[CANCER RESEARCH 44, 1002-1007. March 1984]
`
`Growth Inhibition of Human Tumor Cells in Athymic Mice by Anti-
`
`Epidermal Growth Factor Receptor Monoclonal Antibodies‘
`
`Hideo Masui,’ Tomoyuki Kawamoto, J. Denry Sate,’ Bonnie Wolf, Gordon Sato,‘ and John Mendelsohn‘
`Cancer Center. 04958. University or California, San Diego, La Jolla. California 92093
`
`ABSTRACT
`
`Monoclonal antibodies (MoAbs) were raised against epidermal
`growth factor (EGF) receptors on a human epiderrnoid carcinoma
`cell line, A431. Administration of anti-EGF receptor MoAbs inhib-
`ited tumor formation in athymic mice by A431 cells and by
`another epidermal carcinoma cell line, T222. when one of the
`same MoAbs was used in therapy against Li-7 (a human hepa-
`toma) and HeLa cells (a cervical carcinoma). tumor growth was
`not affected. The number of EGF receptors on A431 cells was
`about 100-fold higher than on T222, Li-7, and HeLa cells, sug-
`gesting that the number of EGF receptors may not be an
`important determinant in suppressing tumor growth. Three anti-
`EGF receptor MoAbs were used in the present studies. MoAbs
`528 (immuncglobulin G223) and 225 (immunoglobulin G1) are
`capable of competing with EGF for receptor binding and inhibit
`proliferation of A431 cells in culture. The other MoAb, 455
`(immunoglobuiin G1), is incapable of blocking the binding of EGF
`to its receptors and has no effect on the proliferation of cultured
`A431 cells. All three MoAbs inhibited A431 tumor growth in
`athymic mice, indicating that the antibody isotype and the site of
`binding on the EGF receptor are not the determinants of antipro-
`liferative activity in vivo. The observation that MoAb against the
`receptor for EGF is cytostatic rather than cytocidal in vitro against
`A431 cells, yet completely prevents tumor growth in vivo, sug-
`gests that some host animal responses also may be involved in
`the antitumor effect. MoAbs against growth factor receptors
`could provide useful immunotherapeutic agents.
`
`lNTRODUCTlON
`
`There have been many attempts at passive immunotherapy of
`malignant tumors, but there are only a few reports in which the
`administration of conventional antisera inhibited tumor growth in
`experimental animals or in cancer patients (26). Thus, passive
`immunotherapy has not been established as a treatment for
`human cancer. The maior reasons are the difliculties in identifying
`tumor-associated antigens and in preparing homogeneous anti-
`body in large quantities by conventional methods.
`Development of the hybridoma technique by Kohler and Mil-
`stein (14, 15) has overcome these difficulties, and experimental
`cancer immunotherapy has been revived in recent years using
`MoAbs.“ Several recent reports demonstrated that MoAbs inhibit
`
`Received May 31.19B3:accepted December 2,1983.
`‘This research is conducted in part by the Clayton Foundation for Research.
`California
`‘Recipient of Grant CA 23052. To whom requests for reprints should be
`addressed.
`“Present address: MOl9Cul&l'G€|'l81icS, Cityof Hope Medicalcenter. 1500 E.
`Duane Road. Duane, CA 91010.
`‘ Recipient of NIH Grants CA 33397, GM 17702. and CA 19731.
`5 Recipient of Grant CA 23052. Clayton Foundation Investigator.
`“The abbreviations used are: MoAb. monoclonal antibody; EGF, epidermal
`
`the growth of human tumor xenografts in athymic mice (8, 9, 16,
`27). Furthermore, the results of several clinical studies on the
`treatment of leukemia and lymphoma using MoAbs have been
`published. but in these cases. clinically significant results have
`not been obtained (17, 18. 20, 23-25), with one exception. This
`was a report on the treatment of a human B-cell lymphoma with
`anti-idiotype antibody, in which the patient has been free from
`disease for more than 1.5 years (t 9).
`In most of these studies, MoAbs against tumor—associated
`antigens have been used for treatment. Another type of lvloAb
`which might be used for cancer immunotherapy is an antibody
`against plasma membrane receptors for growth factors. it is well
`known that the proliferation of tumor cells in culture is controlled
`by various growth factors. and a similar controi mechanism is
`postulated for the control of tumor cell growth in vivo (2).
`Therefore, MoAbs against growth factor receptors, which could
`block access of growth factors to their receptors, may provide
`useful therapeutic agents. Recently, Trowbridge and Domingo
`(29) reported that treatment with anti~transferrin receptor MoAb
`can inhibit tumor formation by a human melanoma cell line in
`athymic mice.
`A431 ceils, a human epidermoid carcinoma cell tine, express
`an unusually large number of EGF receptors on the cell surface
`membrane (1 to 3 x 10°/cell) (4, 7). and addition of EGF to the
`culture medium inhibits the proliferation of these cells in culture
`(1. 6). We have developed MoAbs against the EGF receptor
`using partially purified EGF receptors from A431 cells as antigen
`(13). In this paper, we report that the administration of anti-EGF
`receptor MoAbs inhibits tumor formation in athymic mice by 2
`human epidermoid carcinomas, A431 and T222. However,
`growth of 2 other xenografts is not affected.
`
`MATERIALS AND METHODS
`
`Animals. BALB/c athymic mice 6 to 8 weeks of age were used in the
`present studies. These mice were bred and maintained at the Athymic
`Mouse Colony Facility at the University of Calilomia. San Diego. The
`procedures for breeding and maintenance have been published previ-
`ously (22).
`Human Tumor Cells. A431. T222, Li-7, and HeLa cells were main-
`tained in a mixture of DME and l-lam‘s F-12 medium (Grand Island
`Biological Co.), containing 15 mm 4-(2-hydroxyethyi)-1-piperazineeth~
`anesulfonic acid buffer and 5% newborn calf serum. at 37°C in 5%
`COz295°/o air atmosphere.
`The Li-7 human hepatoma tumor line was established originally at the
`Cancer Center Research Institute, Tokyo, Japan (10). This tumor was
`obtained after 50 passages and has been transplanted serially in this
`facility for 22 passages over the past 3 years. The human lung epidermoid
`carcinoma (T222) was established in our laboratory by inoculating fresh
`tumor tissue from surgery and was used after 47 to 50 passages in the
`
`growth factor; DME. Dulbecco‘s modified Eagle‘s medium; PBS. phosphate-bufh
`ered saline.
`
`1002
`
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`
`present studies. Cell lines were established from these tumors in our
`laboratory and were cultured as described above.
`Anti-EGF Receptor MoAbs. in the present studies. we used 3 anti-
`EGF receptor MoAbs, referred to as Nos. 528, 225. and 455, raised
`against the EGF receptor on A431 cells, derived from a epidermoid
`carcinoma of the vulva. The development and characterization of these
`murine MoAbs have been published elsewhere (Footnote 7; Ref. 13).
`MoAb 528 has the isofype lgG2a and competes with EGF for binding to
`its receptors. when MoAb 528 is added to culture medium, proliferation
`of A431 cells is inhibited. MoAb 225 of the lsotype lgG1 also competes
`with EGF binding to receptors and inhibits proliferation of cultured A431
`cells. In contrast, MoAb 455, also of the isotype lgG1, is incapable of
`blocking EGF from binding to its receptors and does not affect growth
`of A431 cells in culture.
`Hybridorna cells producing MoAbs were cultured in a 2:1:1 RPMl-
`l640:DME:Harn's F-12 mixture containing 15 mu 4-(2-hydroxyethyi)—1-
`piperazineethanesulfonic acid, insulin (10 pg/ml), 10 uM mercaptoethanol,
`and 10 um ethanolamine. To raise large amounts of MoAbs, cells were
`harvested and injected i.p. into athymic mice (107 cells/animal). After 2
`to 3 weeks. ascites fluid was collected, centrifuged to remove floating
`cells, and kept frozen until purification. MoAbs were purified by ammo-
`nium sulfate fractionation followed by DEAE-cellulose column chroma-
`tography. The purity of immunoglobulins in these preparations was more
`than 90%, as judged by sodium dodecyl sulfatezpoiyacrylamlde gel
`electrophoresis. Ascites fluid from one animal provided 3 to 5 mg of
`purified MoAb.
`A murine-murine hybridoma (AR8229), which produces MoAb against
`a haptene (tyrosyi arsenate). obtained from Hybritech. lnc., was used as
`a control.
`Assay of Tumor Growth in Athymic Mice. A431 cells, T222 celis. Li-
`7 cells. or HeLa cells from stock cultures were trypsinized and washed
`with serum-free DME:Ham's F-12 (1:1) medium by oentrifugation. Tumor
`cells were injected so into athymic mice at the specified closes in 0.2
`ml of medium. Tumor size was measured twice weekly using the formula,
`1/2 x 1r x length x width x height (cm). in some experiments with i.l-7
`hepatoma, T222 epidermoid carcinoma, and A431 epidermoid carci-
`noma, ceils for injection into mice were obtained from tumor xenografts.
`in these experiments, tumors of 2- to 4-g weight were removed. minced
`finely with scissors, and washed with DME:Ham‘s F-12 medium, and a
`portion was injected s.c. into athymic mice through an 18-gauge needle.
`For treatment. varying quantities of MoAb were dissolved in 0.5 mi of
`PBS. pH 7.4, and injected i.p. into mice. Initially, injections were given 3
`times per week, but identical results were obtained in later studies using
`a twice weekly schedule. Control animals received 0.5 ml of PBS. Each
`experimental group consisted of 5 or 6 athymic mice unless otherwise
`described.
`
`’”l-EGF Binding Assay. The number of EGF receptors on A431 cells,
`T222 cells, Li-7 cells, and Heta cells was determined by the method
`described previously (13). Briefly, receptor grade EGF (Collaborative
`Research) was labeled with “I (Amersham) by the chloramine T method
`(11). The specific activity was 2.5 x 105 cpm/ng. Cells fixed with 0.2%
`p~tormaldehyde were plated in wells of microtiter plates containing glass
`fiber filters (V & P Enterprises, San Diego, CA) and washed with PBS
`containing 0.25% gelatin and 1% albumin. Varying concentrations of “'51-
`labeled EGF plus unlabeled EGF were added to the wells. After incubation
`for 2 hr at 37°C. the cells were washed with PBS containing 0.25%
`gelatin and 5% newborn calf serum, and radioactivity bound to filters
`was measured with a 7 counter. Nonspecific binding was determined by
`adding 100-fold excess unlabeled EGF. Scatchard analysis was used to
`determine the number of EGF receptors and K,, values.
`Clearance of "’l-labeled MoAb in Athymic Mice Carrying A431
`Tumors. ‘2’l—labeled MoAb 528 was prepared from purified MoAb 528
`by the procedures described previously (13). A431 cells were injected
`s.c. into athymic mice. when small tumors had formed. "sl-labeled MoAb
`528 (10 ;tCi/0.6 149) was injected i.p. into a group of 5 animals. The same
`amount of mi-labeled MoAb 528 plus 1.0 mg of unlabeled MoAb 528
`was injected into a second group of 2 animals. Ten pl of blood were
`
`MARCH 1984
`
`Immunotherapy of Human Tumor Xenografts
`
`taken from the tail veins on successive days and mixed witht ml of 0.1%
`albumin solution. and trichloroacetic acid was added. Acid-precipitable
`radioactivity was counted in a 7 counter. The first group of mice were
`killed. tumors and muscle were removed from each animal and weighed,
`and the tissue radioactivity was measured in a -y counter. The specific
`radioactivity of tumor and muscle was calculated for each animal.
`
`RESULTS
`
`Tumorigenicity of Human Tumor Cells in Athymic Mice. in
`initial experiments, the tumorigenicity of human tumor cells in
`BALB/c athymic mice was tested by injecting varying numbers
`of cells so (Table 1). inoculation of more than 10“ A431 cells
`was needed to produce tumors consistently, and 107 cells were
`selected for the experiments conducted in this study. l-iel.a cells
`were less tumorigenic than were A431 cells, requiring an inocu-
`lation of more than 5 x 10‘ cells for tumor formation. The
`
`tumorigenicity of T222 human epidermoid carcinoma ceils and
`Li-7 human hepatoma cells was also tested, and it was found
`that inoculation of 10” cells was required for tumor formation.
`f=urthermore, tumor growth was too slow for a useful growth
`assay.
`in feasible conduction of immunotherapy experiments
`using T222 and Li-7 cells. an inocuium of finely minced tissue
`was used in subsequent experiments. The inoculum consisted
`of 0.2 mi of minced tumor tissue. Since therapy against T222
`cells and Li-7 cells was analyzed in a system using minced tumor
`and HeLa cells in a system using cultured cells, A431 experi-
`ments were carried out with both systems.
`Growth inhibition of A431 Cells in Athymic Mice by Admin-
`istration of Anti-EGF Receptor MoAbs. Three monoclonal an-
`tibodies against the EGF receptor were assayed for their capacity
`to suppress the growth of A431 cells in athymic mice (Chart 1).
`Treatment 3 times weekly with ascites fluid containing either
`MoAb 528 or 225 completeiy inhibited tumor formation when
`treatment was started on the day of tumor. cell inoculation. After
`stopping the MoAb treatment, the mice were observed for 2
`months, and no tumors formed in these animals. Administration
`of EGF (30 pg/animal, 3 times weekly) did not affect the tumor
`growth. Treatment with ascites fluid containing control MoAb
`from ARB229 did not affect the growth of A431 cells in athymic
`mice (Chart 2). Also, treatment with mouse serum immunogiob~
`ulin did not affect the tumor growth (data not shown).
`.
`Next, the capacity of MoAb treatment to modify the growth of
`developed tumors was tested. Seven days after injection of
`
`Table 1
`Tumorigenicity of A431 and HeLa cells in athymic mice
`Cells were inoculated so as described in ‘Materials and Methods.‘ Tumor size
`was measured 2 weeks after inoculation. Each group given injections of A431 cells
`consistedofsanlrnalsandeachgroupthatreceivedHeLaceltsconslstedof2
`animals.
`
`Cells
`A431
`
`No. of cells
`inoculated
`10’
`3.3 x 10‘
`10‘
`3.3 x 10°
`
`.
`
`Tumors
`formed’
`6/6
`6/6
`2/6
`0/6
`
`Tumor size?’
`(cm’)
`2.2 :1: 0.6
`1.3 1 0.3
`0.3 :9: 0.2
`0.0
`
`Hour
`
`1.4
`2/2
`5 X 10’
`0.4
`2/2
`10"
`0.2
`t/2
`x 10°
`0.0
`0/2
`10‘
`tumors‘ formed per total number of animals studied.
`VVVV
`"Average :t SE. of tumor size for A431 cells and the average size for HeLa
`’
`cells.
`
`5
`
`1003
`
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`
`inhibition of tumor formation was obtained by treatment with
`MoAb 528 at a 0.2-mg dose. whereas the same effect with
`MoAb 225 and 455 therapy required a 2-mg dose. A significant
`suppression of tumor formation was evident at 0.2-mg doses of
`MoAbs 225 and 455 (Table 2). When these experiments were
`repeated. there was some variation in the rate of tumor growth.
`but similar responses to MoAb treatment are always obtained.
`The results of these experiments demonstrate clearly that anti-
`EGF receptor MoAbs, with different characteristics in terms of
`igG isotypes and capacity to block EGF binding,
`inhibit the
`growth of A433 tumors in athymic mice.
`In a parallel experiment, minced A431 tumor tissue was inoc~
`ulaled into athymic mice, and animals were treated with MoAb
`528.
`inhibition required higher antibody concentrations with
`minced tumor than with cuitured cells. Complete inhibition of
`
`4.0
`
`l
`
`i
`
`i
`
`i
`
`O
`
`21
`
`
`
`3.05V)on
`
`E ‘
`
`g 2.0
`E
`
`1.0
`
`we
`
`7
`
`'
`
`14
`DAYS
`
`Charts. Effect of anti-EGF receptor MoAb on the growth of A431 cells in
`athymic mice. A431 cells were injected s.c. in athymic mice. Seven days after the
`tumor cell inoculation, the treatment with 0.5 ml at MoAb 528 asoites fluid was
`started. The treatment schedule is shown by the arrows. 0. control group; 0,
`treated group. Bars, S.E.
`
`Table 2
`Inhibition of tumor formation by A431 cells in animals treated with am‘i—EGF
`receptor MoAbs
`In Experiments 1. 2, and 3. A431 cells (1 0"/animal) were injected s.c. Treatment
`was started simultaneously. and thereafter. animals were treated twice a week.
`Sixteen days alter tumor cell inoculations. treatment was stopped. and tumor size
`was measured. in Experiment 4, minced tumor was inoculated s.c. instead of a
`singlecelsuspensim.sridueaunentwid1MoAbwasmesarneasabove. After20
`days.ueahnmtwasstopped.andhmwrsizewasmeaswed.Whennfincedunnr
`wasused,asmalresiduealweysremamedatmesheofhjecflm(seetextfor
`explanation).
`Experi~
`ment
`1
`
`Dose
`(mg)
`
`c
`0.0002
`0.002
`0.02
`0.2
`2.0
`
`Tumor for—
`mation"___
`6/6
`6/6
`6/6
`4/6
`0/6
`0/6
`
`Tumor size
`_ lcrrtflwww Wflw
`‘
`k
`3 8 : 0.8”
`2.4 1 0.4
`2.4 t 0.7
`1.1 i 0.5 (0.01 < p < 0.05)
`0
`0
`
`Treatment
`523 lgG
`
`2
`
`3
`
`225 igG
`
`455 igG
`
`0
`0.002
`0.02
`0.2
`2.0
`
`0
`0.02
`0.2
`2.0
`
`4
`
`528 lgG
`
`0
`0.02
`0.2
`2.0
`‘ Number of animals in which tumors
`" Average :t S.E.
`
`we
`615
`5/5
`3/5
`0/5
`
`5/5
`6/6
`3/5
`0/6
`
`6/6
`4/4
`6/6
`0/6
`
`- 1.9 3: 0.4
`3.7 1 0.5
`2.8 1: 0.6
`0.3 t 0.2 (0.05 < p < 0.1)
`0
`
`2.4 : 0.5
`5.6 1 1.3
`0.8 1: 0.4 (0.02 < p < 0.05)
`0
`
`8.4 1 1.7
`5.6 t 2.2
`1.0 :t 0.3 (0.01 < p < 0.02)
`0.1 1 0.0 (p < 0.001)
`total number of animals studied.
`
`CANCER RESEARCH VOL. 44
`
`H. Masui et al.
`
`0.2 0.0
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`
`
`TUMORSIZE
`
`0
`
`7
`
`---------—--.u-----nn——-—-—¢in—-4:-/,4’-u
`I3
`16
`20
`23
`80
`DAYS
`
`of A431 cells in athymic mice by anti-EGF receptor
`Chart 1. Growth
`MoAb. A431 cells were injected s.c. into BALB/c athymic mice (107 ceis/animal).
`ForthetreatmentwithMoAbs.0.5mlofascites(whichoontained2to3mgot
`anti-EGF receptor immunoglobulin) was injected i.p. into animals simultaneously
`with the tumor cell inoculation and thereafter as indicated by the arrows. one group
`of animals was treated with 30 pg of EGF in 0.2 ml of PBS. The same volume of
`PBS was injected into the control animals. Each group consisted of 5 or 6 animals.
`ThehmorsizewasmeasmedasdesaibedmTable1.Asdteswhidicontah1ed
`anti»EGF receptor MoAb was prepared in the following way. Hybridorna cells (2 to
`3 x 10’ mils/animal) were injected i.p. into BALS/c athymic mice. Ascites was
`oouecled2to3weeksaflerinouiladonendcerimh:ged.andu1esupanatams
`were frozen until usage. 0. control group: O, EGF-treated group; A and I. group
`treated with antl~EGF receptor MoAbs 528 and 225, respectively. Bars. S.E.
`
`lZ.0
`30.0
`8.0
`6.0
`4.0
`2.0
`
`
`
`TUMORSlZE
`
`0
`
`2
`
`4
`
`6
`
`I0
`
`l2
`
`l4
`
`8
`DAYS
`
`Chart 2. Effect of MoAb ARB229 against the tyrosyl arsenals haptene on the
`growmotA431oellsmamymicnice.Expaimmtalprocedureswaedesaibedh
`thelegendofChart1.AsdtesfliidcontainhgMoAbARB229wesi1jectedes
`treatment. 0. control group: O. ascites fluid (0.5 ml). Bars, S.E.
`
`A431 cells, tumors had grown to an approximate volume of 1 cu
`cm. At this juncture, the tumor-bearing animals were divided into
`2 groups displaying similar size distributions of tumors, and
`treatment with ascites fluid containing MoAb 528 was started
`on a twice weekiy schedule. As shown in Chart 3, this treatment
`regimen reduced tumor growth significantiy. As previously, the
`same treatment abolished tumor formation completely when
`started at the time of tumor cell inoculation (data not shown).
`in the experiments described above, ascites fluid was used as
`the source of Mo/lbs. To quantitate the amount of antibody
`needed for inhibition of tumor formation. experiments were per-
`formed with purified immunoglobulin.
`Cultured A431 cells were inoculated into athymic mice; the
`animals were treated with various amounts of purified MoAb
`528, 225, or 455, and the effects upon tumor formation were
`tested. Because experiments with T222 cells and t.i~7 cells used
`minced tumor, rather than a suspension of cultured cells, the
`effect of MoAb treatment on growth of A431 minced tumor was
`examined also (Table 2). Treatment with increasing concentra
`tions of MoAbs resulted in progressive reduction in mean tumor
`size in each case.
`
`The inhibitory effect of MoAbs 225 and 455 was somewhat
`less than that of MoAb 528 on tumors from A431 cells. Complete
`
`1 004
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`Immunotherapy of Human Tumor Xenografts
`Tables
`Growth inhibition of human tumor cells in athymic mice by anti-EGF receptor
`MoAb
`
`In viva tumor growth assays were performed according to the procedures
`described in ‘Materials and Methods.” The number of HeLa cells inoculated was 5
`x 10’ in this experiment. For Li-7 cells and T222 cells, minced tumor was used
`(see text). MoAb treatment was started on the day of tumor cell inoculation. and
`thereafter treatment was twice weekly. when minced tumor was used. a small
`residue always remained at the site of injection (see text for explanation).
`No.
`528
`lgG
`treat-
`ment
`0
`0.2
`2.0
`
`Experi-
`ment
`1
`
`Tumor cells
`Hepatoma (u-7)
`
`Tumor
`forma~
`tion‘
`6/6
`5/5
`6/6
`
`Tumor size
`(cm‘')
`
`4.5 : 0.4”
`5.1 ~_+ 1.0
`4.9 1 1.3
`
`tumor fonnation was obtained with the 2.0-mg dosage, whereas
`with tumors from cultured cells, growth was completely sup-
`pressed with only 0.2 mg. We estimate that an inocuium of 0.2
`ml of minced tumor tissue contains. at most, 1 to 2 x 10’ cells,
`which is comparable to the inoculum of cultured A431 cells. Our
`estimate is based on the following considerations: a volume of
`0.2 ml of packed cultured A431 cells contains about 4 x 107
`cells; and microscopic examination shows that approximately 50
`to 70% of the A431 minced tumor consists of connective tissue.
`necrotic tumor tissue. and dead cells. We reason that the weaker
`inhibitory response with tumors from minced tisssue may be
`attributable to the fact that the minced tumor retains some of
`
`the preexisting histological structure of tumor tissue and may be
`capable of producing tumors more easily than a single-cell sus-
`pension. when minced tumor was inoculated s.c.. measurable
`residual tissue always remained even when tumor formation was
`inhibited, probably due to the connective tissue present in the
`inoculum. the residual s.c. mass never exceeded 0.3 cu cm. it
`
`should be noted that growing tumors did not appear in ade-
`quately treated animals, which were observed for 2 months after
`MoAb treatment was discontinued.
`
`‘*5!-labeled Anti-EGF Receptor MoAb in
`Clearance of
`Athymic Mice Carrying A431 Tumors. To determine the clear-
`ance of anti-EGF receptor MoAbs, "51-labeled MoAb 528 was
`injected i.p. into athymic mice carrying A431 tumors, and the
`specific radioactivity in the blood was measured on successive
`days as described in “Materials and Methods” (Chart 4). When
`0.6 pg of ‘Z51-MoAb 528 was administered. most of the acid-
`precipitable radioactivity was cleared from the blood in 2 to 4
`days. when 1 mg of unlabeled MoAb 528 was added as a carrier
`at the time of ‘25l-MoAb 528 iniection. the radioactivity remained
`in the blood for up to 8 days. For the former group of mice, the
`specific radioactivities were determined in the muscle and in the
`tumor 6 days after injection. The ratio of the specific radioactivity
`(tumorzmuscle) was 3.46 1 0.43. The results suggest that anti-
`EGF receptor MoAb is concentrated in the tumors, and the
`clearance data indicate that treatment on a twice weekly sched-
`ule is resonable. when mice were given injections of ‘*5!-MoAb
`528 plus carrier MoAb 528, the specific activity of blood remained
`much higher than that of tumor and muscle even 9 days after
`iniection.
`Growth Inhibition of other Human Tumor Cells by Admin-
`istration ot Anti-EGF Receptor MoAb. As described above,
`
`cmix104)
`
`DAYS
`
`Chart 4. Clearance of radioactivity from the blood of mice given injections of
`0.6 pg (10 pct) of ‘”l~labeled MoAb 528. The procedures are described in ‘Materials
`and Methods.“ Each line shows the radioactivity in the blood of each animal:
`- - - -. mice given injections withoutcenier MoAb 528; ————. mice given iniections
`of 1.0 mg of unlabeled MoAb 528.
`
`2
`
`3
`
`4
`
`HeLa
`
`Lung epidermoid
`carcinoma
`(T222)
`
`Lung epidermoid
`carcinoma
`(T222)
`
`0
`0.2
`2.0
`
`0
`0.2
`2.0
`
`0
`0.002
`0.02
`0.2
`
`6/6
`6/6
`5/5
`
`6/6
`0/6
`0/6
`
`6/6
`5/5
`0/6
`0/6
`
`0.9 1: 0.1
`0.6 x 0.1
`0.8 1 0.1
`
`1.6 .4: 0.2
`0.2 1 0.0 (p < 0.001)
`0.1 r 0.0 (p < 0.001)
`
`1.4 1 0.4
`0.6 t 0.2 (0.001 < p < 0.01)
`0.2 2 0.1 (p < 0.001)
`0.0 (p < 0.001)
`
`VVV"5"l§lurn.l;er of animals in which tumors formed per total number of animelsstddied.
`‘’ Average t SE.
`
`administration of anti-EGF receptor MoAbs inhibited the growth
`of A431 cells in athymic mice. To ascertain whether the effect of
`this MoAb treatment upon tumor xenograft growth is general-
`ized, we tested the effects of MoAb 528 on the growth of Li-7
`human hepatoma cells, HeLa cells. and T222 human lung epi-
`derrnoid carcinoma cells. The results are illustrated in Table 3.
`The growth of Li-7 human hepatoma cells and HeLa cells was
`not affected by this treatment. However, the growth of minced
`T222 tumors was inhibited significantly, even at a 0.002-mg
`dose. The inhibitory effect of MoAb 528 on this tumor growth
`was greater than that with minced A431 tumors (Table 2. Ex-
`periment 4).
`‘”l-EGF fainding Assay. One reason for selectivityin the
`antitumor activities of MoAb treatments might be differences in
`ligand binding to the various target cell populations. One impor-
`tant variable is the number of receptors for EGF on these cells,
`because it is known that A431 cells have a number of EGF
`
`receptors which is far higher than that reported for l-leLa cells.’
`We compared the number of receptors and the apparent disso-
`ciation constants for ""5l-EGF on the cells used in these studies,
`using the procedures described in “Materials and Methods." The
`numbers of receptors on A431. T222. Li-7, and HeLa cells (and
`their Kd values) were: 2.6 X 10‘/cell (K., l.6 x 10‘‘‘ M); 2.9 x
`10‘/cell (K. 4.2 x 10"’ M); 2.8 x 10‘/cell (K, 2.3 x 10‘9 M); and
`2.0 x 10‘/cell (Kg 7.1 x 10‘9 M), respectively. Thus, A431 cells
`have a greater number of receptors for this growth factor than
`do T222, Li-7, and HeLa cells, by a factor of 2 logs.
`Using saturating amounts of "SI-MoAb 528. the number of
`binding sites for MoAb was compared between A431 cells and
`T222 cells.
`in this experiment. the numbers of sites on A43t
`cells and T222 cells were 2.0 x 10°/Cell and 4.0 x 10‘/cell.
`respectively, demonstrating that A431 cells had 50 times more
`
`’ J. D. Sato, A. 0. Le. J. Mendelsohn, J. Polikofl. G. H. Sato, and T. Kawamoto.
`Characterization of mouse monoclonal antibodies to EGF receptors of A431 human
`epiderrnoid carcinoma cells, submitted for publication.
`
`MARCl-l 1984
`
`1 005
`
`Downloaded from cancerresaacrjournals.org on October 20, 2015. © 1984 American Association for Cancer
`Research.
`
`APOTEX EX. 1021-004
`
`

`
`H. Masai et al.
`
`binding sites for MoAb 528 than did T222 cells. These data
`suggest that the number of EGF receptor sites on tumor cell
`surface membranes does not account for differences in MoAb
`
`antitumor activity against the various cell lines examined in these
`studies.
`
`DESCUSSEON
`
`The results of our present studies show that the growth of
`A431 and T222 tumor xenografts in athyrnic mice is remarkably
`inhibited by anti-EGF receptor MoAbs. when MoAb 528 is used
`in therapy against Li-7 hepatoma or HeLa cells, under identical
`conditions, tumor growth remains unaffected. Both A431 cells
`and T222 cells were derived from epidermoid carcinomas. HeLa
`cells were established from a cervical cancer and were consid-
`ered to be epidermoid carcinoma cells (5). However. a reexami-
`nation of the original slides revealed that the tumor is an ade-
`nocarcinoma (12). The results suggest that tumor growth sup-
`pression by MoAbs against EGF receptors may be limited to
`epldermoid carcinomas. The fact that A431 cells (vulva source)
`and T222 cells (lung gource) both respond to MoAb therapy
`indicates that the tissue origin of the EGF receptors used to
`raise MoAbs is not a relevant factor in the antitumor effect.
`
`Presently. we are testing the effect of these MoAbs on the
`growth of a variety of human tumor xenografts in athymic mice
`to clarify these points.
`The number of EGF receptors on A431 cells is approximately
`100-fold higher than that on Ll-7 and HeLa cells, but the receptor
`number on T222 cells is comparable to that on Li-7 and HeLa
`cells. This suggests that the EGF receptor number on the cell
`surface membranes is not an important determinant in tumor
`growth inhibition.
`Among the 3 MoAbs utilized in our studies, 2 are capable of
`competing with EGF for receptor binding and of inhibiting A431
`cells in culture; the other is incapable of blocking EGF binding to
`its receptors and has no significant effect on the growth of
`cultured A431 cells.’ Despite the different in vifro characteristics,
`when administered to mice all 3 MoAbs inhibited the tumor
`formation by A431 cells. This suggests that binding of MoAbs to
`EGF receptors, and not blocking of EGF from binding to its
`receptors,
`is the essential process leading to suppression of
`tumor growth in the animals. However, it is also possible that
`binding of MoAbs to EGF receptors could alter the actual function
`of the receptors, resulting in tumor growth inhibition.
`Recently, Koprowski and coworkers (8. 28) proposed that only
`MoAbs of isotype lgG2a can inhibit the growth of human tumors
`in athymic mice. As described in our studies. we found that not
`only MoAb of isotype lgG2a (No. 528), but also MoAbs of lgG1
`isotype (Nos. 225 and 455) were capable of inhibiting tumor
`growth. This difference may be due to the fact that MoAbs used
`in their studies were raised against tumor-associated antigens,
`whereas the lvloAbs in our studies were raised against a growth
`factor receptor. lhese 2 types of MoAbs might exert antitumor
`activity by different mechanisms.
`Data from cell culture studies in our laboratory, as well as
`many others, suggest that malignant cells have specific require-
`ments for cell proliferation. These include some growth-promot-
`ing agents, such as hormones. growth factors, and transport
`proteins, that often are needed or may be essential for selected
`cell types (r'.e., EGF, transferrin. and insulin) (2). Furthermore,
`studies in which essential hormones, growth factors. and nu-
`
`trients are withheld from cultured cells have suggested that
`tumor cells are more likely to die when deprived of substances
`essential for growth than are normal cells (21). EGF is known to
`stimulate proliferation of various types of cells in culture, but
`when added to the culture medium of A431 cells, it results in
`growth inhibition.
`in comparison to the studies with MoAbs
`conducted in athymic mice inoculated with A431 cells, EGF
`therapy has no effect on tumor growth. This failure to inhibit
`growth of A431 cell tumors by EGF is possibly attributed to the
`short half-life of EGF in animals (3). In contrast, anti-EGF receptor
`MoAbs have a longer half-life of about 3 days, under the exper-
`imental conditions in our studies (Chart 4). Thus. MoAbs may
`provide an important method for controlling access of malignant
`cells to growth factors and hormones required for proliferation.
`in cell culture. MoAbs 528 and 225 inhibit proliferation of A431
`cells, but they do not kill the cells, since the cell number increases
`slightly in the presence of these MoAbs.’ when mice that re-
`ceived iniections of A431 cells were treated with MoAb, more
`than 90% of the inoculated cells probably were killed by the
`antibody. The data in Tables 1 and 2 suggest that, if 10“ or more
`cells of the 107 cell inoculum had survived the in vivo MoAb
`treatment, tumors would have formed. The mechanism by which
`MoAbs suppress tumor growth in animals remains unclear, but
`annihilation of A431 tumors by complement—mediated lysis is
`unlikely, because cultured A431 cells are not lysed in the pres-
`ence of these MoAbs and complement. Since cytotoxic T-cells
`are absent in athymic mice, natural killer cells, antibody-depend-
`ent killer cells (K), and/or macrophages may have contributed to
`the inhibition of tumor formation in the animals. Further experi-
`ments should clarify the mechanisms by which anti-receptor
`lvioAbs prevent tumor cell growth and explore refinements which
`could lead to direct application in the treatment of human cancer.
`
`ACKNOWLEDGMENTS
`
`we thank M. A. Zuibach for processing the manuwlpt and H. Matsunaga and
`his colleagues of the Athymic Mouse Colony Facility for provision and maintenance
`of athymic mice. We are grateful to Dr. W. Desmond (Hybrlteoh. La Jolla, CA) for
`providing ARB229 hybridoma.
`
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`
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