millipore filter (Millipore Corporation,
`Bedford, MA) (25-mm diameter). Absolute tumor cell counts
`often exceeded 300 cells/ ml. Leptomeningeal neoplasia alters
`CSF protein, glucose, and LDH isoenzymes, and these param—
`eters were also used as a measure of disease. LDH isoenzyme
`1 (LD1) is the predominant form in CSF from healthy patients
`(44). LDH isoenzyme 5 (LD5) is the most common form to be
`elevated in malignancy (70). The ratio LD5/LD1 in CSF serves
`as a marker of leptomeningeal disease when it is greater than
`0.15 (62). Indirect measures of tumor response included serial
`neurological examinations and gadolinium-enhanced MRI
`scanning of the brain and complete spine.
`
`RESULTS
`
`Eight patients with leptomeningeal spread of systemic neo-
`plasia (six patients with breast carcinomas, one patient with
`melanoma, and one patient with leukemia) received a total of
`10 treatments of intraventricular immunotoxin covering a
`1000—fold increase in drug dose from 1.2 to 1200 Hg. The
`patient data is presented in Table 1.
`
`Pharmacokinetics
`
`The calculated volumes of distribution of 99mTc-DTPA and
`454A12—1'RA ranged from 5.4 to 58.1 ml
`(Tables 2 and 3).
`99mTc—DTPA’ is an extracellular marker with low capillary
`permeability, and, therefore, its clearance from CSF approxi-
`mates bull< CSF flow (47). The early phase (oz-phase) half-life
`of 99mTc-DTI’A in ventricular CSF averaged 49 i 19 minutes,
`and the late phase (B-phase) half—life averaged 304 i 78
`minutes. In seven of the eight patients, the clearance of 99‘“Tc—
`DTPA was less than 18.3 ml per hour (Table 2), which is the
`rate of bulk flow of CSF in healthy patients (65). This finding
`suggests that there was some obstruction to CSF flow before
`treatment in most of our patients.
`The clearance curves for 99mTc—DTPA and 454A12-rRA,
`obtained simultaneously, are illustrated in Figure 2 for two
`patients. In all patients tested, the clearance of the immuno—
`toxin was more rapid than the clearance of 99mTc-DTPA,
`averaging approximately 24—fold greater (Table 2). The or
`phase half—life of 454A12—rRA in ventricular CSF averaged
`44 i 21 minutes, and the B—phase half-life averaged 237 i 86
`minutes. The bioactivity of CSF samples in inhibiting protein
`synthesis of K562 erythroleukemia cells in culture qualita—
`tively tracks the concentration of immunotoxin. Good inhibi—
`
`TABLE 1. Patients Treated with Intraventricular 454A12-rRA“
`
`
`
`
`
` Pfifim Diagnosis fig: KPS Previous Therapy
`
`
`|
`Breast cancer
`66
`40
`MRM, 5—FU, RT, IT MTX
`
`[Inorgzgfifgm
`1.2
`3.8
`12.0
`
`38.0
`38.0
`
`I i i i
`
`
`
`
`
`
`
`
`
`
`2
`3
`
`Breast cancer
`ALL
`
`52
`27
`
`30
`80
`
`MRM, 5—FU, cytoxan, adriamycin, IT MTX, IT thiotepa
`L»asparaginase, daunorubicin, vincristine, prednisone,
`mitoxantrone, Ara<C, RT, IT MTX
`120.0
`MRM, S—FU, cytoxan, adriamycin,
`IT MTX
`4O
`44
`Breast cancer
`4
`120.0
`Tamoxifen,
`IT MTX
`60
`63
`Breast cancer
`5
`380.0
`MRM, 5—FU, RT, tamoxifen, MTX
`40
`52
`Breast cancer
`6
`380.0
`Interleukin—2
`80
`27
`Melanoma
`7
`
`8 1200.0 Breast cancer 58 70 MRM, S—FU, cytoxan, adriamycin
`
`
`
`
`
`a KPS, Karnofsky performance score; MRM, modified radical mastectomy; 5-FU, 5-fluorouracii; RT, radiation therapy; IT, intrathecal; MTX,
`methotrexate; ALL, acute Iymphocytic leukemia; Ara-C, cytosine arabinoside.
`
`Neurosurgery, Vol. 41, N0. 5, November 1997
`
`IMMUNOGEN 2300, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2300, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`t
`I
`
`Intraventricular lmmunotoxin Therapy
`
`1043
`
`"3’1""
`
`TABLE 2. Cerebrospinal Fluid Pharmacokinetics of Technetium-99m-Diethylenetriamine Penta-acetic Acid‘1
`99mTcDTF'A
`_
`R
`two
`W26
`Vd
`Ventricular
`AUCHUmbm/
`Patient No.
`(min)
`(min)
`(ml)
`Clearance (ml/h)
`AUCWEdees,
`
`1b
`0.98
`55
`334
`5.4
`3.6
`0.25
`2
`0.84
`50
`389
`15.8
`6.3
`0.52
`3
`0.99
`23
`147
`6.2
`10.5
`0.86
`4
`0.64
`33
`286
`16.6
`5.6
`0.44
`5
`0.88
`66
`350
`10.0
`4.2
`0.24
`6
`0.94
`31
`270
`14.1
`12.6
`0.50
`7
`0.93
`.57
`273
`36.0
`20.7
`1.07
`8
`0.91
`77
`380
`27.4
`11.0
`0.61
`
`'1 95"“Tc-DTPA, technetium-99m-diethylenetriamene penta—acetic acid; R, ratio cr/(a + B), the fractional contribution of the early phase; two,
`half-life of cut-phase; tl/ZB/ half-life of B-phase; Vd, volume of distribution; AUC, area under the curve.
`b Analysis is of 9""‘Tc-DTPA coinjected with 12 pg of immunotoxin.
`TABLE 3. Cerebrospinal Fluid Pharmacokinetics of lmmunotoxin 454A12-rRA‘1
`454A12rRA
`Dose
`R
`twat
`W213
`V,I
`Ventricular
`AUC(|umbar)/
`lT Clearance/
`AUCWEMCIGS)
`Patient No.
`(ug)
`(min)
`(min)
`(ml)
`Clearance (ml/h)
`AUCWHMCICS)
`Tc Clearance
`(nmoI/L-h)3
`
`l
`1.2
`1.00
`37
`25.1
`27.9
`-—b
`—C
`0.2
`'1
`3.8
`1.00
`54
`12.4
`9.6
`—b
`I
`2.2
`|
`12
`1.00
`57
`18.0
`|3.1
`0.08
`3.6
`5.1
`2
`38
`0.93
`21
`10.6
`14.3
`0.08
`2.3
`14.8
`3
`38
`1.00
`30
`14.9
`20.9
`0.32
`2.0
`10.1
`4
`120
`NE
`53.1
`3.0
`0.22
`12.6
`10.0d
`20
`5
`120
`0.91
`72.0
`2.3
`0.92
`29.3
`36.0
`28
`6
`380
`0.92
`72.2
`1.4
`0.99
`29.3
`47.8
`68
`7
`380
`1.00
`296
`310.2
`2.0
`0.92
`21.5
`58.1
`77
`8
`1200
`0.84
`
`" R, ratio nr/(n + .6}, the tractional contribution of the early phase; twat, half—life of iii-phase; hm“, haif—life of [S phase; VJ, volume of
`distribution; AUC, area under the curve; IT, immunoloxin; Tc, technetium; NE, not evaluated.
`b Lumbar concentrations below detection.
`“Ratio could not be calculated because data on technetium-99m diethylenetriamine penta—acetic acid Coinjected with 1.2 and 3.8 Mg
`immunotoxin were incomplete.
`dVd was constrained to the value from the technetium-99m diethylenetriamine penta-acetic acid studies to get convergence at reasonable
`physiological values.
`
`162
`325
`
`164
`
`[ion by. ventricular and lumbar CSF is evident for several
`hours after inlraventricular injection (Fig. 2). In some ventric-
`ular CSF samples obtained within 4 hours of 454A'12—1'RA
`treatment, bioactivity could still be detected even after 1000-
`to 10,000-fold dilution of the CSF. In addition, no 454A12¢RA
`degradation was detected by Western blot analysis of serial
`Ventricular CSF samples obtained from two patients for a
`period of 24 hours after inlraveutricular injection (data not
`shown).
`
`Toxicity
`Delineating CNS drug toxicity is difficult because of pro—
`gressive leptomeningeal neoplasia (19, 35). Drug toxicity
`needs to be immediate or qualitatively distinct or severe to
`differentiate it from progression of leptomeningeal
`tumor,
`which can cause a wide variety 'of neurological signs and
`symptoms.
`No acute or chronic drug toxicity was identified in patients
`Who received a dose less than or equal to 38 ug of 454A12-rRA
`by intraventricular injection. In patients who received a close
`
`greater than or equal to 120 pg of drug, an early inflammatory
`reaction occurred in the CSF, with an increase in the white
`blood cell count and CSF protein, which peaked within 12 to
`24 hours and resolved during 1 week (Fig. 3). This response
`consisted of primarily polymorphonuclear leukocytes, and
`there was no CSF eosinophilia. The inflammatory reaction
`was more pronounced at 454A12—rRA doses of 380 and 1200
`pg. These three patients experienced headache, vomiting, and
`diminished levels of consciousness associated with elevated
`intracranial pressure. Treatment with steroids and external
`ventricular CSF drainage relieved the symptoms. In Patient 4,
`who died 34 hours after a dose of 120 ng, no CSF inflamma-
`tory reaction was observed for a period of 24 hours. Autopsy
`revealed definite cerebellar tonsillar herniation with compres-
`sion of the medulla and associated focal acute tonsillar hem—
`orrhages. The immunotoxin injection was administered into
`the left lateral ventricle in this patient, and the ependyma and
`choroid plexus were examined bilaterally. Focal
`loss of
`ependymal cells was disclosed on both sides, somewhat more
`prominently on the right; there was little or no subependymal
`
`
`
`-—:—-.-———r-..._...
`
`
`.x_.-..—__.T.._.._____.
`
`Neurosurgery, Vol. 47, No. 5, November 1997
`
`
`
`IMMUNOGEN 2300, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
`l I I l
`
`PATIENT 5
`. ..............1.[.. -- mo
`“WU, .—.1 .—-—v—r
`
`1044
`
`Laske et al.
`
`
`
`2
`9
`H
`Hm
`E:
`22
`333
`5.x“
`03
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`3m
`3.:
`DE
`\.m
`m u
`ti
`i4
`
`I.
`
`10
`
`2
`9
`r;
`k\
`ms
`2%
`3mm“
`Son
`oi
`tcj
`3
`3%
`0&102
`\
`m o
`“
`5
`E
`
`100
`
`(I)
`\
`8
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`508
`CL
`0
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`
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`. .I ...
`4
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`10
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`2
`HOUHSAFTEFI INJECTION
`
`101......
`D
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`4
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`20
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`HOURS AFTER INJECTION
`
`104
`
`..,_._
`... .... ...___..
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`IEI"‘1'l'I""'-.
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`:
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`II§
`$l§
`3%
`3
`\l
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`100
`
`a
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`10
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`3
`53
`J:
`s
`g
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`
`..I...|...|._._. 114.. l o
`2
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`12
`HOUI-IS AFTE/I INJECTION
`
`
`
`_... .,_
`T'rl—r‘ .
`.1 .-- .
`.:I .1
`'l
`.
`l
`8
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`1\
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`
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`
`“/5INHIBITONOFPROTEINSYNTHESIS
`
`SYNTHESIS
`
`FIGURE 2. Line graphs of CSF
`pharmacokinetics. CSF clearance after
`intraventricular administration of 38 pg
`(Patient 3, Panels A and B) and 120 Mg
`(Patient 5, Panels C and D) of
`454A12-rRA coinjected with 346 ”Ci
`and 200 ”Ci of 99mTc-DTP‘A,
`respectively. CSF samples were obtained
`from the Ommaya reservoir (Panels A
`and C) and the lumbar drain (Panels B
`and D) and assayed for radioactivity
`(I— —-I), immunotoxin concentration by
`enzyme-linked immunosorbent assay
`(0—0), and bioactivity with K562
`erythroleukemia cells (A-----A).
`Exponential decay curves were fit to the
`ventricular data. The bioassays were
`conducted with patient CSF serially
`diluted 10-fold.
`
`100
`
`'50 %INHIBITONOFPROTEIN
`
` 1.......
`|_....l.....
`.1...t0
`20
`24
`12
`16
`4
`8
`HOUIIS AFTER INJECI ION
`
`l
`
`
`
`
`
`reaction. The choroid plexus appeared normal bilaterally, and
`the floor of the fourth ventricle Sustained no changes. The
`brain stem compression seemed to be the proximate anatomic
`cause of this patient’s coma and death. Herniation was con—
`sidered to be secondary to extensive tumor infiltrating the
`cerebellum and the associated edema.
`
`In Patient 8, who received the highest close (1200 pg), after
`the early CSF inflammatory response (Fig. 3),
`there was a
`prolonged deterioration in mental status. She became coma—
`tose during the 3rd week after treatment and died 3 weeks
`after treatment. The results of autopsy revealed persistent
`leptomeningeal tumor, but,
`in addition, disclosed multiple
`focal areas of subpial vacuolation with swollen axons in the
`brain stem and spinal cord (Fig. 4/1). These lesions were
`detected in the spinal cord, medulla oblongata, pons, and
`midbrain only adjacent to the subarachnoid space, penetrat—
`ing approximately 1 mm deep, and only focally (never com-
`pletely encompassing the entire circumference of a transverse
`section). They were not related to the presence or absence of
`overlying tumor. There was no similar involvement in the
`supratentorial tissues. In our opinion, neither the localization
`nor the extent of these changes explain a toxic encephalopa-
`thy. The patient became progressively obtunded before death,
`but no proximate anatomic cause of death was determined.
`The results of autops