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`PHIGENIX
`PHIGENIX
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`September 1995
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`Notes
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`Biol. Pharm. Bull. 18(9) 1279—4282 (1995)
`
`1279
`
`Immunoselective Cell Growth Inhibition by Antibody-Adriamycin
`Conjugates Targeting c-erbB—2 Product on Human Cancer Cells
`
`Shinya SUZUKI,* Megumi TANAKA, Takashi MASUKO, and Yoshiyuki HASHIMOTO
`Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Tahoku University, Aobayama, Sendai
`980L77, Japan. Received March 29, 1995; accepted June 7, 1995
`
`Monoclonal antibodies targeting c-erbB-Z protooncogene product p185 were conjugated with adriamycin via a
`pH—sensitive spacer. The resultant antibody-adriamycin conjugates showed immunoselective binding, internalization
`and cytotoxicity to p185—positive human breast cancer cell SKBr-3 and gastric cancer cell MKN-7, but not to normal
`human lymphocytes.
`
`Key words
`
`adriamycin; monoclonal antibody; internalization
`
`Adriamycin (ADM) is a popular anti-neoplastic drug
`because of its high toxicity to solid tumors. However,
`its therapeutic application has been limited by its severe
`side effects. Since such side effects are caused mainly
`by nonspecific adsorption by normal tissues,“ this cause
`could be reduced by conjugating ADM with a target
`selective vehicle such as an anti-cancer antibody.
`c—erbB-Z is a protooncogene which encodes EGF re-
`ceptor-like membrane protein p185.” Amplification and
`overexpression of the c—erbB~2 gene has been shown in
`many human cancers, including 30% of lung, breast, ovary
`and stomach adenocarcinomas and where there is a 50- to
`lOO-fold increase in pl 85 expression as compared with the
`normal cell level (reviewed in”). Thus, p185 is thought to
`be a suitable antigen for the immunotargeting therapy of
`human cancers. In this regard, we have here conjugated
`the monoclonal antibody (mAb) targeting pl 85 with ADM
`and demonstrated the cancer~specific cytotoxicity of the
`antibody-ADM conjugate.
`
`MATERIALS AND METHODS
`
`mAbs mAbs Sv2~6l and SER4 (both IgGl), which
`recognize c—erbB-2 product p185,” and an isotype-
`matched control mAb B3 (IgGl), which recognizes rat
`gp125 antigen,“ were used in this report. mAbs were
`purified from the ascites fluid of mice by 50% (NH4)ZSO4
`precipitation followed by protein—G affinity chromatog-
`raphy.
`cAA-
`cis-Aconityl-ADM-Coupled Antibody (cAA-Ab)
`Ab was prepared according to the method of Shen et a1.”
`as follows. Briefly, ADM hydrochloride (Kyowa Hakko
`Co., Tokyo) was mixed with cis-aconitic anhydride at a
`molar ratio of l 20.75 in 0.1 M NaHCO3, and maintained at
`pH 9 at 0°C for 15min. The resultant cis-aconityl ADM
`(cAA) was precipitated by the addition of 1M HCl and
`collected by centrifugation (10000 X 9, 10min). cAA was
`then resolved in H20, adjusted to pH 7 with 1M NaOH,
`and then activated by mixing it with l-ethyl-3—(3-dimeth.
`ylaminopropyl)-carbodiimide at
`21 mol
`ratio of l : 1.5
`following stirring at
`r.t.
`for 30 min. The activated
`cAA was mixed with purified mAb in 0.1 M NaH2P04 (pH
`9) at a mol ratio of 25 :1 and incubated at It keeping
`pH 9 with 1M NaOH for 3h. Finally, cAA—Ab was
`separated from low molecular materials by Sephadex G25
`* To whom correspondence should be addressed.
`
`column chromatography equilibrated with phosphate—
`buffered saline (PBS), and was sterilized by filtration
`with a millipore membrane of 0.22 pm pore size. IgG
`and cAA content were determined on the basis of the
`absorbance at 280 and 476nm for cAA-Ab.” The mean
`substitution ratio (MSR) of ADM to Ab was calculated
`by the following formula:
`
`MSR (mol ratio) = 0.036 x ODMé/ODND—(OD476 x 0.736)
`
`Binding Analysis of cAA-Ab The binding activity of
`cAA~Ab was determined by indirect immunofluorescence
`analysis using FITC-coupled rabbit anti-mouse immu—
`noglobulin antibody (Dako, Copenhagen, Denmark), in
`which the cell fluorescence was quantified by a flow
`cytometer FACScan (Becton Dickinson, Mountain
`View, CA) with excitation at 488nm and emission at
`515——545 nm. The fluorescence intensity of 10000 viable
`cells was recorded. All determination was done at the same
`
`detection sensitivity, and the mean fluorescence intensity
`of each sample was computed as the relative amount of
`each binding.
`Cell Growth Inhibition Analysis The cell growth
`inhibitory effect of cAA-Ab was analyzed on SKBr~3
`human breast cancer cells and MKN-7 human gastric
`cancer cells as follows. Cells (4 x 104) suspended in 100 pl
`of a standard medium, Dulbecco’s modified Eagles’s
`minimal essential medium (Nissui Pharmaceutical Co.,
`Tokyo) supplemented with 10% heat-inactivated fetal calf
`serum (FCS) (M.A. Bioproducts, Walkersville, MD), were
`mixed with a reciprocal dilution of cAA-Ab (100 pi) and
`incubated at 37 °C for 1h. After being washed with ice~
`cold PBS 3 times, the cells were resuspended in the stan-
`dard medium and distributed in quadruplicate into a 48-
`wcll
`tissue culture plate (Costar) at 1x104cells/500ul
`standard medium/well. After being cultured for 4d, the
`cells in each well were then harvested with actinase/EDTA
`treatment and were counted for viable cell number as
`determined by Trypan blue staining.
`Analysis of the Response of Human Peripheral Blood
`Monocytes
`(PBMC) with Phytohemagglutinin (PHA)
`The cytotoxic activity of cAA—Ab for PBMC was de-
`termined by assaying the inhibition of PBMC proliferation
`with FHA (Sigma) as follows, Human PBMC were freshly
`prepared from blood obtained from a healthy volunteer
`© 1995 Pharmaceutical Society of Japan
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`1280
`
`by Ficol (Pharmacia) gradient centrifugation. The PBMC
`(2.0 x106) were then mixed with various concentrations
`of cAA-Ab in 200,111 of standard medium at 37 °C for 1 h.
`After being washed twice with ice—cold standard medium,
`the cells were seeded at 2.5 x 105 cells/well in quadruplicate
`into a Falcon flat—bottomed 96—well tissue culture plate
`and cultured in 200 pl of standard medium containing
`PHA at 20 ,ug/ml. After 3 d of culture, the cells were pulsed
`with [3H]thymidine (Amersham Lab., Buckinghamshire,
`England) at 0.5 ,uCi/well for 4h, and then harvested. The
`radioactivity of the cells was measured by standard liquid
`scintillation counting.
`
`RESULTS AND DISCUSSION
`
`We have successfully prepared three cAA-Abs by the
`method described in the above section. The resultant
`cAA-Abs
`from Sv2-6l-IgG, SER4-IgG and B3~IgG
`(Sv2-6l—cAA, SER4-cAA and B3—cAA) contained ADM
`at a MSR of 7.89, 6.85 and 9.18, respectively.
`Binding Analysis of cAA-Abs We first examined the
`binding activity of cAA—Ab as compared with intact Ab.
`Representative binding curves of intact Ab and cAA-Ab
`
`
`
`
`
`MeanfluorescenceIntensity
`
`120
`
`mC
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`I
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`.001
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`.01
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`.1
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`1
`
`10
`
`Antibody Concentration ( ug/ml)
`
`Fig. 1. Binding of Intact Antibody and cAA-Antibody
`SKBr-3 cells (2 x 105) in 100 pl ofstandard medium were mixed with the indicated
`concentration of intact antibodies or cAA-antibodies and were then analyzed for
`their binding as described in the Methods section. Mean fluorescence intensity of
`the cells is shown. (0), Sv2v6l-IgG; (A), Sv2—6I—cAA; (Q), B3-IgG; (A), B3—cAA.
`
`Vol. 18, No. 9
`
`in increasing concentrations were demonstrated on SKBr-3
`in Fig. 1, and the same type of analysis was done on the
`other Abs and cAA-Abs, on MKN—7 and PBMC. From
`these analyses, maximal binding, which suggests the
`relative binding capacity on the cell, and the concentra—
`tion resulting in 1/2 of maximal binding (titer), which
`suggests binding affinity, were read and then summariz-
`ed in Table 1._As shown in Table l, the relative binding
`capacities are 1.3~»1.6V times higher in Sv2-6l-related
`epitopes than in the SER4aepitope for both SKBr—3 and
`MKN-7 cells. Both anti~p185 Abs and cAA—Abs showed
`significant binding to SKBr-3 and MKN-7 with titers of
`1.8—4.6x10"8M, but showed no detectable binding to
`PBMC. If binding aflinities (titers) were compared between
`intact IgG and corresponding cAA-Ab, no significant
`change (less than a 2 times increase in titer) was observed
`in either Sv2-61-cAA or SER4—cAA, suggesting that the
`modification of ADM had no inhibitory effect on antibody
`binding.
`Cell Growth Inhibition Analysis We next examined the
`cell growth inhibitory effect of cAA—Abs targeting p185
`(SER4-cAA and Sv2-61~cAA), the non-targeting control
`B3-cAA, and free cAA on both p l 85—positive target cancer
`cells and negative normal PBMC.
`As shown in Fig. 2, SER4—cAA and Sv2—61-cAA sig-
`Table 1. Binding Characteristics of Intact Antibodies and cAA-Abs
`Ti
`r
`M
`in in
`(XIOISMY)
`Target cell
`3:1)“; g
`
`SKBr-3
`2.6
`1 17
`MKN7
`2.8
`83
`SKBr-3
`1.8
`93
`MKN7
`2.7
`65
`PBMC
`ND
`ND
`SKBr-3
`2.8
`75
`MKN7
`4.6
`60
`SKBr—3
`4,5
`67
`MKN7
`4.6
`43
`PBMC
`ND
`ND
`SKBr~3
`ND
`ND
`MKN7
`ND
`ND
`PBMC.
`ND
`ND
`
`Sv2—6l-IgG
`Sv2-6l-IgG
`Sv2~6l-cAA
`Sv2-61-cAA
`Sv2~61-cAA
`SER4-IgG
`SER4-IgG
`SER4—eAA
`SER4-cAA
`SER4-cAA
`BS-cAA
`BS—cAA
`BB-cAA
`
`l and the
`a) Antibody concentration resulting in 1/2 of maximal binding in Fig.
`same type of figures (data not shown).
`b) Mean fluorescence intensity. ND: not
`detected; no detectable specific binding was observed.
` 100
`
`7:34
`
`\
`
`.01
`
`.1
`
`i
`
`10
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`.1
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`1
`
`10
`
`so
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`so
`
`40
`20
`
`o.
`
`01
`
`c:o._
`‘5
`
`0U ~
`
`5
`2‘:
`g
`o‘—
`6
`7'3
`U
`
`cAA Concentration (ttg /ml)
`Fig. 2, Cell Growth Inhibitory Effect of cAA—Abs on SKBr—S and MKN—7 Cells
`
`Cells were treated with the indicated concentration of cAA~Ab or free cAA as described in the Methods section. The percentage of cell densities as compared with
`that of the non-treated control culture is shown, Symbols represent the average values from four determinations. All SD. of the average values were less than 14% of
`
`the control. a, SKBr-3; b, MKN-7; (O), BLCAA; (A), SER4-cAA; (j), Sv2~61~cAA; (0), free cAA.
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`September 1995
`
`1281
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`Sv2-61-cAA alone
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`sum-cm + as tgG
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`Sv2-61—cAA + Sv2—61-lgG
`W
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`Control)
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`CellSurfaceLevelofcAA-Ab(%of0Time
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`0
`
`30
`
`60
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`90
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`120
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`100
`20
`4o
`60
`80
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`W o
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`SETH-CM alone
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`SER4~cAA + as -lgG
`
`SEEM-0AA , SEMIgG
`
`Cell Growth ("/o of Control)
`
`Time After Washlng (mln)
`
`Fig. 3. Competitive Inhibition of Cell Growth Inhibitory Effect of
`cAA—Ab by Intact Antibody
`MKN—7 cells were treated with cAAaArb at'0;2pgreAA/ml (7+7.5;¢g IgG/ml)
`in the absence or presence of intact antibodies (final concentration of 500 rig/ml),
`and were analyzed for growth activity as in Fig. 2. The columns and bars represent
`the mean value from four determinations and the SD. of the mean, respectively.
`
`
`
`
`
`3H-Thyrnidlnelncorporatlon(%ofControl)
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`120
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`100
`BO
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`60
`40
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`20
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`0
`.01
`
`/U
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`\O
`
`1
`1
`cAA Concentration (pg lml)
`
`10
`
`Fig. 4. Effect of cAA-Ab on FHA-Response of PBMC
`PBMC was treated with the indicated concentration of cAA-Ab or free cAA
`and was analyzed for proliferation with FHA as described in the Methods section.
`The percentage of [3H]thymidine incorporation as compared with that of the
`non-treated control cells is shown. The symbols represent average values from four
`determinations All SD. of the average values were less than 8.3%. (O), B3£AA;
`(A), SER4—cAA; (Cl), Sv2-6l-CAA; (0) free cAA.
`
`nificantly inhibited the growth of both SKBr~3 and
`MKN-7 cancer cells
`in a' dose dependent
`fashion.
`Meanwhile, B3-cAA showed only 50% or less inhibition
`even at the maximal concentration tested. For example
`(Fig. 2a),
`the [C50 of SER4-cAA for SKBr-3 cell was
`0.25 fig cAA/ml, which was 20 times lower than that of
`the control B3-cAA (ICE,o corresponding to Sag/ml).
`Sv2-61-cAA also showed lower
`IC50 (0.75 pg/ml) as
`compared with B3-CAA. Though the data is not shown,
`these anti—p185-cAA-Abs showed 10—30 times higher
`1C5O to plSS-negative human bladder cancer T24.
`These cell growth inhibitory effects of anti-p185
`cAA-Abs were inhibited by the addition of excess amounts
`(over 65-fold) of intact corresponding antibodies, but not
`by an unrelated antibody, B3 (Fig. 3). Figure 3 also shows
`that neither the intact SER4—IgG nor the Sv2—61—IgG
`inhibits cell growth, even at a high (500 pg/ml) con-
`centration. These results suggest that the effect of cAA-
`
`Fig. 5. Modulation of Cell Surface SER4—cAA on SKBr-3 and
`MKN—7
`Intact cellsr(closed symbols) or i'onnalinvfixed cells (open symbols) were mixed
`with SER4-cAA at 20 pg lgG/rnl in a standard medium at 4°C for 2h. After being
`washed twice with ice-cold PBS, the cells were rewincubated in the standard medium
`,at 37 °C. At the indicated time after incubation, the cells were washed with icewld
`PBS once and processed for indirect immunofiuorescence analysis as in Fig. l. The
`percentage of the mean fluorescence intensity as compared with the 0 time value
`is shown as an index of cAA-Ab level on the cell surface. (0, O), SKBr-3;
`(A, A), MKN-7.
`
`Ab is dependent on both antibody selectivity and the drug
`effect of cAA in the conjugate.
`The effect of cAA-Abs on p185-negative normal human
`PBMC was also analyzed to confirm the specificity. As
`shown in Fig. 4, all cAA-Abs showed no significant
`inhibition of the PHA-response of PBMC. Only free cAA
`showed a somewhat inhibitory effect. These show that the
`nonspecific eifect of anti-p185 cAA—Abs on normal human
`PBMC is negligible in the tested concentration.
`The IC50 of free cAA in the PHA-response analysis
`(Fig. 4) was higher than that in the cell growth inhibi-
`tion analysis (Fig. 2). This seems to be due to a differ-
`ence in native sensitivity to cAA between the normal
`PBMC and cancer cells. In the same type of analysis, free
`intact ADM showed a 2.5—7 times greater effect than free
`cAA (data not shown).
`Internalization of cAA-Ab by Target Cell Since the
`pH-sensitive cis-aconityl spacer in cAA-Ab is thought to
`be cleaved in an intracellular acidic compartment, such
`as the endosomes or lysosomes, resulting in the release
`of free ADM into cytosol, endocytosis (internalization)
`of cAA—Ab by target cells after its initial binding is an
`important
`factor
`in expressing its cytotoxic activity.
`Thus, we finally demonstrated the internalization of
`cAA-Ab by SKBr-3 and MKN-7 in Fig. 5. Cells were first
`coated with SER4-cAA at 4 °C and then incubated at 37 °C
`to allow the internalization of the cell surface-bound
`SER4-cAA. In the case of SKBr—3, the level of cell surface
`SER4-cAA decreased by 25% in 10min and thereafter
`increased back to 90% of the initial level. By contrast, in
`the case of MKN-7, it continuously decreased to 54% of
`the initial level during 2h of incubation (Fig. 5). In both
`cell types, the fixed cells showed only a slight decrease
`in cell surface SER4-cAA. In conjunction with the results
`that
`the total cAA—Ab content
`in the cells remained
`unchanged during the same type of analysis as in Fig. 5,
`as determined using 125I—labeled cAA-Abs, and the
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`1282
`
`intra.
`immunofluorescence observation showed their
`cellular localization (data not shown), the decrease in cell
`surface cAA-Ab substantially represents the internaliza-
`tion, but not thesimple dissociation, of cAA-Abs.
`The increase of SER4-CAA in SKBr-3 cell surface,
`following an initial decrease after 30 min incubation (as
`described above) may suggest
`that cAA-Abs are first
`internalized and then recycled to the cell surface. How-
`ever,
`in MKN—7, no such apparent “recycle” was ob—
`served. Though we have no explanation for this different
`internalization pattern between SKBr-3 and MKN~7, it
`should concern the effect of cAA-Ab. The correlation
`between the cytotoxic activity of cAA-Ab and its
`intracellular fate has now been under investigation.
`Conclusion
`It is noteworthy that anti—p185 cAA—Abs
`inhibit cancer cell growth at a concentration resulting in
`
`Vol. 43, No. 9
`
`no inhibitory effect on normal cell activity. Taken together,
`anti-p185 cAA-Abs will be a good immunotargeting drug
`for p185-expressing human tumors.
`
`REFERENCES
`
`1) Speth P. A., Van Hoesel Q. G., Haanen 0, Clin. Pharmacokinet,
`15, 15—31 (1988).
`L
`'
`2) Di Fiorre P. P., Segatto 0., Aaronson S. A., Methods Enzymol.,
`198, 272—277 (1991).
`3) Masuko T., Sugahara K., Kozono M., Otsuki T., Akiyama T.,
`Yamamoto T., Toyoshima K., Hashimoto Y., Jpn. J. Cancer Res,
`80, 10~14 (1989).
`4) Hashimoto Y., Masuko T., Yagita H., Endoh N., Kanazawa 1.,
`Tazawa J., Jpn. J. Cancer Res, 74, 818—821 (1983).
`Shen W. C., Ryser H. J. P., Biochim. Biophys. Acta, 102, 1048‘
`1054 (1981).
`
`5)
`
`
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