(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:83)(cid:85)(cid:76)(cid:79)(cid:3)(cid:24)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:27)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:21)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`
`Genentech 2131
`Hospira v. Genentech
`IPR2017-00737
`
`

`

`ANTIBODY TARGETING 0F HER~2/neu
`
`were radiolabeled with 1 mCi Na'3‘1 (Amersham) by the lodeen
`method. Mice received i.p. injections of 10 pg of l"1—4D5 (specific
`activity, 1.8 pCi/pg) and 10 pg of ”SI-DA4—4 (specific activity, 1.4 pCi/
`pg),,‘Atg6,. 18, 24, 48, 72, and 120 h postinjection the animals were
`sacrificed, and their tumors and normal organs were harvested, washed,
`weighed, and analyzed for both l3'1 and ”‘1 activity. The i.p. route was
`chosen for these studies because preliminary experiments demonstrated
`virtually identical muMAB biodistributions after i.p. and i.v. injections
`but less mouse-to~mouse variability with the i.p. route, presumably
`because of occasional extravasations after tail vein injections. Results
`were expressed as the percentage injected dose of radioactivity per gram
`of tissue. Data were corrected for radioactive decay and spillover from
`the '3'] to the ”’1 channel. Groups of five mice were utilized to generate
`each data point, and their mean values were used to construct biodis-
`tribution curves for tumors and normal organs. The area under the
`blood activity versus time curves were calculated assuming a model with
`first-order absorption and elimination. Tumorzorgan ratios of radioac—
`tivity and localization indices were also determined. Localization in-
`dices were calculated from the ratio of specific:irrelevant antibody taken
`up by tumor (or organ) and normalized for simultaneous blood levels:
`
`% lD/Gm ”’I-4D5/% lD/Gm ”’1—DA—4—4 in tumor or (organ)
`% ID/Gm ”’I-4D5/% ID/GM ”’1-DA4—4 in blood
`
`Flow Cytometry. The specificity of binding of muMABs 7C2 and
`4D5 to the HER-Z/neu gene product was assessed by indirect immu-
`nofluorescence. Untransfected 3T3 cells do not express the HER-Z/
`neu gene product on their surface and were used as negative controls.
`3T3 cells transfected with the HER-Z/neu oncogene and SKBR3 cells
`express high surface densitities of the p185 protein and were used as
`positive cell lines. Cells (5 x 10’) of each type were incubated with
`saturating concentrations of muMABs 4D5, 7C2, or DA4—4 for 30 min
`on ice, washed three times, incubated with a 1:20 dilution of a fluores-
`ceinated, affinity-purified, goat anti-mouse immunoglobulin reagent
`(TAGO, lnc., Burlingame, CA) for 30 min, washed three times, and
`then fixed in 1% paraformaldehyde. Cells were analyzed on a FACS 11
`cell sorter (Becton Dickinson, Inc., Sunnyvale, CA).
`Cellular Radioimmunoassay. muMABs 7C2 and 4D5 were labeled
`with Na”’1 (Amersham, Arlington Heights, IL) by the Iodo-Gen
`method as previously described (18). Briefly, 100 pg of antibody were
`added to 0.5 mCi of Nam] in a glass tube coated with 10 pg lodo-Gen
`(Pierce, Rockford, IL) for 10 min at room temperature. Free Na'zsl
`was removed by chromatography on a PD-10 column (Pharmacia,
`Piscataway, NJ), and eluted fractions were pooled and stored at 4°C.
`The immunoreactivities of x2"‘l-7C2 and ”51-4D5 using NlH3T3 HER-
`2/neu cells were 84% and 79%, respectively, by linear extrapolation to
`binding at infinite antigen excess (19) (data not shown). The avidities
`of ”’I-7C2 and '”I-4D5 as determined by Scatchard analysis were 4.48
`x 10’ L/M and 1.55 x 109 L/M, respectively (data not shown).
`NIH3T3 HER—Zlneu cells were washed with cold serum-free RPMI
`1640, pelleted by centrifugation, and cooled on ice for 45 min. ”’1-
`muMABs were then added to cell pellets in a ratio of 0.1 pg protein
`(200,000 cpm)/10‘ cells and incubated for 1 h. Cells were washed twice
`with cold RPMI 1640, plated in microtiter wells (1 x 10" cells/well),
`and warmed to 37'C in a humidified C02 incubator. After 0, 1, 4, 10,
`and 24 h the cultures were assayed for surface membrane-bound,
`intracellular, and supernatant radioactivity in a Beckman Gamma 5500
`counter (Palo Alto, CA) as previously described (20). Cell surface
`radioactivity was determined by elution of antibody from the cell
`membrane by a brief (S-min) exposure to a low-pH buffer (pH 1-2).
`The cells were subsequently pelleted by centrifugation at 300 x g and
`removed from the microtiter wells with cotton swabs to determine
`intracellular cpm. Culture supematants (0.2 ml) were treated with 0.6
`ml 25% TCA to precipitate protein—bound us1 released from the cell
`surface. This was separated from TCA-soluble supernatant radioactivity
`by centrifugation, and both fractions were assayed in a gamma counter.
`Immunoelectron Microscopy. NIH3T3 HER-Z/neu cells were grown
`to confluence in 96-well flat—bottomed plates and then incubated with
`muMAB 7C2 (64 pg/ml) for 45 min at 4'C. The cells were washed
`twice with cold media, and then a 1:2 dilution of a horseradish perox—
`idase-conjugated, monovalent, Fab’ goat anti-mouse immunoglobulin
`antiserum was added for an additional 45 min. The plates were washed
`again and incubated at 37'C for 0, 15, 30, or 240 min and fixed in 2%
`glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate (pH 7.4).
`Cells were then reacted for 15 min with 3,3’~diaminobenzidine (Sigma,
`St. Louis, M0) at 0.5 mg/ml in 0.05 M Tris buffer, pH 7.6, containing
`0.0009% hydrogen peroxide. After two washes in 0.1 M cacodylate,
`cells were postfixed in 1% osmium tetroxide and dehydrated in graded
`ethanol. Cell layers were then carefully processed into Epon 812, and
`blocks were sectioned en face or in cross-section and examined in a
`Philips 201 transmission electron microscope operated at 60 kV. Con-
`trol experiments demonstrated no detectable binding of isotype-
`where V,‘ is the mean tumor volume on day x and V0 is the mean tumor
`volume on the day of treatment.
`matched irrelevant antibodies DA4—4 and UPC10 to 3T3/HER-2/neu
`or SKBR3 cells or of anti-HER-Z/ueu antibodies 7C2 or 405 to
`At 0, 24, 48, 72, 144, and 216 h the mice were anesthetized and
`untransfected 3T3 cells by this immunoperoxidase technique.
`imaged for 10 min with a 400T General Electric gamma camera
`Antibody Biodistributions.
`lmmunodeficient 4—6-week-old male
`equipped with a high-energy collimator. The camera was peaked for
`BALB/c x C57/Bl beige/nude mice (Life Sciences, St. Petersburg, FL)
`I"I using a symmetric window of 38 keV. The activity within each
`were used for all in vivo experiments, since preliminary studies dem-
`mouse image was determined, and mean values were used to construct
`onstrated a higher tumor implantation rate (100% versus 80%) and
`whole-body time-activity curves for I“1.4D5 and I"I-DA4-4. Tumors
`greater synchrony of tumor growth than that observed with standard
`were outlined as regions of interest with a dedicated ADAC DPS-3300
`nude mice (data not shown). Animals received s.c. flank injections of 1
`computer, and the intraregional activity was measured in order to
`x 106 NIH3T3 HER-Z/neu cells. Palpable tumors (24—280 mm’; mean,
`generate tumor time-activity curves. Data were corrected for radioactive
`60 mm’) developed after 10 days. For biodistribution experiments,
`decay by serially imaging an ”'1 standard. All animals were given
`muMAB 4D5 and isotype-matched control muMAB DA4-4 (300 pg)
`potassium iodide as described above.
`1917
`
`Potassium iodide was added to the drinking water of mice 72 h prior
`to injecting radiolabeled antibody and was continued for 2 weeks in
`order to block thyroid uptake of free iodine.
`Autoradiography. To assess the homogeneity of radioactivity deliv—
`ered to tumor cells at a microscopic level, s.c. nodules were excised
`from 15 mice 18 h after i.p. injection of 10 pg (15-20 pCi) of radioio-
`dinated antibodies and processed for autoradiography. Excised tumors
`were bisected, embedded in OCT compound (Miles, 1nc., Elkhart, IN)
`and frozen in a dry ice—ethanol bath. Full-thickness 6-pm cryosections
`were cut, fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1
`M cacodylate (pH 7.4), dehydrated in graded ethanol, and air dried.
`”'1—4D5 (or u’1—DA4~4) was detected by clipping slides in Kodak NTB-
`2 nuclear emulsion (International Biotechnologies, Inc., New Haven,
`CT), exposing for 1 week at 4‘C, developing in Dektol (Kodak), fixing,
`and staining. A Nikon Microphot microscope was used to enumerate
`silver grains and perform photomicrography.
`Radioimmunotherapy and Radioimmunoscintigraphy. For therapy ex-
`periments, muMABs 4D5 and DA4—4 (0.6-1.0 mg) were radioiodinated
`with Na”'1 (6—10 mCi) to specific activities of 7.2-9.3 pCi/pg. Follow—
`ing PD-10 column chromatography, >98% of the I"I in the resulting
`radioimmunoconjugates was protein bound, as demonstrated by cellu-
`lose polyacetate electrophoresis. Groups of 4-7 tumorbearing mice
`each received injections of 400 pCi (range, 392—432 pCi) of IJ'1-
`muMABs in 0.6 ml PBS in Experiment 1 (45 pg/mouse) and 700 pCi
`(range, 667-705 pCi) in Experiment 2 (100 pg/mouse). Additional
`control groups received either unlabeled muMAB 405 (45 pg/mouse
`in Experiment 1; 100 pg/mouse in Experiment 2) or PBS. Tumors
`were measured twice weekly in 3 dimensions with a precision caliper,
`and the mean volumes were plotted versus time to generate growth
`curves. The percentage increase in tumor size was calculated as
`
`V, — V0
`V0
`
`% increase =
`
`x 100
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:83)(cid:85)(cid:76)(cid:79)(cid:3)(cid:24)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:27)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:21)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on April 5, 2018. © 1992 American Association for Cancer Research.
`
`

`

`a
`
`too + SurfaceCPM
`‘0' SupernatantCI’M
`80 + IntracellularCPM
`EU 60
`
`
`
`40
`
`20
`
`0
`
`E.
`
`2
`s“
`
`0
`
`4
`
`16
`12
`3
`Time (hours)
`
`20
`
`24
`
`b 25 + TCASol.CPM
`--
`'TCAPrecip.CPM
`
`ANTIBODY TARGETING OF HER-2/neu
`
`a?
`
`En.
`
`UT
`
`:
`‘6p.
`
`0
`
`4
`
`12
`8
`16
`Time (hours)
`
`20
`
`24
`
`Fig. 3. Intemalization and catabolism of antibody by SKBR3 cells. Cells were
`pulse labeled with ”51-405 and incubated at 37°C for 0, 1, 4, 10, or 24 h before
`assaying for surface membrane-bound, intracellular, and supernatant cpm (a).
`Culture supernatants were subsequently precipitated with 25% TCA to distinguish
`intact usI-‘tDS (TCA-precipitable) from low-molecular-weight catabolites (TCA-
`soluble) (b).
`
`RESULTS
`
`Antibody Specificity. Indirect immunofluorescence with flow
`cytometry demonstrated intense, specific binding of the 4D5
`and 7C2 antibodies to cell lines known to express high surface
`antigen densities of the p185 HER-Z/neu gene product (3T3/
`HER-Z/neu cells, SKBR3 and SKOV3) but no significant bind-
`ing to cell lines lacking oncoprotein expression (e.g., untrans-
`fected 3T3 cells). Fig. 1 shows typical results from one of five
`experiments performed (using 3T3/HER-2/neu cells).
`Cellular Radioimmunoassays. Intemalization and catabolism
`of lz5I-4D5 and ‘251-7C2 by NIH3T3 HERJ/neu, SKBR3, and
`SKOV3 cells were evaluated in vitro using a cellular radio-
`immunoassay. Pulse-labeled cultures were assayed after 0, l, 4,
`10, and 24 h of incubation at 37°C for surface membrane-
`bound, intracellular, and supernatant radioactivity. Three sep-
`arate experiments yielded similar results. There was a steady
`loss of surface cpm from NIH3T3 HER-Z/neu cells, with a
`reciprocal increase in supernatant cpm for both 4D5 and 7C2
`(Fig. 2, a and b). Intracellular cpm peaked at 22% after 4 h for
`
`Number
`
`Cell
`
`Fluorescence Intensity (Logw)
`Fig. l. Fluorescein-activated cell sorter analysis of binding of anti-HER-Z/neu
`antibodies to untransfected NIH 3T3 cells and to 3T3 cells transfected with the
`HER-Z/neu oncogene. ----- , 4D5 on untransfected cells; — — — —, 7C2 on
`
`untransfected cells; —. 4D5 on HER-Z/neu transfected cells; ----- , 7C2 on
`transfected cells;
`. control antibody UPCIO on transfected cells.
`
`4D5 and 17% after 1 h for 7C2. After 24 h the percentage of
`total radioactivity remaining cell associated (i.e., cell surface
`plus intracellular cpm) was 46% and 56% for 4D5 and 7C2,
`respectively. Conversely, after 24 h 54% and 44% of total
`culture cpm were found within the supernatant for 4D5 and
`7C2, respectively. Supematants were treated with 25% TCA to
`determine the proportion of supernatant radioactivity contrib-
`uted by intact antibody (TCA precipitable) and by small-molec-
`ular-weight metabolites (TCA soluble). TCA-soluble cpm pro-
`gressively increased during the 24-h incubation period for both
`4D5 and 7C2, reaching peak values of 44% and 39% of total
`culture cpm, respectively (Fig. 2, c and (1). Hence, approxi-
`mately 44% of 125I—4D5 and 39% of ”51-7C2 were internalized,
`degraded, and exocytosed by the tumor cells in 24 h. The same
`pattern of internalization and catabolism was observed when
`these antibodies were incubated with human breast (SKBR3;
`Fig. 3) and ovarian (SKOV3; not shown) carcinoma cell lines,
`which also overexpress the HER-Z/neu oncoprotein.
`Immunoelectron Microscopy. The endocytic pathway of
`muMAB 7C2 was studied by immunoelectron microscopy in
`vitro. NIH3T3 HERéZ/neu cells were incubated with saturating
`concentrations of 7C2 for 30 min at 4'C, washed three times,
`and then incubated for 30 min with a monovalent, horseradish
`peroxidase—conjugated Fab’ goat anti-mouse immunoglobulin
`antiserum. Cells were washed again with cold buffer and then
`warmed to 37°C for 0, 15, 30, or 240 min before processing for
`electron microscopy as described in “Materials and Methods.”
`At 4'C anti-HER-Z/neu antibodies remained circumferentially
`distributed on the surface of cells (Fig. 4a), but when cells were
`warmed to 37°C endocytosis rapidly ensued. Within 15-30 min
`of incubation at 37°C,
`large numbers of peroxidase-labeled
`1918
`
`too
`
`“0
`
`2
`A.
`0 so
`
`l- 40
`a?
`20
`
`0
`
`o
`
`d
`
`a
`
`l6
`1:
`s
`Time (hours)
`
`:0
`
`24
`
`“-0-
`penal-MC
`+ 33".“ CPM PM
`—.— IntracelluluCPM
`
`too
`
`
`
`3°
`
`2
`a.
`0 5°
`0
`l- ‘0
`.e
`10
`
`
`
`s
`
`is
`11
`a
`Time (hours)
`
`to
`
`14
`
`0
`
`o
`
`c
`
`%TotalCPM
`
`%TotalCPM
`
`oaxtzis
`Time (hours)
`
`20 u
`
`0
`
`to
`II
`4 Wu
`lime (hours!
`
`50“
`
`Fig. 2. Intemalization and catabolism of antibody by NIH3T3 HER-Z/neu
`cells. Cells were pulse labeled with ”‘I-4D5 (a, c) or ‘z’I-7C2 (b, d) and incubated
`at 37‘C for 0, l, 4. 10, or 24 h before assaying for surface membrane-bound,
`intracellular, and supernatant cpm (a, 1)). Culture supematants were subsequently
`precipitated with 25% TCA to distinguish intact u’I-muMABS (TCA-precipitable)
`from low-molecular-weight catabolites (TCA—soluble) (c, d).
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:83)(cid:85)(cid:76)(cid:79)(cid:3)(cid:24)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:27)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:21)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on April 5, 2018. © 1992 American Association for Cancer Research.
`
`

`

`ANTIBODY TARGETING 0F HER—Z/neu
`
`time points evaluated. The tu-
`the normal organs at all
`morznormal organ ratios of radioactivity ranged from 5:1 to
`10:1 and were most favorable 24—72 h postinjection (Table 1).
`The superior localization of l3'1-4D5 within tumors was a result
`of specific binding to the p185 oncoprotein, as illustrated in
`Fig. 5b. Up to 8-fold more anti-HER-Z/neu muMAB than the
`irrelevant antibody lzsI-DA4—4 accumulated in the tumors.
`However, the percentage injected dose of radioactivity per gram
`of tissue of '3'1-4D5 decreased precipitously from 25% at 24 h
`to 5% at 120 h postinjection. The area under the blood activity
`versus time curve was determined for both conjugates and
`revealed that ”SI-DA4-4 remained in circulation approximately
`3 times longer than '3‘1-4D5 (data not shown). Localization
`indices for the tumor and normal organs are presented in Table
`
`%ID/Gm
`
`0
`
`25
`
`75
`50
`Time (Hrs)
`
`—._ Tumor
`Blood
`Kidney
`Liver
`Muscle
`Spleen
`LungStomach
`Thyroid
`
`
`
`Fig. 4. Immunoelectron microscopy documenting endocytosis of anti-HERZ/
`neu antibodies by N1H3T3 cells transfected with the HER-Z/neu oncogene.
`Indirect immunoperoxidase cytochemistry was used to study the internalization
`of muMAB 7C2 as described in “Materials and Methods.“ (1, muMAB 7C2 was
`densely distributed on the surface of N1H3T3 HER-Z/neu cells at time 0. Within
`15—30 min of incubation at 37‘C large numbers of peroxidase-labeled “coated
`pits” (b, arrows), “coated vesicles." and uncoated endocytic vesicles (c) were
`detectable. Large numbers of peroxidase-labeled dense body lysosomes became
`evident between 30 and 240 min of incubation (1!). Bars, 1 pm (a, c, d) or 0.1 pm
`(0)-
`
`“coated pits,” “coated vesicles,” and uncoated endocytic vesicles
`were detectable (Fig. 4, b and c). Large numbers of peroxidase-
`labeled dense body lysosomes became evident between 30 and
`240 min of incubation (Fig. 4d). Similar results were observed
`with other anti-HER-Z/neu antibodies and other p185—express~
`ing cell lines (e.g., SKBR3; data not shown).
`In Vivo Biodistribution. The biodistribution of muMABs 4D5
`
`%ID/Gm
`
`0
`
`25
`
`100
`
`125
`
`7 5
`50
`Time (Hrs)
`Fig. 5. a, biodistribution of ”'1—4D5 in beige/nude mice bearing HER-Z/neu—
`expressing tumors. Groups of five mice bearing s.c. N1H3T3 HER-Z/neu tumor
`grafts received simultaneous injections of 10 ug of I"1-4D5 (18 uCi) and 10 ug
`of I”l-DA4—4 (l4 uCi). At 6, 24, 48. 72, and 120 h postinjection the animals were
`sacrificed, and their tumors and normal organs were harvested, weighed, and
`assayed for ”'1 and ”’1 activity. The biodistribution curves for '3'1-4D5 within
`tumor. blood, and normal tissues are shown. b, comparative localization of ”'1-
`4D5 and 'z’l-DA4-4 within tumors as a function of time.
`
`Table 1 Tumormarmal organ ratios for "'l—405
`The tumor:normal organ ratios of activity for ”'1—4D5 were calculated at 6,
`24, 48, 72. and 120 h after the injection of radioimmunoconjugate into beige/
`nude mice bearing 3T3/HER~2/neu tumors. Groups of five mice were used to
`generate mean values for each time point. Data were normalized for tissue weight
`and corrected for radioactive decay.
`
`and 7C2 were evaluated in beige/nude mice bearing s.c.
`N1H3T3 HER-Z/neu tumor grafts. Animals received simulta-
`neous i.p. injections of 10 pg each of ”'1-4D5 and ”SI-DA4-4.
`120
`72
`48
`24
`6
`Organ
`1.21
`1.58
`1.35
`1.47
`0.43
`Blood
`At 6, 18, 24, 48, 72, and 120 h postinjection the mice were
`5.49
`10.40
`9.20
`10.56
`2.60
`Kidney
`sacrificed, and their tumors and organs were harvested, washed,
`5.50
`7.14
`6.36
`6.42
`1.54
`Liver
`weighed, and assayed for ”'1 and '251 activity. Specific localiza-
`4.01
`8.29
`6.77
`7.85
`2.15
`Spleen
`4.21
`5.55
`5.25
`6.37
`1.74
`Lung
`tion of antibody to tumors was documented in four separate
`6.79
`20.78
`20.65
`1 7.95
`3.77
`Stomach
`experiments. A representative study is shown in Fig. 5a. The
`4.12Thyroid 6.93 10.81 8.86 9.69
`
`
`
`
`
`concentration of l"1—4D5 within tumor exceeded that in any of
`1919
`
`Time (h)
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:83)(cid:85)(cid:76)(cid:79)(cid:3)(cid:24)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:27)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:21)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on April 5, 2018. © 1992 American Association for Cancer Research.
`
`

`

`ANTIBODY TARGET’ING 0F HER-Z/neu
`
`2. Specific uptake of mI-4D5 within tumors was seen as early
`as 6 h postinjection (localization index = 2.2) and reached a
`maximum value of 6.6 at 24 h. The biodistribution of muMAB
`
`7C2 paralleled that of 4D5, with up to 28% injected dose of
`radioactivity per gram of tumor accumulating within tumors at
`36 h, and tumor:normal organ radioactivity ratios of 5:1 to 30:1
`(data not shown).
`Autoradiography of Tumor Nodules. To assess the heteroge-
`neity of antibody distribution within tumor nodules, tumors
`from 15 mice in 3 biodistribution experiments were processed
`for autoradiography 18 h after injection of l3’I-4D5, as de-
`scribed in “Materials and Methods.” A heterogeneous distri-
`bution of autoradiographic silver grains was observed in tumors,
`with the heaviest grain densities occurring in perivascular lo-
`cations (Fig. 6).
`Radioimmunotherapy and Imaging. In order to assess the
`therapeutic efficacy of radioiodinated anti-HER-Z/neu mu-
`MABs in our animal model, beige/nude mice bearing estab-
`lished NIH3T3 HER-Z/neu tumor grafts received i.p. injections
`of 400 uCi of ”'1—4D5 (45 ug/mouse). Groups of control mice
`received either 400 pCi
`l"I-DA4-4, unlabeled 4D5 (45 ug/
`mouse), or PBS. Tumor volumes were measured twice weekly,
`and the mean values were plotted versus time to construct
`growth curves. No significant difference in tumor growth was
`observed between animals treated with unlabeled 4D5 and PBS
`
`Table 2 Localization indicesfar ”‘I-4D5
`The localization indices for l"l-4D5 were calculated 6, 24, 48, 72. and 120 h
`after the simultaneous injection of I"1—405 and ”’I-DA4-4 into tumor-bearing
`beige/nude mice, as described in “Materials and Methods.” Groups of five mice
`were used to generate mean values for each time point. Data were corrected for
`radioactive decay and spillover from the ”'1 to the lzsI channel.
`Time (h)
`
`Organ
`Tumor
`Kidney
`Liver
`Spleen
`Lung
`Stomach
`Thyroid
`
`6
`2.17
`0.96
`1.50
`1.07
`1.15
`0.75
`0.98
`
`24
`6.60
`1.09
`1.46
`1.10
`1.09
`1.59
`1.23
`
`48
`4.14
`1.08
`1.27
`0.97
`1.05
`2.08
`1.12
`
`72
`5.31
`1.06
`1.22
`0.97
`1.07
`2.16
`1.09
`
`120
`3.50
`1.48
`1.23
`2.02
`1.14
`2.51
`1.22
`
`8 weeks. None of the 10 mice mice treated with unlabeled 4D5
`or PBS survived for 8 weeks.
`
`Serial gamma camera imaging was performed as described in
`“Materials and Methods” to assess the abilities of ”‘I—4D5 and
`
`”'I-DA4-4 to image tumor grafts (Fig. 9). Excellent imaging
`of HER—Z/neu-expressing tumors was reliably achieved using
`'3'1—4D5, with optimal resolution being obtained between 8 and
`72 h after injection of l3‘I-4D5. Specific localization of radio-
`activity to tumors was not observed using l"I-DA4-4, but mice
`retained radioactivity longer when receiving injections of this
`antibody. The biological I”; of l"I-DA4-4 was 6.5 days, ap—
`proximately twice that of ”'I—4D5 (3.3 days; Fig. 10a). Tumor
`time-activity curves for ”‘I-4D5 and ”'I-DAM were generated
`by serially measuring the activity within a region of interest
`constructed around the tumor image (Fig. 10b). As anticipated,
`a higher peak activity within the tumor was achieved with the
`anti-HER-Z/neu conjugate. However, this antibody appeared
`to be cleared from the tumor more rapidly than the irrelevant
`conjugate. The protracted clearance of the irrelevant antibody
`from tumor sites most likely resulted from the lack of specific
`endocytosis and catabolism of I31I-DA4-4 by tumor cells, as
`well as from the increased blood flow to rapidly growing tumors
`in mice given the control reagent.
`
`(Fig. 7). However, mice treated with ”'1-4D5 showed marked
`tumor growth retardation. Animals given 13'1-DA4-4 also ex-
`perienced tumor growth delays, although the degree of inhibi-
`tion was significantly less pronounced than in the ”'1-4D5
`treated group (Fig. 7). The different regimens were also com-
`pared by calculating the percentage increase in tumor size at
`various days posttreatment, relative to the initial tumor volume.
`Table 3 demonstrates that '3‘1-4D5 was 20—fold more effective
`than I3'1-DA4-4 in retarding tumor growth 24 days after anti-
`body injection and was 75-fold more efficacious than unlabeled
`4D5. Although the effect of ”'1-4D5 was striking,
`tumors
`eventually progressed in all animals. Tumor regrowth, which
`generally occurred 3 weeks after therapy, was not due to the
`selection of an antigen-negative cell clone, since grafts contin-
`ued to overexpress the HER-2/neu protein when evaluated by
`immunohistochemistry (data not shown). Rather, treatment
`failure most
`likely resulted from the inability to deliver a
`sufficiently lethal radiation dose to the tumor. No fatal toxici-
`ties were observed in mice treated with 400 uCi of 13‘I-4D5 or
`'3‘I-DA4-4; all deaths at this dose level were due to tumor
`progression.
`In an attempt to achieve curative tumor ablations, we re-
`peated the radioimmunotherapy experiment using a higher l3‘I
`activity (700 rather than 400 uCi/mouse; Fig. 8). This higher
`dose of ”'I-4D5 caused tumor growth retardation similar in
`magnitude to that seen in mice given 400 [1C1 of the conjugate
`(Fig. 7) and did not prevent eventual tumor progression (Fig.
`8). Furthermore, increasing the radiation dose of the control
`reagent, '3'I-DA4-4, to 700 uCi increased the nonspecific total
`body irradiation in recipient mice dramatically, resulting in
`significant tumor regressions in this control group (Fig. 8). At
`the higher dose level, fatal hematological toxicity was observed
`in two of five recipients of the l3‘I-DA4-4 reagent but in none
`of the four mice treated with 13‘I-4D5. Hematological parame—
`ters reached nadir values 14 days posttherapy and returned to
`normal by day 21 in surviving mice (data not shown). Two of
`‘0. W. Press, .1. F. Eary. C. C. Badger, P. J. Martin, F. R. Appelbaum. R.
`four mice treated with 700 uCi l3'I-4D5 survived >8 weeks,
`Levy. R. Miller. S. Brown, W. B. Nelp. K. A. Krohn, D. Fisher, K. B. De Santes.
`B. Porter, P. Kidd, E. D. Thomas, and I. D. Bernstein, unpublished results.
`whereas only one of five mice treated with ”'I-DA4-4 survived
`1920
`
`DISCUSSION
`
`Breast and ovarian adenocarcinomas account for 36% of all
`
`adult female malignancies and cause over 50,000 deaths/year
`in the United States alone (21). Although considerable progress
`has been made in treating these diseases, approximately 30%
`of breast cancer patients and 60% of ovarian cancer patients
`cannot be cured with current therapeutic approaches. Patients
`whose tumors amplify and overexpress the HER-Z/neu gene
`have an especially poor prognosis (7) and constitute a group
`for whom new treatment strategies are needed. One novel
`approach for such patients might involve therapy with immu-
`noconjugates targeting the HER-Z/neu gene product. We have
`recently tested an analogous approach in 14 patients with
`relapsed non-Hodgkin’s lymphoma using radioiodinated mono-
`clonal antibodies directed against B-lymphocyte differentiation
`antigens. Preliminary results have been encouraging; all 14
`lymphoma patients have had objective responses, including 11
`complete responses, with remission durations ranging from 4
`to 38+ months (22).‘
`The HER-Z/neu oncoprotein is an attractive target for anti-
`body-mediated therapy for several reasons. Overexpression oc-
`
`(cid:82)(cid:81)(cid:3)(cid:36)(cid:83)(cid:85)(cid:76)(cid:79)(cid:3)(cid:24)(cid:15)(cid:3)(cid:21)(cid:19)(cid:20)(cid:27)(cid:17)(cid:3)(cid:139)(cid:3)(cid:20)(cid:28)(cid:28)(cid:21)(cid:3)(cid:36)(cid:80)(cid:72)(cid:85)(cid:76)(cid:70)(cid:68)(cid:81)(cid:3)(cid:36)(cid:86)(cid:86)(cid:82)(cid:70)(cid:76)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:73)(cid:82)(cid:85)(cid:3)(cid:38)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:3)(cid:53)(cid:72)(cid:86)(cid:72)(cid:68)(cid:85)(cid:70)(cid:75)(cid:17)(cid:3)
`(cid:70)(cid:68)(cid:81)(cid:70)(cid:72)(cid:85)(cid:85)(cid:72)(cid:86)(cid:17)(cid:68)(cid:68)(cid:70)(cid:85)(cid:77)(cid:82)(cid:88)(cid:85)(cid:81)(cid:68)(cid:79)(cid:86)(cid:17)(cid:82)(cid:85)(cid:74)
`(cid:39)(cid:82)(cid:90)(cid:81)(cid:79)(cid:82)(cid:68)(cid:71)(cid:72)(cid:71)(cid:3)(cid:73)(cid:85)(cid:82)(cid:80)(cid:3)
`Downloaded from cancerres.aacrjournals.org on April 5, 2018. © 1992 American Association for Cancer Research.
`
`

`

`ANTIBODY TARGETING OF HER-Z/neu
`
`0 1.131405
`o 1-1310A4-4
`A Unlabeled405
`A No Treatment
`
`(1
`
`10
`
`20
`
`30
`
`Day
`
`14
`
`n 12
`5
`V 10
`g '8
`6
`4
`g
`a 20
`
`3>
`
`Fig. 7. Radioimmunotherapy of NIH3T3 HER-Z/neu tumor grafts. Groups of
`five to seven beige/nude mice bearing well-established s.c. tumors received injec-
`tions of 400 yd "'1—405. 400 uCi "'1-DA4—4, unlabeled 4D5. or PBS. Tumor
`volumes were measured twice weekly, and the mean values were plotted versus
`time to generate growth curves.
`
`imally tolerated doses of radioimmunoconjugate were used (700
`uCi). At least two mechanisms may have contributed to our
`inability to permanently eradicate tumors in this model system.
`First, the inhomogeneous deposition of ”'1-4D5 within tumors
`demonstrated by autoradiography may have resulted in the
`sublethal irradiation of tumor cells distant from capillaries,
`allowing subsequent regrowth of tumors. Second, rapid intra-
`tumoral antibody catabolism may have compromised the effi-
`cacy of this approach by diminishing the retention of l3'1
`targeted to tumors by the 4D5 antibody. Cellular radioimmuno-
`assays and immunoelectron microscopy demonstrated prompt
`internalization of I"1—4D5 after binding to cells, followed by
`intracellular routing to lysosomes where degradation and deiod-
`ination occurred, followed by exocytosis of the radiolabel. Sim-
`ilar studies have recently been published by other workers (30).
`In vivo biodistribution studies demonstrated that the anti-HER-
`2/neu conjugate reached a 6—7-fold higher peak concentration
`within tumors than the irrelevant antibody but was also cleared
`more rapidly from tumors than the nonbinding, nonintemalized
`”‘l-DA4-4 antibody.
`Several strategies could be attempted to improve radiation
`delivery to HER-2/neu-expressing tumors. A fractionated treat-
`ment schedule might permit delivery of a higher cumulative
`
`curs in approximately one-third of breast and ovarian cancers
`but not on normal adult tissues (23). In addition, antigen density
`appears to be uniform throughout a given tumor, so the anti-
`body could theoretically be distributed to all regions of the
`malignancy (9). Furthermore, excellent concordance has been
`observed between the level of expression within primary and
`metastatic or recurrent lesions (8, 9, 12). Finally, the likelihood
`of developing an antigen-negative cell clone may be reduced in
`this setting, since continued expression of p185 appears to be
`a prerequisite for maintaining the malignant phenotype in cells
`transformed by HER-Z/neu (14).
`In this report, we have demonstrated the ability of a radio-
`iodinated anti-HER-Z/neu muMAB to retard the growth of
`HER-Z/neu-expressing tumors implanted in immunocompro—
`mised mice. Several mechanisms may be responsible for the
`antitumor effect including down-regulation of p185 expression
`(24) and radiation-induced genetic damage from I"l (25—27).
`Antigenic modulation of the HER-Z/neu protein may deprive
`the cell of an important oncog