Surg Today Jpn J Surg (1996) 26:501-507 @ SURGERYTODAY © Springer-Verlag 1996 Effectiveness of an Adriamycin Immunoconjugate that Recognizes the C-erbB-2 Product on Breast Cancer Cell Lines HIROMITSU JINNO, MASAKAZU UEDA, KOHJI ENOMOTO, TADASHI IKEDA, PSARRAS KYRIAKOS, and MASAKI KITAJIMA Department of Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Abstract: Adriamycin (ADM) was chemically conjugated to a murine monoclonal antibody, A0011, which recognizes the c-erbB-2 product, via a disulfide bond using N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) and 2-iminothiolane (2-IT). The molar ratio of ADM to the monoclonal antibody ranged from 15:1 to 25:1 and enzyme-linked immunosorbent assay (ELISA) showed that the binding activity of the conju- gate was almost retained. We compared the efficacy of A0011 alone, ADM alone, the A0011-ADM conjugate, and a non- specific murine IgM-ADM conjugate, against the human breast cancer cell lines SK-BR-3, MDA-MB-361, MCF-7, and BT-20. The A0011-ADM conjugate was observed to be ten times more cytotoxic to the cell lines overexpressing the c-erbB.2 product, namely, SK-BR-3 and MDA-MB-361, than free ADM, but it showed weak cytotoxicity against the cell lines with a low level of c-erbB-2 product expression, namely, MCF-7 and BT-20. However, free A0011 and nonspecific murine IgM-ADM conjugate showed no cytotoxicity toward any of the four cell lines, while the addition of a tenfold molar excess of A0011 inhibited conjugate cytotoxicity. These data suggest that conjugate cytotoxicity is antibody-mediated. Moreover, conjugate cytotoxicity at 10-6M was correlated with antigen volume, and the data were fitted to the regression equation y = -11.631ogX + 116.38 where the correlation coefficient = 0.950. Our results indicate that targeting therapy aiming at the c-erbB-2 product may be useful in the treatment of breast cancers overexpressing the c-erbB-2 product. Key Words: Adriamycin, breast cancer, c-erbB-2, immuno- conjugate, targeting therapy Introduction The c-erbB-2 protooncogene encodes a 185-kDa cell surface glycoprotein belonging to the tyrosine kinase Reprint requests to: M. Ueda (Received for publication on Feb. 6, 1995; accepted on Nov. 7, t995) receptor family, 1 and its product is known to be exten- sively homologous to the epidermal growth factor re- . ceptor (EGFR). 2 Amplification and/or overexpression of the c-erbB-2 protooncogene has been found in can- cers of the breast, 3 ovaries, 4 stomach, 5 and lung, 6 and has been correlated with a poor prognosis. Moreover, artifi- cial overexpression of c-erbB-2 in NIH 3T3 cells has been shown to result in malignant transformation. 7,a These findings indicate that overexpression of c-erbB-2 may play a role in malignant tumor development. As breast cancer patients with overexpression of the c-erbB-2 product have a higher rate of recurrence and lower overall survival, 9 treatment involving not only surgery but also multidisciplinary therapy is required. As part of the multidisciplinary treatment, we devel- oped targeting therapy aimed at the c-erbB-2 product. We selected the c-erbB-2 product as the target for two reasons: first, because it exists on the cell membrane; and second, because it is expressed on malignant cells and almost never found in normal tissues~ In our previous studies, we conjugated B4G7, a mu- rine monoclonal antibody which recognizes human EGFR to gelonin, a 60S ribosome-inactivating protein, and examined its cytotoxic effect. The B4G7-gelonin conjugate killed EGFR-hyperproducing squamous carcinoma cells but not EGFR-deficient small cell lung cancer cells/° When this conjugate was injected intraperitoneally into nude mice bearing EGFR- hyperproducing cancer cells, it suppressed solid tumor growth/~ However, since gelonin is not a human protein and cannot be used clinically, we developed targeting therapy using an antineoplastic agent, pepleomycin, and determined its in vitro cytotoxicity/2 Although Adriamycin (ADM) is a clinically effective drug for treating breast cancer, its usage is limited by its dose- dependent side effects of bone marrow suppression and cardiac toxicity/3,~4 During the last decade, to minimize these side effects and increase its preferential concen- tration at the tumor site, ADM has been linked to a
`
`IMMMUNOGEN 2096, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`502 H. Jinno et al.: Immunoconjugate Against Breast Cancer variety of antibodies which react with tumor-associated antigens. 15-22 We conjugated ADM to a murine mono- clonal antibody, A0011, which reacts with the c-erbB-2 product via a disulfide bond. The cytotoxicity of this A0011-ADM conjugate was determined against four breast cancer cell lines that overexpress the c-erbB-2 product at various levels, and compared with free ADM and nonspecific murine IgM-ADM conjugate at match- ing doses. A0011 alone was also tested. The A0011- ADM conjugate was found specifically to kill breast cancer cell overexpressing c-erbB-2 and thus, we evalu- ated this new immunotherapy using an overexpressing oncogene as the target. Materials and Methods Chemical Reagents A0011 is an IgM murine monoclonal antibody that immunoprecipitates 15 amino acids (HTANRPEDECVGEGL) in the extracellular domain of the c-erbB-2 product without any homology to other transmembrane receptors. The A0011 used in this study was purchased from Iwaki Glass, Tokyo, Japan, and Adriamycin was donated by Kyowahakko, Tokyo, Japan. N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), 2-iminothiolane (2-IT), poly-IMysine, glutaral- dehyde, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma, St. Louis, MO, USA; routine polyclonal IgM was purchased from Cappel, West Chester, PA, USA; and alkaline phosphatase conjugated goat antimouse IgM F(ab')2 was purchased from Zymed, San Francisco, CA, USA. was mixed with 2-IT at a final concentration of 5 raM. This mixture was incubated at 4°C for 90min, and the excess reagent was removed by passage through a Sephadex G-50 column (Pharmacia, Uppsala, Sweden) equilibrated with 2 M NaC1/PBS. 22 ADM i mg/ml in dis- tilled water was mixed with equimolar amounts of SPDP solution, being 50mM in ethanol. The mixture was adjusted to pH 7.4 with triethylamine followed by incubation at room temperature for 6h. Conjugation of ADM to Antibody The 2-iminothiolane (2-IT) thiolated antibody in 2M NaC1/PBS was added to 10 equivalents of modified ADM. The reaction mixture was incubated at 4°C over- night, then centrifuged at 10000 × g, and the non- conjugated ADM was removed from the mixture by passage through a Sephadex G-50 column. The conju- gate was dialyzed against 10raM sodium phosphate buffer at a pH of 7.4. The molar ratio of antibody to ADM was determined by absorbance at 495 nm (e495 = 8030) for ADM, and at 280nm (lmg/ml = 1.4 absor- bance units) for the antibody. To correct for the over- lapping absorption of ADM at 280nm, the following formula was used: antibody (mg/ml) = [A280 - (0.72 × A495)]/1.4. The conjugate was finally sterilized by pas- sage through a 0.22-~m filtration membrane (Millex- GV; Millipore, Bedford, MA, USA). To ensure that the ADM was conjugated to the antibody via a disulfide bond, the conjugate was treated with tenfold molar excess dithiothreitol (DTT), a reducing reagent, at room temperature for 30min. Reduced conjugate was applied to a Sephadex G-50 column and the absorbance of each fraction at 280 and 495 nm was measured. Cell Lines The four breast cancer cell lines used for in vitro binding and cytotoxic studies, namely, SK-BR-3, MDA- MB-361, MCF-7, and BT-20, were obtained from the American Type Culture Collection, Rockville, MD, USA. All cell lines except MCF-7 were maintained in RPMI 1640 medium. MCF-7 was maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 100U/ml penicillin, and 100mg/ml streptomycin. Cells were grown at 37°C in a humidified atmosphere with 5% CO2 in air. Preparation of the AOOll-ADM Conjugate Antibody and AD M Modification A volume of lmg/ml of antibody in 10mM sodium phosphate buffer (PBS) containing 50mM triethy- lamine, 50 mM NaC1, and 1 mM EDTA, at a pH of 8.0 ELISA The binding activity of A0011 and its conjugate was determined by ELISA using SK-BR-3 breast cancer cell lines. SK-BR-3 cells, 1 × 104 cells/well, were incubated at 37°C overnight in a 96-well flat-bottomed plate precoated with 50mg/ml ploy-L-lysine. The wells were washed twice with PBS and fixed with 0.025% glutaral- dehyde in PBS for 15min at room temperature, fol- lowed by blocking with 0.2% gelatin, 0.1% fetal bovine serum, and 100mM glycine/PBS for lh at room tem- perature. Serially diluted A0011 or its conjugate was added to the wells at 37°C. Each point was done in quadruplicate. After lh, the wells were washed twice with 0.05% Tween 20, 0.1% gelatin in PBS and incu- bated for an additional lh with alkaline phosphatase conjugated goat antimouse IgM (1:2500). The wells were then washed five times with Tween/PBS, and I mg/ ml p-nitrophenol phosphate was added. The absor- bance (405nm) of the wells was measured in a
`
`IMMMUNOGEN 2096, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`H. Jinno et al.: Immunoconjugate Against Breast Cancer 503 ~ ~ ~, ~ 10 15 5 1.2 1.0 0.8 0.6 0.4. ~_~ 0.2 0 a , I 0 15 FRACTION NUMBER Fig. la,b. Gel filtration analysis of the A0011-Adriamycin (ADM) conjugate, a A0011 with absorbance at 280nm and ADM with absorbance at 495 nm linked via a disulfide bond eluted from a Sephadex G-50 column as a single peak. b After Q z ,~ rn n- O m ,~ 1.2 1.0 0.8 0.6 0.4 0.2 5 10 FRACTION NUMBER reaction with a tenfold molar excess of dithiothreitol (DTT), the A0011-ADM conjugate was eluted from a Sephadex G-50 column as separated peaks. Ope n circles, 280 nm; solid circles, 495 nm microplate reader l h later. The binding activity of A0011 to the other three breast cancer cell lines, MDA- MB-361, MCF-7, and BT-20, was investigated as described above. Statistical Analysis Statistical analysis was performed using Student's t test. All values are expressed as means _+ SE. Cytotoxicity In Vitro The pharmacological activity of the conjugate was mea- sured by the MTT assay. Briefly, exponentially growing cells, 1 × 104 cells/well, were transferred to a 96-well plate and incubated at 37°C overnight in complete medium. Subsequently, the cells were exposed for 15 min to the following serially diluted reagents: A0011- ADM conjugate, nonspecific murine IgM-ADM conju- gate, A0011 alone, and ADM alone. Quadruplicate determinations were done for each dilution. The cells were then washed with PBS and resuspended with com- plete medium. After 3 days incubation, the cells were washed twice with PBS and a fresh mixture of MTT, 0.4% in PBS, and sodium succinate, 0.1M in PBS, was added to each well, followed by incubation at 37°C for 3 h. At the end of this incubation, 150 ~1 dimethyl sulfox- ide (DMSO) was added to each well to dissolve the MTT formazan. The absorbance of each well was mea- sured in a microplate reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. Competitive Inhibition of the Conjugate Effects by AO011 SK-BR-3 cells, 1 × 104/well, were exposed to a mixture of conjugate (10-6M) and a zero- to tenfold molar ex- cess of A0011. The cytotoxic effect was determined as described above. Results Preparation of the AOOll-ADM Conjugate ADM was conjugated to A0011 via a disulfide bond. First, ADM was coupled to SPDP, a heterobifunctional reagent, and the monoclonal antibodies (MAbs) were thiolated by 2-IT. Excess 2-IT was removed by passage through a Sephadex G-50 column. The thiolated MAbs were subsequently reacted with modified ADM, which led to the formation of a disulfide bond as a linker arm between ADM and the MAbs. Unconjugated ADM was removed by passage through a Sephadex G-50 col- umn, but unconjugated MAbs could not be separated from the conjugates. Contamination may result in underestimating, but not overestimating the cytotoxic effect of the conjugates. Based on spectrophotometric analysis, the ADM:MAb molar ratio achieved ranged from 20 to 25, and the protein yields varied from 50% to 80%. Disulfide bond cleavage can be easily achieved by treatment with a reducing agent such as dithiothreitol (DTT). Regarding the untreated A0011-ADM conju- gates, the 280nm absorbance for antibody peaked si- multaneously with the 495nm absorbance for ADM (Fig. la). In the case of reduced conjugates with tenfold molar excess DTT, antibody absorbance showed the same curve as nonreduced conjugate, but the ADM absorbance peak shifted to the right (Fig. lb).
`
`IMMMUNOGEN 2096, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`504 H. Jinno et al.: Immunoconjugate Against Breast Cancer Ill ~ 5 z < m rr 4 O ¢1 m 3 < LM -~ 2 I'- < .J uJ 1 ee 0~ BT-20 MCF-7 MDA-MB-361 SK-BR-3 Fig. 2. Enzyme immunoassay analysis of A0011 binding to the various cells 8 LIJ z 6 < ell I:I: Ill 4 < iiJ >_ ~- ~ < .J UJ er ..... " ...... ' ................. ' CONCENTRATION (M) Fig. 3. Antigen binding activity of the A0011 and A0011- ADM conjugate. Open circles, A0011; solid circles, A0011- ADM ELISA The binding activity of A0011 to the four breast cancer cell lines was investigated by ELISA (Fig. 2). A0011 bound to the breast cancer cells in proportion to c-erbB- 2 amplification. A0011-ADM conjugate retained most of its binding activity as compared with unconjugated 10011 (Fig. 3). Cytotoxicity In Vitro The cytotoxic activity of the A0011-ADM conjugate, A0011 alone, ADM alone, and murine IgM-ADM conjugate was assessed on SK-BR-3, c-erbB-2 over- expressing cells, using the MTT assay (Fig. 4). The A0011-ADM conjugate inhibited cell growth dose- dependently and was significantly more potent than equivalent amounts of ADM alone (P < 0.01). The cytotoxicity (ICs0) was 6 × 10-6M for ADM and 7 × 10-7M or A0011-ADM conjugate. Nonspecific murine IgM-ADM conjugate did not show any cytotoxicity at a high concentration of 10-6M, and A0011 alone did not affect cell growth. The cytotoxicity of the conjugate was also evaluated against several breast cancer cell lines with various levels of c-erbB-2 expression (Fig. 5). The c-erbB-2 overexpressing cells, MDA-MB-361, were killed dose-dependently as in the case of SK-BR-3. Against MCF-7 and BT-20, which did not overexpress the c-erbB-2 product, the cytotoxicity of A0011-ADM conjugate was not recognized at the high concentration of 10-6M. The cytotoxicity (IC~0) of ADM alone against MDA-MB-361, BT-20, and MCF-7 was 4 × 10-6M, 7.5 × 10 -7 M, and 9 x 10 -7 M, respectively. Competitive Inhibition of the Conjugate by Free AO011 A0011-ADM conjugate showed dose-dependent cyto- toxicity against SK-BR-3 cells and the number of surviv- ing cell at 10-6M conjugate was almost 40%. To determine whether the effect of A0011-ADM conjugate was dependent on A0011, a zero- to tenfold molar ex- cess of A0011 was mixed with 10-6M conjugate before addition to the SK-BR-3 cells (Fig. 6). A0011 showed competitive inhibition of cytotoxicity dose-dependently and the number of surviving cells at 10-6M conjugate with 5-fold molar excess A0011 rose to 80%. Conjugate mixed with more than a tenfold molar excess of A0011 did not kill the SK-BR-3 cells. > tU Correlation between Cytotoxicity and Binding Activity The correlation between cytotoxicity and the binding activity (shown in Fig. 2) of conjugate at 10-6M was 120 ,00 .0 j 60 - ,0 -J 20 0 ~ , ~ ~ i ,,,I , ....... ~ ........ i 10 "8 10 -7 10 B6 ADM (M) Fig. 4. Cytotoxicity of the A0011-ADM conjugate, nonspe- cific IgM-ADM conjugate, and ADM against SK-BR-3. Each data point represents an average of four determinations. Open circles, IgM-ADM; solid circles, ADM; squares, A0011-ADM; *P < 0.01
`
`IMMMUNOGEN 2096, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`H. Jinno et al.: Immunoconjugate Against Breast Cancer 505 0 110 ' '°°1 '° 80 a0 ~01 ~0~ 0 I i i li II , , , , ,,,,I 1~ 8 lff 7 15 6 , , = , , ,,,! ADM (M) Fig. 5. Cytotoxicity of the A0011-ADM conjugate on the vari- ous cells. Open squares, BT-20; solid squares, MCF-7; open circles, MDA-MB-361; solid circles, SK-BR-3. *P < 0.01 g LU --I U/ o 100 90 80 70 60 50. 40'. 30 90 10 0 I I, I i ~ i 2 4 6 8 10 12 AO01 1/CONJUGATE CONCENTRATION RATIO Fig. 6. Competitive inhibition of the A0011-ADM conjugate activity by free A0011. Each data point represents and aver- age of four determinations analyzed. Against breast cancer cells with high binding activity, A0011-ADM conjugate revealed more power- ful cytotoxicity at the concentration of 10-6M. The re- gression equation of the formula y = 116.38 - 11.63 logX where the correlation coefficient -- 0.950 was fitted to the data (Fig. 7). Discussion In this study we described the preparation of anti-c- erbB-2 product-ADM immunoconjugate via a disulfide bond and evinced its specific and potent cytotoxicity to breast cancer cells overexpressing c-erbB-2. Despite the difficulty involved in directly demonstrating that ADM is covalently conjugated to antibody by a disulfide bond, our conclusion was supported by the following findings: 1. This bond is known to be easily cleaved under reduced conditions and, while ADM conjugated with the antibody via a disulfide bond eluted from gel filtration as a void volume, in the case of the reduced conjugate with excess DTT, ADM and the antibody eluted from gel filtration separately. 2. Nonspecific murine IgM-ADM conjugate showed no cytotoxicity against any of the four breast cancer cell lines, and the A0011-ADM conjugate was not as effective against cell lines which did not overexpress the c-erbB-2 product. Assuming that ADM was not linked to the antibody, these conjugates ought to have shown the same cytotoxicity as equimolar free ADM. These findings confirm the conjugation between ADM and the antibody. The c-erbB-2 product was selected as the target for the following reasons: First, it exists on the cell mem- brane. Second, it is strongly expressed in malignant cells, but poorly expressed in normal adult epithelial cells other than the renal tubuli. 23 Third, there have been reports concerning ligands for the c-erbB-2 prod- uct 24,25 which have been detected in the conditioned medium of c-erbB-2-positive cancer cells and found to induce tyrosine phosphorylation and stimulate cell growth. The c-erbB-2 product is a growth factor recep- tor that could play an important role in the autocrine cancer cell prolifieration. In other words, breast cancer 110 100 90 g " 80 LU _~ "-t ~ 7O ~, -1 UI O 8O 50 MDA-MB-361 ~ ~ SK-BR-3 40 ~ I ~ I = I = I = I ~ I ~ I 0 1 2 3 4 5 6 7 RELATIVE ABSORBANCE Fig. 7. Correlation between cytotoxicity and the binding ac- tivity of the conjugate (10-6M). The regression equation of the formula y = -11.630x + 116.38, where the correlation coefficient -- 0.950, was fitted to the data
`
`IMMMUNOGEN 2096, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`506 H. Jinno et al.: Immunoconjugate Against Breast Cancer cells with overexpression of the c-erbB-2 product may proliferate rapidly, which could lead to a high malig- nancy. Thus, using the c-erbB-2 product as the target, we were able to attack cancer cells with high malignant potential selectively. It was recently reported that soluble c-erbB-2 protein was found in the circulating blood of breast cancer pa- tients. 26 With in vivo application of the A0011-ADM conjugate, soluble receptors may cause the formation of immune complexes that inhibit the cytotoxic effect and lead to renal impairment; however, soluble c-erbB-2 protein was very small in quantity and probably differ- ent in conformation from the c-erbB-2 product on the cell membrane. The effectiveness of targeting therapies using gelonin, ~°,11,27,28 Pseudornonas exotoxin, 29 and ricin 27 which have been developed in the last decade is well documented; however, clinically these toxins are im- practical because they are not human proteins. As a single agent, ADM is the most effective anticancer drug in the treatment of breast cancer, with an overall re- sponse rate of 43% in previously untreated breast can- cer patients, 14 but it is associated with dose-dependent toxic effects, including bone marrow suppression, car- diac toxicity, and alopecia. In fact, it has been reported that leukopenia occurs in 68%-80% of treated patients, and the incidence of cardiomyopathy reached 20% at total doses above 550 mg/mm2? 3 Thus, in order to utilize the anticancer activity of ADM, but minimize its harm- ful side effects, we employed ADM as the warhead of the immunoconjugate. ELISA showed that the binding activity of the A0011-ADM conjugate was equal to that of A0011. A0011-ADM conjugate was more effective than an equivalent amount of free ADM to the c-erbB-2- overexpressing cells. In other words, the ADM activity of the conjugate was almost completely preserved. In conclusion, this conjugation procedure did not affect either the binding activity of the conjugate or the cyto- toxic activity of ADM. Hence, as a link between ADM and the antibody, we selected a disulfide bond. A0011-ADM conjugate exhibited specific cytotoxic- ity to SK-BR-3 and MDA-MB-361 breast cancer cell lines which overexpress the c-erbB-2 product, and its cytotoxicity was antibody-mediated, as shown by the following four findings: 1. Nonspecific murine IgM-ADM conjugate did not show any cytotoxicity to cells overexpressing the c- erbB-2 product. 2. A0011-ADM conjugate was not cytotoxic to MCF-7 or BT-20, which did not overexpress the c-erbB-2 product. 3. The effect of the A0011-ADM conjugate was com- petitively inhibited by free A0011. 4. The cytotoxicity of the conjugate was correlated with the binding activity of A0011. These characteristics will cause the selective in vivo accumulation of conjugate in tumor tissue and decrease its side effects. Moreover, the cytotoxicity of the A0011- ADM conjugate was specific because the conjugate did not kill BT-20 cells overexpressing the EGF receptor, which is very similar to the c-erbB-2 product. 2 A0011, being a murine IgM monoclonal antibody, is large in size, which causes high imunogenicity and diffi- culty in penetration into the core of a solid tumor mass. Thus, for clinical application, an IgG type antibody or smaller fragments including Fab should be developed. In summary, A0011-ADM conjugate demonstrated specific cytotoxicity to breast cancer cells over- expressing the c-erbB-2 product. The results of this study therefore indicate that targeting therapy using the c-erbB-2 product as a target may be useful for treating potentially malignant breast cancers with c-erbB-2 overexpression. Acknowledgments. The authors wish to thank Toshiaki Miura and Norifuni Hirota for their excellent technical assistance and advice in the laboratory. This work was supported in part a grant-in-aid from the Japanese Ministry of Education, Cul- ture, and Science. References 1. Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto T (1986) The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 232:1644- 1646 2. Coussens L, Yang-Feng TL, Liao YC, Chen E, Gray A, McGrath J, Seebug PH, Libermann TA, Schlessinger J, Francke U, Levinson A, Ullrich A (1985) Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal loca- tion with neu oncogene. Science 230:1132-1139 3. Slamon D J, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL (1987) Human breast cancer correlation of relapse and sur- vival with amplification of the HER-2/neu oncogene. Science 235:177-181 4. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SC, Keith DE, Levin WJ, Stuart SC, Udove J, Ullrich A, Press MF (1989) Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science 244:707-712 5. Fukushige S, Matsubara K, Yoshida M, Sakai M, Suzuki T, Semba K, Toyoshima K, Yamamoto T (1986) Localization of a novel v- erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line. Mol Cell Biol 6:955-958 6. Kern JA, Schwartz DA, Nordberg JE, Weiner DB, Greene MI, Torney L, Robinson RA (1990) p185 "eu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res 50: 5184-5191 7. Fiore PPD, Pierce JH, Kraus MH, Segatto O, King CR, Aaronson SA (1987) erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science 237:178-182 8. Hudziak RM, Schlessinger J, Ullrich A (1987) Increased expres- sion of the putative growth factor receptor p185 ~m causes trans- formation and tumorigenesis of NIH 3T3 cells. Proc Natl Acad Sci USA 84:715%7163 9. McCann AH, Dervan PA, O'Regan M, Codd MB, Gullick WJ,
`
`IMMMUNOGEN 2096, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`H. Jinno et al.: lmmunoconjugate Against Breast Cancer 507 Tobin BMJ, Carney DN (1991) Prognostic significance of c-erbB- 2 and estrogen receptor status in human breast cancer. Cancer Res 51:3296-3303 10. Ozawa S, Ueda M, Ando N, Abe O, Minoshima S, Shimizu N (1989) Selective killing of squamous carcinoma cells by an immunotoxin that recognizes the EGF receptor. Int J Cancer 43:152-157 11. Hirota N, Ueda M, Ozawa S, Abe O, Shimizu N (1989) Suppres- sion of an epidermal growth factor receptor-hyperproducing tu- mor by an immunotoxin conjugate of gelonin and a monoclonal anti-epidermal growth factor receptor antibody. Cancer Res 49:7106-7109 12. Osaku M, Ueda M, Ando N, Shinozawa Y, Hirota N, Shimizu N, Abe O (1991) Targeted killing of squamous carcinoma cells by a monoclonal antibody-pepleomycin conjugate which recognizes the EGF receptor. Anticancer Res 11:1951-1956 13. Carter SK (1975) Adriamycin -- A Review. J Natl Cancer Inst 55:1265-1274 14. Young RC, Ozols RF, Myers CE (1981) The anthracycline antineoplastic drugs. N Engl J Med 305:139-153 15. Braslawsky GR, Edson MA, Pearce W, Kaneko T, Greenfield RS (1990) Antitumor activity of adriamycin (hydrazone-linked) immunoconjugates compared with free adriamycin and specificity of tumor cell killing. Cancer Res 50:6608-6614 16. Dillman RO, Shawler DL, Johnson DE, Meyer DL, Koziol JA, Frincke JM (1986) Preclinical trials with combinations and conju- gates of T101 monoclonal antibody and doxorubicin. Cancer Res 46:4886-4891 17. Galun E, Shouval D, Adler R, Shahaar M, Wilchek M, Hurwitz E, Sela M (1990) The effect of anti-c~-fetoprotein-adriamycin conju- gate on a human hepatoma. Hepatology 11:578-584 18. Hurwitz E, Levy R, Maron R, Wilchek M, Arnon R, Sela M (1975) The covalent binding of daunomycin and adriamycin to antibodies, with retention of both drug and antibody activities. Cancer Res 35:1175-1181 19. Pimm MV, Jones JA, Price MR, Middle JG, Embleton MJ, Baldwin RW (1982) Turnout localization of monoclonal antibody against a rat mammary carcinoma and suppression of tumour growth with Adriamycin-antibody conjugates. Cancer Immunol Immunother 12:125-134 20. Yeh MY, Roffler SR, Yu MH (1992) Doxorubicin: monoclonal antibody conjugate for therapy of human cervical carcinoma. Int J Cancer 51:274-282 21. Yang HM, Reisfeld RA (1988) Doxorubicin conjugated with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xe- nografts in nude mice. Proc Natl Acad Sci USA 85:1189-1193 22. Greenfield RS, Kaneko T, Daues A, Edson MA, Fitzgelald KA, Olech LJ, Grattan JA, Spitalny GL, Braslawsky GA (1990) Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker. Cancer Res 50:6600- 6607 23. Mori S, Akiyama T, Yamada Y, Morishita Y, Sugawara I, Toyoshima K, Yamamoto T (1989) C-erbB-2 gene product, a membrane protein commonly expressed on human fetal epithelial cells. Lab Invest 61:93-97 24. Holmes WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, Park JW, Yansura D, Abadi N, Raab H, Lewis GD, Shepard HM, Kuang WJ, Wood WI, Goeddel DV, Vandlen RL (1992) Identifi- cation of heregulin, a specific activator or p185 ~rb~2. Science 256:1205-1210 25. Lupu R, Colomer R, Kannan B, Lippman ME (1992) Character- ization of a growth factor that binds exclusively to the erbB-2 receptor and induces cellular responses. Proc Natl Acad Sci USA 89:2287-2291 26. Mori S, Mori Y, Mukaiyama T, Yamada Y, Sonobe Y, Matsushita H, Sakamoto G, Akiyama T, Ogawa M, Shiraishi M, Toyoshima K, Yamamoto T (1990) In vitro and in vivo release of soluble c-erbB-2 potein from human carcinoma cells. Jpn J Cancer Res 81:489-494 27. Colombatti M, Nabholz M, Gros O, Bron C (1983) Selective killing of target cells by antibody-ricin A chain or antibody- gelonin hybrid molecules: comparison of cytotoxic potency and use in immunoselection procedures. J Immunol 131:3091-3095 28. Lambert JM, Senter PD, Yau-Young A, Blatter WA, Goldmacher VS (1985) Purified immunotoxins that are reactive with human lymphoid cells. J Biol Chem 260:12035-12041 29. Fitzgerald D J, Willingham MC, Pastan I (1986) Antitumor effect of an immunotoxin made with Pseudomonas exotoxin in a nude mouse model of human ovarian cancer. Proc Natl Acad Sci USA 83:6627-6630
`
`IMMMUNOGEN 2096, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
`
`