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`Radioimmunotherapy Targeting of HER2/neu Oncoprotein on Ovarian Tumor Using
`Lead-212-DOTA-AEl
`(cid:160)
`Eva Horak, Frank Hartmann, Kayhan Garmestani, Chuanchu Wu, Martin Brechbiel, Otto A. Gansow, Nicholas F. Landolfi
`and Thomas A. Waldmann
`J Nucl Med.(cid:160)(cid:160)
`
`1997;38:1944-1950.
`
`This article and updated information are available at:
`
` http://jnm.snmjournals.org/content/38/12/1944
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`Information about reproducing figures, tables, or other portions of this article can be found online at:
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`(cid:160)
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`
`
` is published monthly.The Journal of Nuclear Medicine
`
`SNMMI | Society of Nuclear Medicine and Molecular Imaging
`1850 Samuel Morse Drive, Reston, VA 20190.
`(Print ISSN: 0161-5505, Online ISSN: 2159-662X)
`
`© Copyright 1997 SNMMI; all rights reserved.
`
`IMMUNOGEN 2086, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
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`
`from preexisting
`
`variant
`
`cells within a
`
`results
`22. Fidler U. Kripkc ML. Metastasis
`malignant
`tumor. Science 1977:197:893-895.
`tumor
`relationships of intravaseular
`23. Liotta LA, Kleineman J. Saidcl GM. Quantitative
`cells,
`tumor vessels and pulmonary métastasesfollowing tumor
`implantation. Cancer
`Res 1974:34:997
`1004.
`24. Terranova VP. Hujanen ES. Martin CiR. Basement membrane and the invasive activity
`of metastatic tumor cells. J Mall Cancer
`Insl 1986:77:311-316.
`25. Fishman AJ. Khaw BA. Strauss HW. Quo vadis radioimmune
`1989:30:1911-1915.
`Inhibition of
`Ito Y. Tanaka K. Sugioka Y.
`Iwamoto Y. Nomizu M. Yamada-Y.
`angiogenesis.
`(umour growth and experimental metastasis of human tibrosarcoma
`cells
`
`26.
`
`imaging. J NucÃ(cid:173)Med
`
`(YIGSR).
`
`form of the laminin sequence Tyr-lle-Gly-Ser-Arg
`HT1080 by a multimene
`Br./ Cancer 1996:73:589-595.
`27. Hill RP. Metastasis.
`In: Tannock IF. Mill RP. eds. The basic science of oncologi; 2nd
`ed. New York: McGraw-Hill;
`1992:178-195.
`In:
`28. Hanna N. Role of natural killer cells in host defense against cancer metastasis.
`Nicolson GL. Milas L, eds. Cancer
`invasion and metastasis:
`hiologic and therapeutic
`aspects. New York: Raven Press; 1984:309-319.
`29. Greenberg AH. Egan SE. Jarolin L. et al. Natural killer cell regulation of implantation
`and early lung growth of H-/-«.v-transformed10Tl/2fibroblasts
`in mice. Cancer Rex
`1987:47:4801-4805.
`
`Radioimmunotherapy Targeting of HER2/neu
`Oncoprotein on Ovarian Tumor
`Using Lead-212-DOTA-AEl
`
`Eva Horak, Frank Hartmann, Kayhan Garmestani, Chuanchu Wu, Martin Brechbiel, Otto A. Gansow, Nicholas F. Landolfi
`and Thomas A. Waldmann
`Metabolism Branch and Inorganic and Radioiinmune Chemistry Section, Radiation Oncology Branch, National Cancer
`Institute. National
`Institutes oj Health, Bethesda, Maryland; and Protein Design Labs, Inc., Mountain View, California
`
`radioimmuno-
`lead f12Pb)
`toxicity and efficacy of
`The specificity,
`therapy were evaluated in nude mice bearing the SK-OV-3 human
`ovarian tumor cell
`line expressing the HER2/neu proto-oncogene.
`Methods: The therapeutic agent used was the tumor-specific anti-
`HER2/neu monoclonal antibody AE1 conjugated to 212Pb, 212Bi
`being the daughter and thus the source of the alpha-particle and
`beta emissions. A bifunctional derivative of
`tetraazacyclodode-
`canetetraacetic acid (p-SCN-Bz-DOTA) was used to couple 212Pb
`to the anti-HER2/neu monoclonal antibody AE1. The chelating agent
`did not alter
`the binding affinity to its antigenic target or
`the
`pharmacokinetics and tissue distribution of the AE1 antibody. Tox
`icity and therapeutic efficacy of 212Pb-AE1 were evaluated in nude
`mouse ascites or solid tumor models, wherein SK-OV-3 cells were
`administered i.p. or s.c., respectively. Results: The dose-limiting
`acute toxicity after i.v. administration of 212Pb-AE1 was bone mar
`row suppression, which was observed at doses above 25 ^Ci.
`Therefore, doses of 10 and 20 ¿¿Ciwere used in efficacy trials. The
`i.p. administration of 212Pb-AE1 3 days after i.p. tumor inoculation
`led to a significant (p2 = 0.015) prolongation of tumor-free survival.
`In a second model,
`i.v. treatment with 212Pb-AE1 3 days after s.c.
`tumor inoculation prevented subsequent
`tumor development
`in all
`animals treated with 10 or 20 ¿¿Ciof 212Pb-AE1 (p2 = 0.002
`compared to control groups). This efficacy in the adjuvant setting
`was antibody specific because treatments with equivalently labeled
`control antibody or unlabeled AE1 antibody or no treatment were
`less effective. The rate of growth of small (mean tumor volume, 15
`mm3) SK-OV-3 tumors was modestly inhibited. However,
`tumor
`growth was not inhibited in mice bearing larger (mean tumor volume,
`146 mm3) SK-OV-3 tumors by the administration of a single dose of
`10 or 20 juCi of 212Pb-AE1. Conclusion: Lead-212-AE1 as an intact
`radiolabeled monoclonal antibody may be of only modest value in
`the therapy of bulky solid tumors due to the short physical half-life of
`212Pb and time required to achieve a useful tumor-to-normal
`tissue
`ratio of radionuclide after administration. However, the radiolabeled
`monoclonal antibody may be useful
`in therapy of
`tumors in the
`adjuvant setting. Furthermore, 212Pb may be of value in select
`situations,
`including treatment of leukemia, intercavitary therapy or
`strategies that target vascular endothelial cells of tumors.
`
`Received Jul. 23, 1996; revision accepted Feb. 3, 1997.
`For correspondence
`or reprints contact: Thomas A. Waldmann, MD. Metabolism
`Branch, National Cancer
`Institute, National
`Institutes of Health, Building 10. Room
`4N115, Bethesda. MD 20892.
`
`Key Words: HER2; neu proto-oncogene; alpha-emitting radionu-
`clides; monoclonal antibody therapy; ovarian tumor
`J NucÃ(cid:173)Med 1997; 38:1944-1950
`
`targeted to cell surface antigens have
`Lonoclonal antibodies
`been used to treat patients with a variety of cancers. However,
`unmodified monoclonal antibodies have been relatively ineffec
`tive (7). One of the factors
`in this low therapeutic
`efficacy is
`that most of the monoclonal
`antibodies used are not effective
`cytocidal agents against human neoplastic cells. Furthermore,
`in
`most cases,
`the antibodies were not directed
`against
`a vital
`structure on the surface of malignant
`cells, such as a growth
`factor receptor
`involved in tumor cell proliferation and survival.
`In an attempt
`to circumvent
`these problems,
`researchers
`have
`augmented
`the cytotoxic
`action of monoclonal
`antibodies
`by
`arming them with toxins or radionuclides
`(2-6). Furthermore,
`cell surface antigenic
`targets, especially receptors
`for growth
`factors,
`have been defined
`for more
`effective monoclonal
`antibody
`action.
`In particular,
`the HER2/neu
`receptor,
`the
`product of the c-erbB-2 proto-oncogene,
`has been the target of
`monoclonal
`antibody therapeutic
`trials (7-12). The HER2/neu
`oncogene
`(erbB-2)
`encodes
`a Mr 185,000
`transmembrane
`phosphoglycoprotein.
`The \\ER2lneu
`gene is overexpressed
`in
`20%-30% of adenocarcinomas
`of the breast, ovary,
`lung and
`stomach and has been linked to poor prognosis
`(13-15). A
`series of anti-HER2/neu monoclonal
`antibodies has been gen
`erated that
`includes antibodies without biological
`activity,
`as
`well as those with agonist action in terms of tyrosine phosphor-
`ylation
`of HER2/neu
`(7-12).
`Preclinical
`efficacy
`in mice
`bearing HER2/neu-expressing
`human
`tumor
`xenografts
`has
`been demonstrated with certain antibodies directed toward this
`receptor.
`to the HER2/neu
`antibodies
`unmodified murine
`Although
`oncoprotein inhibited tumor growth in certain cases,
`in general
`it was not sufficient
`to cure animals of established tumors. This
`limited
`efficacy
`led to alternative
`approaches
`that
`include
`the humanization
`of an anti-HER2/neu
`antibody (12,16),
`the
`development
`of a humanized
`bispecific F(ab')2 fragment
`for
`
`1944
`
`Tin-:JOURNALOFNUCLEARMKDICINK•Vol. 38 •No. 12 •December 1997
`
`IMMUNOGEN 2086, pg. 2
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`
`retargeting cytotoxic T cells (17,18), a disulfide stabilized
`anti-HER2/neu Pseitdomonas immunotoxin (//)
`and a disul-
`fide-stabilized anti-HER2/neu Fv-beta-lactamase fusion protein
`for the activation of a cephalosporin doxorubicin prodrug (¡9).
`In addition, anti-HER2/neu antibodies have been used as
`carriers of toxic agents, including radionuclides. De Santéset al.
`(10) demonstrated that animals treated with 400-700 ju,Ciof
`131I-anti-HER2/neu monoclonal
`antibodies manifested
`a
`marked inhibition of the growth of large tumors. In general,
`131I-labeledmonoclonal antibodies have been relatively inef
`fective due to limitations of this radionuclide as a therapeutic
`agent.
`In light of the limitations of radioiodine, metallic radionu
`clides that can be linked to antibodies may provide a better
`choice. Antibodies may be armed with beta- or alpha-emitting
`radionuclides. Future development of radionuclide-conjugated
`monoclonal antibodies may focus on radionuclides that emit
`alpha particles, which may be the most effective agents at
`killing tumor targets without damaging distant normal tissues
`(2-6).
`Our present studies focused on the radionuclide 2l2Pb, which
`has a 10.6-hr half-life. Lead-212 is the parent of 212Bi,which is
`the source of an alpha particle as well as beta and gamma
`emissions.
`In this study, we used 2l2Pb-tetraazacyclodode-
`canetetraacetic acid-AEl
`(2l2Pb-DOTA-AEl ) directed toward
`SK-OV-3 tumors in our therapeutic trials. The goals of this
`study were:
`to determine specificity and pharmacokinetics,
`including biodistribution of the DOTA chelate AE1 anti-HER2/
`neu monoclonal antibody in our experimental tumor model; to
`define the toxicity of the 2l2Pb-DOTA-AEl monoclonal anti
`body conjugate and, thereby, establish the maximum tolerated
`dose; and to define the efficacy, specificity and toxicity of 2l2Pb
`radioimmunotherapy of a human HER2/neu-expressing tumor
`SK-OV-3 in nude mice.
`
`MATERIALS AND METHODS
`Cell Lines and Monoclonal Antibodies
`The SK-OV-3 cell line (ATCC HTB 77), derived from a human
`ovarian adenocarcinoma, was used. AE1 is an IgG2a murine
`monoclonal antibody directed against the extracellular domain of
`the HER2/neu receptor. The previously described anti-Tac, an
`IgG2a murine monoclonal antibody that binds to the human
`IL-2Ralphasubunit, but not to murine SK-OV-3cells, was used as
`a negative control antibody (20).
`Radioiodination of Monoclonal Antibodies
`Radioiodinations of AE1 for bindability studies with I25I were
`performed using a modification of the chloramine-T method;
`the
`resultant
`specific
`activity was 4.0-6.5
`/u,Ci//xg. Ninety-seven
`percent of the iodinated AE1 was precipitable with 20% trichloro-
`acetic acid.
`Conjugation of the Chelate to Monoclonal Antibodies
`To permit labeling with 2l2Pbor SXY,the monoclonal antibody
`AE1, as well as anti-Tac, which served as the control antibody for
`all studies, was first conjugated with 2-(p-SCN-Bz)-DOTA
`(21,22). Forconvenience, the chelate will be referredto as DOTA.
`Typically, 8-10 mg of antibody at a concentration of about 5
`mg/ml were dialyzed against bicarbonate buffer, pH 8.6, for 6 hr.
`The antibody preparation was then conjugated to l4C-(p-SCN-Bz)-
`DOTA as described (21,22). The average number of DOTA
`chelates per molecule of antibody was 1.2.determinedas described
`previously (22). Each lot of chelated AE1 antibody was compared
`with the unmodified antibody and was shown to have an unaltered
`binding capacity using a competitive binding assay (Fig. 1).
`Briefly, 5 X IO5 SK-OV-3 cells were incubated on ice with
`
`AE-1[j
`
`1 B eft
`
`1
`
`
`
`DDOTA-AE-1aDD
`
`eoOxEa
`
`u600-500-400-300-200-100-0_•
`
`10.1
`
`•1
`
`1
`
`10100000antibody
`1000
`concentration
`
`•
`
`(ng)
`
`FIGURE 1. Assay of the effect of AE1 and DOTA-AE1 on the binding of
`125I-AE1 to SK-OV-3 cells. •unmodified competitor; D, DOTA-AE1 com
`petitor.
`
`increasing concentrations of unmodified (AE1) or chelated
`(DOTA-AE1) antibody (ranging from 0 to 75,000 ng/vial or from
`0 to 2,688 ng/vial, respectively) in the presence of a constant
`nonsaturatingamount of 125I-AE1antibody (6.25 ng/vial). After 3
`hr of incubation,cells were centrifugedthrough an oil cushion, and
`cell-bound radioactivity was determined.
`
`Procedure for Radiolabeling of Chelated Antibody
`by
`Carrier-free SXY was purified from metal contaminants
`column extraction chromatography and incorporated into the che
`lated antibody as described (23).
`Lead-212 was eluted from a 224Ra/2l2Pbgenerator with 1 ml of
`2.0 M HC1. The solution was passed through a column (2 X 20
`mm) of MP-50 resin, pre-equilibrated with 2.0 M HC1 to remove
`any breakthrough of 224Ra. The solution was then evaporated to
`dryness with the addition of 0.5 ml of concentrated HNO,. The
`2l2Pb activity was dissolved in 0.1 M HNO,, with the pH adjusted
`to —4.0with 3 M NH4OAc. Chelated antibody was added to the
`2l2Pb solution and allowed to react at 35°Cfor 45 min. Five
`microliters of 0.1 m EDTA were added to scavenge any free
`radionuclide. Radiolabeled antibody was purified by high-perfor
`mance liquid chromatography using a size-exclusion column and
`4-morpholinepropanesulfonic
`acid/Cl buffer as the mobile phase ( 1
`ml/min flow rate). Radiolabeling yields were 65%-75%, and more
`than 99% of the 2l2Pb was protein-bound before administration.
`
`in
`
`Animals
`Female 6-8-wk-old athymic-NCR-nude mice were kept
`microisolation units on sterilized water and food.
`Pharmacokinetics and Tissue Distribution of
`Radiolabeled AE1
`To define the rate of entry of AEl antibody into a HER2/neu-
`expressing tumor, mice bearing established s.c. injected SK-OV-3
`tumors on their shoulder were injected i.v. with the 2-ju.Ci dose of
`ssY-labeled monoclonal antibody. Seventeen mice were injected
`with the specific anti-HER2 antibody (K8Y-DOTA-AE1), and two
`mice were injected with an irrelevant (*sY-DOTA-anti-Tac) anti
`body. Three of the mice injected with specific antibody were killed
`at 2, 6, 10 and 24 hr, and two mice were killed at 48 hr following
`infusion; their organs were removed for weighing and radioactivity
`determination using a gamma ray counter.
`Toxicity of Lead-212
`Lead-212 was used as the radionuclide in the immunotherapeutic
`studies. The emissions of 2l2Pb, the decay product daughters and
`the details of the decay events as they relate to radioimmuno
`therapy have been described in the literature and are outlined in
`Figure 2 (24).
`tolerated dose of the
`To establish the toxicity and maximal
`2l2Pb-DOTA-AEl,
`healthy,
`tumor-free mice were injected with
`
`RADIOTHERAPYOFHER2/Ntu-ExpRESsiNGTUMOR•Horak et al.
`
`1945
`
`IMMUNOGEN 2086, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
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`
`by on October 28, 2014. For personal use only.
`
`tumor occur
`was tumor-free survival (time from treatment until
`rence). Data were evaluated according to the methods of Kaplan
`and Meier (25) and Mantel and Haenszel (26). For the third and
`fourth studies,
`the observed parameter was the change in tumor
`volume calculated as (L X W2)/2, where L is length and W is
`width. The tumor volume for each animal at the time indicated was
`normalized to the tumor volume of the animal that was present on
`the day of therapy.
`
`RESULTS
`SK-OV-3 Murine Model of a HER2/neu-Expressing Human
`Solid Tumor Model
`In our initial studies, we established a murine tumor model
`for human HER2/neu-directed
`alpha particle-emitting
`radioim
`munotherapy
`using the SK-OV-3
`human ovarian tumor cell
`line. The SK-OV-3
`cell
`line expressed
`approximately
`10h
`HER2/neu receptors per cell, as defined by Scatchard analysis
`using the '25I-anti-HER2/neu
`antibody AEl. With this cell line,
`after administration
`of 2 X IO'1cells, tumor occurrence rate was
`over 95%, with a median time to tumor appearance of 8 days
`after s.c.
`injection,
`and the occurrence
`rate was 60%, with a
`median time to appearance of 10-13 days after i.p. inoculation.
`The s.c. administered
`tumor
`remained localized, but grew to a
`diameter of 1 cm in 15-71 days
`(mean, 38 days) and to a
`diameter of 2 cm in slightly over 100 days.
`and to
`cells
`To define
`the radiosensitivity
`of SK-OV-3
`tumor models
`provide
`a baseline
`for comparison with other
`used to evaluate
`radioimmunotherapeutic
`agents, we quanti-
`tated tritiated thymidine uptake and defined the percentage of
`remaining cells still capable of colony formation after external
`irradiation of SK-OV-3 cells at different dose levels (0-10,000
`rad). A dose of 520 rad reduced the colony-forming
`ability of
`irradiated cells to 2% of that observed with nonirradiated cells.
`Biological Activity of DOTA-AE1
`of HER2/neu-ex-
`To be effective
`in radioimmunotherapy
`pressing tumors,
`the chelating agent used to link the radionu-
`clide to the AEl
`antibody should not alter
`the specificity
`or
`binding affinity of the monoclonal
`antibody
`to its antigenic
`target. The binding integrity of DOTA-AEl was assessed by
`comparing it to unlabeled AEl
`in a competitive
`binding assay
`(Fig.
`I).
`In this assay, SK-OV-3 cells were incubated with a
`nonsaturating
`amount of l25I-AEl, along with increasing quan
`tities of either unmodified
`or DOTA-AEl.
`The chelated anti
`body manifested identical binding activity as unmodified anti
`body when evaluated in this competition assay.
`In Vivo Pharmacokinetics and Tissue Distribution of
`Radiolabeled DOTA-AE1
`antibody using
`The stability of a 21l3Pb-labeled monoclonal
`DOTA as a chelating agent has been examined previously and
`shown to be stable under in vivo conditions
`(24). To test the in
`vivo stability of the particular DOTA-AEl
`antibody used in this
`study, we used 88Y rather
`than 2l2Pb,
`in light of
`its longer
`physical
`half-life
`and gamma
`emission. The yttrium-DOTA
`complex has been shown to be and is also generally accepted to
`be inert and stable
`in vitro and in vivo,
`thus providing
`an
`unequivocal
`biodistribution
`standard
`for a radiolabeled
`anti
`body using a chelated metal. We and others have noted that the
`indium complex formed with DOTA was not identical with the
`yttrium complex and,
`therefore, 88Y was used as an accurate
`tracer
`isotope.
`In these pharmacokinetic
`studies, animals with
`established
`s.c. SK-OV-3
`tumors
`(~l
`g) were given
`i.v.
`injections of X8Y-DOTA-AE1 (2 juCi per mouse), serial blood
`samples were collected after
`injection and the fraction of the
`dose remaining
`in the plasma was determined. The terminal
`
`1.52(21%)
`1.80(50%)
`EY=0.511
`(23%)
`0.583 (86%)
`0.860(12%)
`2.614(100%)
`
`FIGURE 2. Decay series of 224Rafor generating therapeutic doses of 212Pb
`and 212Bi, with description of the radioactivity emissions of 212Pb and its
`daughters. The emissions are expressed in MeV.
`
`unmodified AEl or different doses of 2'2Pb-DOTA-AEl. Group I
`received only unmodified AEl antibody, while groups 2, 3 and 4
`received 10, 25 and 40 /¿Ciof 2l2Pb-DOTA-AEl,
`respectively.
`
`Radioimmunotherapy of HER2/neu-Expressing Tumors
`with Lead-212 AE1
`In all radioimmunotherapy experiments, we used female athymic
`nude mice (10-12 wk old). In the initial preliminary therapeutic
`trial, mice were given i.p. injections of 2 X IO6 SK-OV-3 cells.
`Three days later, groups of mice were given i.p.
`injections of
`2l2Pb-AEl
`at dose levels of 10 and 20 /nCi (specific activity,
`0.6-1.5
`/u.Ci//ig), the control
`radiolabeled monoclonal antibody
`(2l2Pb-anti-Tac) at the same dose levels (specific activity, 1.0-2.5
`jj-Ci/iJLg),unlabeled AEl
`(10 jug) or no treatment.
`In the second
`trial, animals were inoculated with 2 X IO6 SK-OV-3 cells s.c. in
`the back of the neck. Three days later, groups of five mice were
`treated i.v. with either 212Pb AEl or 2l2Pb-anti-Tac at dose levels
`of 10 and 20 /¿Ci.Control groups again received either
`i.v.
`unlabeled anti-Tac or no treatment. In the third trial, groups of five
`mice with established small-sized s.c. tumors (studied 14 days after
`s.c.
`tumor
`inoculation when mean tumor volume was 15 mm3)
`were treated with the same agents under the same i.v. protocol,
`whereas
`in the fourth group animals with larger
`(mean tumor
`volume, 146 mm3) tumors were studied with the same agents as
`used in trials 2 and 3. The primary end point in the initial two trials
`
`1946
`
`Tin: JOURNALOFNUCLKARMEDICINE•Vol. 38 •No. 12 •December 1997
`
`IMMUNOGEN 2086, pg. 4
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`
`TABLE 1
`Tissue Distribution of Yttrium-88-AE1 and Yttrium-88-Anti-Tac
`
`in Percentage of Injected Dose per Gram of Tissue
`
`Yttrium-88-AE1 specific antibody
`
`Yttrium-88-anti-Tac
`control antibody
`
`2hr
`
`6hr
`
`10 hr
`
`24 hr
`
`48 hr
`
`24 hr
`
`Specific-to-
`control ratio
`of incorporation
`
`24 hr
`
`
`
`Kidney
`IntestineHeartLung
`
`Femur3.70
`
`SK-OV-3tumorBloodLiverSpleen
`
`±0.8835.57
`
`±5.0511.57
`±0.346.45
`±0.63
`9.57 ±0.35
`
`2.88 ±0.1013.82
`±0.5611.
`
`0.69
`48 ±0.70
`2.31 ±0.207.7030.189.906.02
`
`2.05±±0.2410.35
`
`1.10±0.53
`
`±6.1723.81
`
`0.28±4.13±±26.65
`
`±8.42
`
`±6.019.1
`±6.07
`
`5 ±1.336.13
`±5.70
`±
`±
`±1.46
`6.08 ±
`7.25 ±
`5.79
`±0.35
`0.17
`8.13 ±1.00
`8.44
`±1.02
`0.53
`2.26±9.17
`
`±0.26±0.72±0.622.24±8.50
`0.191.010.37
`
`2.42 ±0.3610.80
`
`2.5810.5410.38±0.14±0.48±
`0.001.720.81
`1.838.806.98
`±9.14
`±3.0510.53
`±8.50
`±
`±
`±3.06
`±0.106.40
`2.25 ±2.350.840.000.20
`1.90 ±0.574.341.301.25
`2.37 ±0.4424.6717.386.425.45
`1.05
`0.2026.05
`
`1.94±3.88±3.19±0.73±0.420.004.071.111.391.01
`
`±21.50±6.56
`
`1.34
`1.081.271.15
`
`half-life (beta) of these studies was 86 hr; the fraction intravas-
`cular was 56% with 33% of the i.v. pool catabolized per day as
`assessed by the method of Matthews
`(27).
`tumors with
`The effective
`treatment
`of
`large established
`2l2Pb-AEl
`is dependent
`in part on the physical half-life of 2l2Pb
`(10.6 hr) and the time required to achieve an optimum tumor-
`to-normal
`tissue ratio of radioactivity
`after i.v. administration.
`To address
`this issue, animals with established
`s.c. SK-OV-3
`tumors were given
`i.v.
`injections
`of either
`tumor-specific
`antibody (SXY-AE1 or of an irrelevant antibody X8Y-anti-Tac).
`In Table 1, we show the tissue distribution of XKY-AE1and the
`control antibody KXY-anti-Tac expressed as a percentage of the
`injected dose per gram of tissue in these tumor-bearing mice, as
`well as the ratio of specific-to-control
`antibody incorporation
`into different organs 24 hr after the i.v. infusion. The values are
`expressed as the mean ±s.d. of three mice killed at each time
`point. Within the first 6 hr, there was essentially no enrichment
`of AE1 in the SK-OV-3 tumors compared with normal
`tissues.
`However, by 24 hr, SK-OV-3 tumors manifested an enrichment
`of 88Y-DOTA-AE1,
`yielding tumor-to-normal
`solid tissue ra
`tios of 2.85-11 to 1. Such enrichment was not observed with the
`control monoclonal
`antibody anti-Tac. Furthermore,
`the ratio of
`incorporated specific antibody (AE-1) to control antibody (anti-
`Tac) at 24 hr was 4.07 for the tumor, whereas
`there was no
`enrichment
`(ratios of 1.01-1.39) with the normal organs.
`Radioactivfty-Toxicity Relationship and Maximum
`Tolerated Dose of Lead-212-AE1
`for 2l2Pb-AEl,
`relationship
`To define
`an activity-toxicity
`increasing
`doses
`(10, 25 and 40 /nCi) of 2'2Pb-AEl were
`administered
`i.v.
`into tumor-free
`athymic nude mice, and the
`effects on hematological
`and serum chemistry parameters were
`defined over a period of 180 days. The activity-limiting
`toxicity
`was bone marrow suppression in animals receiving a more than
`25-/u,Ci dose of lead-radiolabeled AE1 (Fig. 3). Specifically, all
`animals receiving the 40-jnCi dose of 2l2Pb-labeled monoclonal
`antibody died within 6-10 days of therapy, manifesting a mean
`weight
`loss of 25.8%. Pathological
`examination
`of the tissues
`revealed significant
`lymphoid depletion and a marked reduction
`in the size of the spleen (which, on histological
`examination,
`was associated with decreased extramedullary
`hematopoiesis),
`an acellular bone marrow and pulmonary congestion. Four of
`the five animals
`in this group developed
`septicemia, with
`bacterial
`infections affecting the various tissues. All 16 animals
`receiving 25- and 10-/H.CÃ(cid:141)doses of 212Pb monoclonal
`antibody
`i.v. survived, but manifested
`transient granulocytopenia,
`lym-
`phocytopenia
`and modest
`thrombocytopenia
`(Fig. 3). The
`animals
`receiving the 25-/U.CÃ(cid:141)dose developed a nadir
`in their
`hematopoietic
`elements
`in the period spanning days 4-11, with
`
`range in
`leukocyte
`a leukocyte nadir of 400 cells/ju.1 (normal
`mice, 2,600-10,700
`/nl), lymphocyte
`levels of approximately
`250 (normal
`lymphocyte
`range in mice, 1,430-9,940
`jul) and
`thrombocytopenia with a nadir of approximately
`170,000 ¡JL\
`(normal platelet
`count
`range, 592,000-2.97
`X 106//Ltl). The
`platelet and total white blood cell levels returned to the normal
`range by day 20, whereas
`the lymphocyte
`levels were normal
`
`Platelets
`
`ilì*
`
`20
`
`40
`
`100 120 140 160 180
`80
`60
`Days post I.v. therapy
`
`WBC
`
`-20
`
`20
`
`40
`
`100 120 140 160 180
`80
`60
`Days post i.v. therapy
`
`Lymphocytes
`
`-20
`
`:
`40
`
`20
`
`I
`I
`I
`I
`I
`l
`i
`100 120 140 160 180
`80
`60
`Days post i.v. therapy
`
`unlabeled AE1
`
`lOuCi AE1
`
`25uCiAE1
`
`FIGURE 3. The effect of i.v. administered unlabeled AE1 (•).10 ¿¿Ciof
`212Pb-AE1 (O) and 25 /xCi of 212Pb-AE1 (*) on platelet, white blood cell
`(WBC) and lymphocyte levels.
`
`RADIOTHERAPYOKHER2/NEU-ExpRESSiNGTUMOR•Horak et al.
`
`1947
`
`IMMUNOGEN 2086, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Downloaded from
`
`jnm.snmjournals.org
`
`by on October 28, 2014. For personal use only.
`
`90-1
`
`60-«
`50-
`
`0)1
`
`30-,2
`
`20-
`20o
`10-V—
`
`•— n
`
`—O—Aj,
`
`therapy
`
`E1
`12Pb-iOn
`
`2pb-20u
`
`AE1
`
`2Pb-10M
`
`Z\ anti-irTac
`
`12Pb-20nl
`
`TacIII
`
`notherapy-0-
`
`s?3(AikOE90-80-70-60-50-30-10-•
`AE1-»-
`2'2pb-10uCiAEl—O-
`
`212Pb-20uCiAElI'
`
`anti-Tacu
`
`-*-212Pb-10uCianti-Tac_û^
`
`2i2Pb.20uc¡
`
`50Ci
`
`100200Days
`150
`
`after treatment
`
`i
`
`I
`50
`
`0
`
`i
`
`i
`I
`I
`100200Days
`150
`
`After Treatment
`
`i •
`
`l
`
`FIGURE 4. Tumor-free survivalafter i.p. treatment with 20 /¿Ciof 212Pb-AE1
`(O; n = 4), 10 iiCi of 212Pb-AE1 (*; n
`4), 20 /xCi of ?12Pb-anti-Tac (A; n =
`5), 10 /¿Ciof 212Pb-anti-Tac (A; n •5), unlabeled AE1 (D; n = 5) or no
`treatment (•;n
`10) 3 days after i.p. inoculation with 2 x 106 SK-OV-3 cells.
`f>2 = 0.015, 10- and 20-jiCi AE1 treatment groups compared with the two
`control groups not treated with a radiolabeled monoclonal antibody. p2 =
`0.06, comparison of 212Pb-AE1 to 212Pb-anti-Tac groups.
`
`FIGURE 5. Tumor-free survivalafter i.v. treatment with 20 /¿Ciof 2l2Pb-AE1
`(O; n = 4), 10 /¿Ciof 2l2Pb-AE1 (*; n
`4), 20 /¿Ciof 212Pb-anti-Tac (A; n =
`5), 10 /¿Ciof 212Pb-anti-Tac (A; n
`5), AE1 (D; n = 5) or no therapy (•;n =
`10) 3 days after s.c.
`inoculation with 2 x 106 SK-OV-3 cells. p? •0.002,
`comparison of 10- and 20-itCi AE1 treatment groups with the two control
`groups not treated with a radioactive monoclonal antibody.
`
`irradiation because
`of tumors was not due solely to nonspecific
`four of five animals
`receiving
`10 juCi of 212Pb-anti-Tac
`and
`three of
`five receiving
`20 juCi of
`this control
`radiolabeled
`monoclonal
`antibody developed tumors by day 22 (p2 = 0.003,
`animals receiving 10 or 20 /u.Ciof AEl compared with animals
`receiving the same doses of radiolabeled anti-Tac). The animals
`surviving after
`i.p. and s.c.
`inoculation of the SK-OV-3 tumor
`were killed on day 180, and the tissues obtained at necropsy
`were submitted for histológica! evaluation. At that
`time,
`there
`was no evidence of residual
`tumor or bone marrow abnormality.
`A third trial group of mice with small-sized established s.c.
`tumors
`(studied
`14 days after
`tumor
`inoculation when mean
`tumor volume was 15 mm'1) was treated with the same agents
`using the same protocol
`(Fig. 6). The rate of tumor growth was
`inhibited in the period after
`therapy with 20 ¿z.Ciof 212Pb-
`spccific
`antibody. However,
`no complete
`remissions were
`observed. A fourth group of animals had large (mean tumor
`volume,
`146 mm')
`s.c. SK-OV-3
`tumors when they were
`
`no Tx
`
`AE1
`
`20nCi212PbAE1
`
`lOnCi 212Pb anti-Tac
`
`2CHiCi212Pb anti-Tac
`
`30.0-1
`
`25.0-
`
`15.0
`
`10.0-
`
`5.0-
`
`o 3
`
`10
`
`20
`
`60
`50
`40
`30
`Days after treatment
`
`70
`
`80
`
`90
`
`FIGURE 6. Tumor growth of small s.c. SK-OV-3 tumors (mean tumor
`volume, 15 mm3) after i.v. treatment with 20 /¿Ciof ?12Pb-AE1 (.:*), 10 /¿Ciof
`2l2Pb-AE1 (*), 20 /¿Ciof 212Pb anti-Tac (A), 10 ¿iCiof 212Pb anti-Tac (A),
`unlabeled AE1 (O and no therapy (•).The tumor volume for each animal at
`the time indicated was normalized to the tumor volume for that animal that
`was present on the day of treatment. Mean values for the five animals in each
`group are shown.
`
`after day 60. Seventy-five percent of the mice receiving 25 ju,Ci
`of 2l2Pb-AEl
`or 2l2Pb anti-Tac
`developed
`long-term renal
`toxicity with elevated mean blood urea nitrogen or creatinine.
`The
`levels of blood
`urea nitrogen
`and creatinine
`for all
`surviving mice were 39 and 0.8 mg/dl,
`respectively,
`compared
`with the upper limits of the normal range for mice of 28 and 0.7
`mg/dl,
`respectively.
`Lead-212-AE1 Radioimmunotherapy: Therapeutic Efficacy
`We first evaluated the therapeutic
`efficacy of 2l2Pb-AEl
`in
`an SK-OV-3 tumor model
`featuring direct access of the thera
`peutic agent
`to the tumor-containing
`compartment.
`For
`this
`group,
`I0-l2-wk-old
`female nude mice were given i.p.
`injec
`tions of 2 X IO6 SK-OV-3 cells. Three days later, different
`groups of five mice each received i.p. administered ZI2Pb-AEl
`at doses of IO and 20 juCi, the control
`radiolabeled monoclonal
`antibody (2l2Pb-anti-Tac)
`at
`the same dose levels, unlabeled
`AEl
`(20 fig) or no treatment
`(Fig. 4). Lead-2l2-AEl
`treatment
`resulted in a statistically significant delay in the appearance or
`prevention
`of tumor occurrence when mice received
`10- and
`20-jnCi doses; considered together,
`these mice were compared
`with the two groups of mice receiving either no treatment or
`unlabeled AEl
`(p2 = 0.0 15). In interpreting these exploratory
`studies,
`it should be noted that only 60% of the animals
`in the
`control
`group
`injected
`i.p. with SK-OV-3
`cells developed
`tumors.
`group of animals was used to obtain a more
`A second
`evaluation of the effectiveness
`of 2l2Pb-AEl
`ther
`meaningful
`apy in the adjuvant
`setting.
`In this study trial, nude mice were
`injected with 2 X 10" SK-OV-3 cells s.c.
`in the back of the
`neck. Three days later, mice were treated i.v. with 212Pb-AEl or
`control 2l2Pb-anti-Tac
`at dose levels of 10 and 20 /nCi, treated
`with unlabeled AEl or
`received no treatment. The Kaplan-
`Meier plot depicting
`tumor-free
`survival
`after
`treatment
`re
`vealed no differences
`between the group of animals
`receiving
`no therapy (n = 10) and that receiving unlabeled AEl
`(n = 5);
`all animals
`in both groups developed a tumor by day 20.
`In
`contrast, all animals receiving cither 10 or 20 /iCi of 21"Pb-AEl
`remained tumor-free throughout
`the 180-day study period (Fig.
`5; p2 = 0.002,
`animals
`receiving
`low and high doses of
`2l2Pb-AEl
`compared with those in the nontreated group). We
`conclude that
`this effectiveness
`in preventing the development
`
`1948
`
`Tui JOURNALOF NUCLKARMEDICINE•Vol. 38 •No. 12 •December
`
`1997
`
`IMMUNOGEN 2086, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Downloaded from
`
`
`
`by on October 28, 2014. For personal use only. jnm.snmjournals.org
`
`-D-AE-1
`-•-10mCi212PbAE1
`-O- 25(iCi212PbAE1
`-O- 25tiCi212PbAE1
`-•-anti-Tac
`-A-
`10nCi212Pb anti-Tac
`-û- 25nC¡212Pbanti-Tac
`
`30.0-,
`
`<S25.0
`
`_2
`> 20.0
`kO
`2 15.0
`i== 10.0
`
`I
`
`5.0 H
`
`5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
`Days after treatment
`
`FIGURE 7. Tumor growth of larger (mean tumor volume, 146 mmr!)estab
`lished s.c. SK-OV-3 tumors after i.v. treatment with 25 ^Ci of 21?Pb-AE1 (O),
`25 ,iCi of ?l:>Pb-AE1 (- ), 10 juCi of ?1?Pb-AE1 (»),25 /iCi of pl;;Pb-anti-Tac
`(i,), 10 P.CÃ(cid:141)of ?1?Pb-anti-Tac (A), unlabeled AE1 (IT) and unlabeled anti-Tac
`(•).The tumor volume for each animal at the time indicated was normalized
`to the tumor volume for that animal that was present on the day of treatment.
`Mean values for the five animals in each group are shown.
`
`(Fig. 7). Tumor growth was not
`treated with the same agents
`inhibited in these mice bearing large SK-OV-3 tumors by the
`administration
`of a single doses of 25 /u.Ci of 2l2Pb-specific
`antibody
`as compared
`to those
`receiving
`the nonspccified
`radiolabeled
`anti-Tac
`antibody.
`In particular,
`the size of the
`tumors
`increased
`in all
`treatment
`groups and there were no
`partial or complete
`remissions. Thus,
`Pb-AEl
`did not pro
`vide effective therapy for large established tumors.
`
`DISCUSSION
`armed with toxins or
`antibodies
`The use of monoclonal
`target
`these cytotoxic
`agents
`to
`radionuclides
`to specifically
`tumor cells provides a valuable alternative
`for cancer
`therapy.
`Factors that appear critical
`in developing a