An Anti-a-Fetoprotein Antibody-Daunorubicin Conjugate
`With a Novel Poly-L-glutamic Acid Derivative as
`Intermediate Drug Carrier 1
`
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` at University of Chicago on September 10, 2015
`
`Yutaka Tsukada,2 Yoshinori Kato,3 Naoji Umemoto,3 Yumiko Takeda,3 Takeshi Hara,3 and Hidematsu Hirai2
`
`ABSTRACT-In studies on antitumor antibody-cytotoxic drug
`conjugates as potential antitumor agents with improved tumor
`specificity, daunorubicin [(OM) daunomycin] was conjugated with
`an affinity-purified horse antibody to rat O'-fetoprotein (AFP) with
`a novel derivative of poIY-L-glutamic acid (PLGA) as the inter(cid:173)
`mediate drug carrier. A single masked thiol group first was
`introduced by PLGA, and the thiol group was generated from it
`after the linking of DM to PLGA at the carboxyl groups of PLGA.
`The thiol group was used selectively for binding PLGA-OM to
`antibody that had been modified so as to have the maleimide
`groups. The conjugates (OM:PLGA:immunoglobulin molar ratio,
`19.6:2.8:1 or 11.8:1.1:1) were more potent than OM in in vitro
`cytotoxicity against the AFP-producing rat ascites hepatoma cell
`line AH66. In therapeutic experiments, the conjugates were more
`efficacious in prolonging the lives of AH66 hepatoma-bearing
`OONRYU rats than OM, antibody, a mixture of OM and antibody,
`or a conjugate similarly prepared with normal horse immuno(cid:173)
`globulin.-JNCI 1984; 73:721-729.
`
`In recent years, there has been an increasing amount
`of research on the preJ1aration of antitumor agents with
`improved tumor specificity by covalent linking of
`antitumor drugs to antibodies against tumor-associated
`antigens (1-6). For realization of the potent antibody(cid:173)
`antitumor drug conjugates, conjugation methods must
`be developed by which a considerably large number of
`drug molecules can be linked to one molecule of
`antibody with minimal detrimental effect to the anti(cid:173)
`gen-binding activity of the antibody. Thus methods
`using an intermediate drug carrier (intermediary) were
`investigated. With these methods drugs first are con(cid:173)
`jugated with a macromolecular intermediary, which is
`linked in turn to antibody.
`We have developed a new conjugation method using
`as the intermediary a PLGA derivative that has a single
`active center for linking to antibody. Prior to its
`contact with drug-linked PLGA, the antibody was
`modified to contain functional groups that were reactive
`specifically with the active center of the PLGA deriva(cid:173)
`tive. This method is advantageous over the previous
`methods with the use of a similar intermediate drug
`carrier (7, 8) in that the present method affords con(cid:173)
`jugates that have a well-defined chemical structure and
`that are stable in solutions (being free from high(cid:173)
`molecular-weight conjugates and aggregates resulting
`from
`the binding of more than one molecule of
`antibody to the intermediary and the homo-coupling of
`the antibody). We prepared a DM conjugate by this
`method using aAFP, and the conjugate showed a
`carrier effect of antibody both in in vitro and in vivo
`
`antitumor activIties against the AFP-producing rat
`ascites hepatoma cell line AH66.
`
`MATERIALS AND METHODS
`
`aAFP.-Specific antiserum to rat AFP was produced
`in a horse by weekly sc injections of 1 mg purified AFP
`(9) emulsified in Freund's complete adjuvant. The
`aAFP (10) was purified by affinity chromatography of
`the antiserum on Sepharose 4B coupled to rat AFP
`(11).
`Tumor cells and rats.-The rat ascites hepatoma
`cell line AH66 used in this study was maintained by
`intraperitoneal passage in syngeneic male DONR YU
`rats (12). DONRYU rats were purchased from Nihon
`Rat Co., Saitama, Japan, and were 10-12 weeks old
`(190-210 g) at the beginning of the in vivo experiments.
`Preparation of Ig-PLGA-DM conjugates (text-figs. 1,
`2).-Anti-AFP and control normal conjugates subjected
`to biological studies were prepared twice in different
`batches from PLGA (table I); details are given for the
`first preparation (preparation 1) in table 1.
`Introduction of the 3-(2-pyridyldithio )propionyl group
`to PLGA.-To a stirred solution of Na salt of PLGA
`(average mol wt, 21,000; Sigma Chemical Co., St.
`Louis, Mo.) (PLGA, I; 1.0 g, 47.6 Jlmol) in 0.1 M Na
`phosphate buffer, pH 7.5 (70 ml), was added at room
`temperature a solution of SPDP (Pharmacia Chemicals
`AB, Uppsala, Sweden; 0.3 g, 0.96 mmol) (13) in DMF
`(6 ml) in three portions at 1.5-hour intervals, and the
`mixture was stirred for an additional 1.5 hours. Then
`
`ABBREVIATIONS USED: AFP=O'-fetoprotein; aAFP=affinity-purified
`horse anti-rat a-fetoprotein antibody; CMF-PBS=Ca2+ _Mg2+ -free
`phosphate-buffered saline; DM=daunorubicin (daunomycin); DMF=
`N,N-dimethylformamide; DNP-Cys=dinitrophenylcysteine; DTT=l,4-
`dithiothreitol; EDCI = l-ethy 1-3-( 3-dimethylaminopropy I )carbodiimide
`hydrochloride; Ig=immunoglobulin; 2-ME==2-mercaptoethanol; MST==
`mean survival time(s); nIg==normal horse immunoglobulin; PAGE==
`polyacrylamide gel electrophoresis; PBS==phosphate-buffered saline;
`PLGA==polY-L-glutamic acid; 5MBE==N-succinimidyl m-(N-maleimido)(cid:173)
`benzoate; 5MBU==N-succinimidyl 4-(N-maleimido)butyrate; SPDP==
`N-succinimidyI3-(2-pyridyldithio)propionate.
`
`I Received january 3, 1984; accepted May 18, 1984.
`2 Department of Biochemistry, Hokkaido University School of
`Medicine, Sapporo 060, japan.
`3 Department of Medicinal Chemistry and Biochemistry, Teijin
`Institute for Biomedical Research, Hino, Tokyo 191, japan.
`
`721
`
`JNC), VOL. 73, NO.3, SEPTEMBER 1984
`
`SANOFI-AVENTIS Exhibit 1041 - Page 721
`
`IPR for Patent No. 8,951,962
`
`

`
`
`
`722 Tsukada, Kato, Umemoto, et al. 722 Tsukada, Kato, Umemoto, et al.
`
`Downloaded from
`
`http://jnci.oxfordjournals.org/
`
` at University of Chicago on September 10, 2015
`
`phosphate buffer (pH 7.5) containing 0.5 M NaCI (1.0
`phosphate buffer (pH 7.5) containing 0.5 M NaCI (1.0
`ml), and the mixture was heated at 40°C for 30 minutes
`ml), and the mixture was heated at 40°C for 30 minutes
`
`and dialyzed against 5 mM Na acetate buffer (pH 5.5) and dialyzed against 5 mM Na acetate buffer (pH 5.5)
`
`containing 0.14 M NaCI and I mM EDT A to give the containing 0.14 M NaCI and I mM EDT A to give the
`
`PLGA-DM conjugate having the free PLGA-DM conjugate having the free
`
`thiol group thiol group
`
`HS-PLGA-DM (V). HS-PLGA-DM (V).
`
`Binding of HS-PLGA-DM to 19.-To a solution of Binding of HS-PLGA-DM to 19.-To a solution of
`
`aAFP in 0.1 M Na phosphate buffer, pH 6.5 (23.4 aAFP in 0.1 M Na phosphate buffer, pH 6.5 (23.4
`
`mg/ml, 1.6 ml), was added 50 mM 5MBE (0.1 mI, 5 mg/ml, 1.6 ml), was added 50 mM 5MBE (0.1 mI, 5
`
`JLmol) (14) in DMF, and the mixture was allowed to JLmol) (14) in DMF, and the mixture was allowed to
`
`stand at 25°C for 30 minutes and subjected to gel stand at 25°C for 30 minutes and subjected to gel
`
`filtration on a Sephadex G-25 column (0.8X40 cm) with filtration on a Sephadex G-25 column (0.8X40 cm) with
`
`0.9% NaCI to remove low-molecular-weight materials. 0.9% NaCI to remove low-molecular-weight materials.
`
`To the resulting solution of the maleimide group(cid:173)To the resulting solution of the maleimide group(cid:173)
`
`containing aAFP [VI (lg=aAFP); 9.93 mg/ml, 2.96 ml] containing aAFP [VI (lg=aAFP); 9.93 mg/ml, 2.96 ml]
`
`were added V (0.388 mM of PLGA equivalence, 5.05 were added V (0.388 mM of PLGA equivalence, 5.05
`
`ml) and 0.5 M Na phosphate buffer, pH 6.5 (2 ml), and ml) and 0.5 M Na phosphate buffer, pH 6.5 (2 ml), and
`
`the mixture was allowed to stand at 25°C for 24 hours. the mixture was allowed to stand at 25°C for 24 hours.
`
`The generated precipitate was removed by centrifuga(cid:173)The generated precipitate was removed by centrifuga(cid:173)
`
`tion, and the solution was dialyzed against CMF-PBS tion, and the solution was dialyzed against CMF-PBS
`
`to give the aAFP-PLGA-DM conjugate [VII (lg=aAFP)]. to give the aAFP-PLGA-DM conjugate [VII (lg=aAFP)].
`
`The normal conjugate nlg-PLGA-DM was prepared The normal conjugate nlg-PLGA-DM was prepared
`
`by the same procedure from nIg (10). by the same procedure from nIg (10).
`
`Quantitation of DM and the 2-pyridyldithio end Quantitation of DM and the 2-pyridyldithio end
`
`group (Py-SS-) of PLGA.-The DM content was group (Py-SS-) of PLGA.-The DM content was
`
`determined spectrophotometrically based on absorbance determined spectrophotometrically based on absorbance
`
`at 480 nm [t;, 12,000 (15)]. The content of the 2-at 480 nm [t;, 12,000 (15)]. The content of the 2-
`
`pyridyldithio end group was determined by treatment pyridyldithio end group was determined by treatment
`
`of a sample with DTT followed by measurement of UV of a sample with DTT followed by measurement of UV
`
`absorbance at 343 nm [t;, 8,080 (16)] due to liberated absorbance at 343 nm [t;, 8,080 (16)] due to liberated
`
`pyridine-2-thione. A sample was dissolved in or diluted pyridine-2-thione. A sample was dissolved in or diluted
`to 2-3 ml of 0.1 M Na phosphate buffer (pH 7.0) and
`to 2-3 ml of 0.1 M Na phosphate buffer (pH 7.0) and
`treated with an excess (2 JLI) of 0.5 M DTT in 0.1 M Na
`treated with an excess (2 JLI) of 0.5 M DTT in 0.1 M Na
`
`phosphate buffer (pH 7.0), and absorbance at 343 nm phosphate buffer (pH 7.0), and absorbance at 343 nm
`
`was measured. was measured.
`
`Quantitation of the maleimide group introduced to Quantitation of the maleimide group introduced to
`
`19.-An aliquot of a sample (=2.5 mg) was treated 19.-An aliquot of a sample (=2.5 mg) was treated
`
`with an approximately fiftyfold molar excess of DNP(cid:173)with an approximately fiftyfold molar excess of DNP(cid:173)
`
`Cys in 0.1 M Na phosphate buffer (pH 6.0) containing Cys in 0.1 M Na phosphate buffer (pH 6.0) containing
`
`0.1 M NaCI at room temperature for 0.1 M NaCI at room temperature for
`
`I hour, and I hour, and
`
`unreacted DNP-Cys was removed by gel filtration on a unreacted DNP-Cys was removed by gel filtration on a
`
`Sephadex G-25 column (0.8X40 cm) in CMF-PBS. UV Sephadex G-25 column (0.8X40 cm) in CMF-PBS. UV
`
`absorption at 360 [t;, 17,000 (17)] and 280 nm (EI~, 14.0) absorption at 360 [t;, 17,000 (17)] and 280 nm (EI~, 14.0)
`
`was measured for the determination of the content of was measured for the determination of the content of
`the maleimide group and of the protein.
`the maleimide group and of the protein.
`
`Quantitation of 19 in conjugates.-Quantitation of Quantitation of 19 in conjugates.-Quantitation of
`
`Ig protein was performed by the Bio-Rad protein assay Ig protein was performed by the Bio-Rad protein assay
`
`(Bio-Rad Laboratories, Richmond, CaliL) (18). This (Bio-Rad Laboratories, Richmond, CaliL) (18). This
`
`assay was based on the shift of absorbance maximum assay was based on the shift of absorbance maximum
`
`for an acidic solution of Coomassie brilliant blue for an acidic solution of Coomassie brilliant blue
`
`G-250 from 465 to 595 nm when binding to protein G-250 from 465 to 595 nm when binding to protein
`
`occurred. A standard curve was drawn by the use of occurred. A standard curve was drawn by the use of
`
`nlg, and correction was made with respect to the nlg, and correction was made with respect to the
`
`influence of coexistent PLGA-DM. influence of coexistent PLGA-DM.
`
`Disk PAGE.-Disk PAGE was done according to Disk PAGE.-Disk PAGE was done according to
`
`the method of Davis (19). Electrophoresis was con(cid:173)the method of Davis (19). Electrophoresis was con(cid:173)
`
`ducted in 5% polyacrylamide gel (0.5X5 cm) in 5 mM ducted in 5% polyacrylamide gel (0.5X5 cm) in 5 mM
`
`Tris-38 mM glycine buffer (pH 8.6) at 4 mA/gel for I Tris-38 mM glycine buffer (pH 8.6) at 4 mA/gel for I
`
`hour, and gels were dyed with Coomassie brilliant blue hour, and gels were dyed with Coomassie brilliant blue
`
`G-250 by the method of Burges (20). G-250 by the method of Burges (20).
`
`
`the reaction mixture was dialyzed against water and the reaction mixture was dialyzed against water and
`
`concentrated to approximately 30 ml by evaporation at concentrated to approximately 30 ml by evaporation at
`
`40°C under reduced pressure. 40°C under reduced pressure.
`
`The pH of the solution thus obtained containing a The pH of the solution thus obtained containing a
`
`mixture of Na salts of I and 3-(2-pyridyldithio)(cid:173)mixture of Na salts of I and 3-(2-pyridyldithio)(cid:173)
`
`propionyl PLGA (Py-SS-PLGA, II) was made 8.5 with propionyl PLGA (Py-SS-PLGA, II) was made 8.5 with
`
`0.1 N NaOH, and the mixture was treated with DTT 0.1 N NaOH, and the mixture was treated with DTT
`
`(73.3 mg, 0.476 mmol) at 45°C for 1.5 hours. The pH of (73.3 mg, 0.476 mmol) at 45°C for 1.5 hours. The pH of
`
`the mixture was made 2.0 with I N HCI and allowed to the mixture was made 2.0 with I N HCI and allowed to
`
`stand at 4°C for I hour. The generated precipitate was stand at 4°C for I hour. The generated precipitate was
`
`collected by centrifugation and washed with 0.01 N collected by centrifugation and washed with 0.01 N
`
`HCI to give a mixture of I and 3-mercaptopropionyl(cid:173)HCI to give a mixture of I and 3-mercaptopropionyl(cid:173)
`
`polY-L-glutamic acid (HS-PLGA, III). polY-L-glutamic acid (HS-PLGA, III).
`
`To a suspension of Thiopropyl Sepharose 6B resin To a suspension of Thiopropyl Sepharose 6B resin
`
`(Pharmacia Chemicals AB; 60 ml) in 0.1 M Na phos(cid:173)(Pharmacia Chemicals AB; 60 ml) in 0.1 M Na phos(cid:173)
`phate buffer (pH 6.0) containing I mM EDTA (100 ml)
`phate buffer (pH 6.0) containing I mM EDTA (100 ml)
`was added a solution (pH 6.0) of I and III [prepared by
`was added a solution (pH 6.0) of I and III [prepared by
`the addition of 0.1 N HCI to a solution of I and III in
`the addition of 0.1 N HCI to a solution of I and III in
`
`I N NaOH (7.0 ml)], and the mixture was stirred gently I N NaOH (7.0 ml)], and the mixture was stirred gently
`
`at room at room
`
`temperature overnight to allow temperature overnight to allow
`
`
`the the
`thiol thiol
`derivative III
`
`derivative III to bind to the resin. The resin was to bind to the resin. The resin was
`
`collected by filtration and washed with 0.01 M Na collected by filtration and washed with 0.01 M Na
`
`phosphate buffer (pH 7.5). phosphate buffer (pH 7.5).
`The resin thus obtained was stirred gently at room
`The resin thus obtained was stirred gently at room
`temperature overnight in 0.1 M Tris-HCI buffer (pH
`temperature overnight in 0.1 M Tris-HCI buffer (pH
`
`8.5) containing I mM EDTA (80 ml) with added 2-ME 8.5) containing I mM EDTA (80 ml) with added 2-ME
`(4.7 g) to liberate Na salt of III from the resin, and the
`(4.7 g) to liberate Na salt of III from the resin, and the
`
`mixture was filtered. The filtrate was made acidic (pH mixture was filtered. The filtrate was made acidic (pH
`
`1.8) with 1.8) with
`
`I N HCI, and the precipitate (III) was I N HCI, and the precipitate (III) was
`
`collected by centrifugation and washed with 0.01 N collected by centrifugation and washed with 0.01 N
`
`HCl. HCl.
`
`The polymer III was dissolved in 0.4 M Na phosphate The polymer III was dissolved in 0.4 M Na phosphate
`
`buffer (pH 7.5) containing I mM EDTA (5 ml), and the buffer (pH 7.5) containing I mM EDTA (5 ml), and the
`
`solution was diluted with 0.1 M Na phosphate buffer solution was diluted with 0.1 M Na phosphate buffer
`
`(pH 7.0) containing 1 mM EDTA (40 ml). To the (pH 7.0) containing 1 mM EDTA (40 ml). To the
`
`resulting solution was added a solution of 2-pyridyl(cid:173)resulting solution was added a solution of 2-pyridyl(cid:173)
`
`disulfide (lOS mg, 0.48 mmol) in ethanol (10 ml), and disulfide (lOS mg, 0.48 mmol) in ethanol (10 ml), and
`
`the mixture was. allowed to stand at room temperature the mixture was. allowed to stand at room temperature
`
`for 1 hour and dialyzed against water, concentrated to for 1 hour and dialyzed against water, concentrated to
`
`approximately 20 ml under reduced pressure, and approximately 20 ml under reduced pressure, and
`
`lyophilized to give Na salt of II (84.5 mg). lyophilized to give Na salt of II (84.5 mg).
`
`Binding of DM to Py-SS-PLGA and generation of Binding of DM to Py-SS-PLGA and generation of
`
`the free thiol group.-Na salt of II (75 mg, 5.86 JLmol) the free thiol group.-Na salt of II (75 mg, 5.86 JLmol)
`
`was dissolved in 3% NaCI (50 ml), and the pH was was dissolved in 3% NaCI (50 ml), and the pH was
`
`made 7.0 with 1 N NaOH. To the resulting solution made 7.0 with 1 N NaOH. To the resulting solution
`
`were added DM hydrochloride (Accurate Chemical and were added DM hydrochloride (Accurate Chemical and
`
`Scientific Corp., Hicksville, N.Y.; 42 mg, 74.6 JLmol) Scientific Corp., Hicksville, N.Y.; 42 mg, 74.6 JLmol)
`
`and EDCI (95.2 mg, 0.497 mmol), and the mixture was and EDCI (95.2 mg, 0.497 mmol), and the mixture was
`
`stirred at room temperature for 4 hours and for an stirred at room temperature for 4 hours and for an
`
`additional 4 hours after further addition of EDCI (95.2 additional 4 hours after further addition of EDCI (95.2
`
`mg). Over the whole reaction period, the pH of the mg). Over the whole reaction period, the pH of the
`
`mixture was kept at 6.5-7.5 by the addition of 1 N mixture was kept at 6.5-7.5 by the addition of 1 N
`
`HCl. The mixture was dialyzed against 0.01 M Na HCl. The mixture was dialyzed against 0.01 M Na
`
`phosphate buffer (pH 7.0) and against water to give a phosphate buffer (pH 7.0) and against water to give a
`
`conjugate (Py-SS-PLGA-DM, IV) of DM with II. The conjugate (Py-SS-PLGA-DM, IV) of DM with II. The
`
`solution was concentrated to 11.5 ml at 40°C under solution was concentrated to 11.5 ml at 40°C under
`reduced pressure and dialyzed against 0.01 M Na
`reduced pressure and dialyzed against 0.01 M Na
`
`phosphate buffer (pH 7.0). phosphate buffer (pH 7.0).
`
`To the above solution of IV (0.501 mM of PLGA To the above solution of IV (0.501 mM of PLGA
`
`equivalence, 10 ml) was added 0.5 M DTT in 0.5 M Na equivalence, 10 ml) was added 0.5 M DTT in 0.5 M Na
`
`
`
`JNCI. VOL. 73. NO.3. SEPTEMBER 1984 JNCI. VOL. 73. NO.3, SEPTEMBER 1984
`
`SANOFI-AVENTIS Exhibit 1041 - Page 722
`
`IPR for Patent No. 8,951,962
`
`

`
`An Antlbody-Daunorublcln Conjugate
`
`723
`
`Downloaded from
`
`http://jnci.oxfordjournals.org/
`
` at University of Chicago on September 10, 2015
`
`Ouchterlony test.-The Ouchterlony test was done
`in 1% agar in eMF-PBS. The rabbit anti-horse IgG
`antiserum used was purchased from Miles Laboratories,
`Inc., Elkhart, Ind.
`Purification of conjugates by starch-block electro(cid:173)
`phoresis.-A conjugation product was applied to a
`starch block (1.2XIOX50 cm) equilibrated with IS mM
`Na phosphate-I I mM Na tetraborate buffer (pH 8.4),
`and electrophoresis was conducted at 5 rnA for 22
`hours. The starch block was cut every I cm from one
`end, and each section was extracted two or three times
`with eMF-PBS. After analysis by disk PAGE, Ig(cid:173)
`bound PLGA-DM-rich fractions were combined, con(cid:173)
`centrated by ammonium sulfate fractionation (50% satu(cid:173)
`ration), and dialyzed against eMF-PBS. One conjugate
`product was subjected to the above starch-block electro(cid:173)
`phoresis in three portions.
`
`RESULTS
`
`Preparation of Intermediate Drug Carrier
`
`To introduce a single masked thiol, 2-pyridyldithio
`group to the N-terminal of PLGA, we treated PLGA
`with a twentyfold molar excess of SPDP at pH 7.5
`(text-fig. I). The reactivity of the terminal amino group
`was relatively low, and much of the group remained
`unreacted. The masked thiol group-containing PLGA
`was isolated from intact PLGA by use of Thiopropyl
`Sepharose 6B resin. After conversion of the masked
`thiol group to the free thiol group with DTT, the
`mixture of II and PLGA was allowed to react with the
`resin. The resin contained the active disulfide 2-pyridyl(cid:173)
`dithio groups and reacted with the thiol group of the
`modified PLGA, which was recovered from the resin by
`the disulfide exchange reaction with 2-ME.
`The pure thiol derivative III
`thus obtained was
`converted back to. the 2-pyridyldithio derivative II for
`the protection of 'the thiol group. The 2-pyridyldithio
`functional group also allowed the spectrophotometric,
`
`~SS~COtNHCHCOlnOH
`H{NHCHCOln"OH aN )
`..
`)
`C02H
`C02H
`Py-SS-PLGA, II
`~G~I
`+
`
`HS~CO{NHCHC0Tr10H d
`-L~
`)
`I
`
`I
`I
`
`•
`
`C02H
`HS-PLGA, III
`a, SPOP; b, OTT; c, Thiopropyl Sepharose 68 resin;
`2-ME; d, 2-pyridyldisulfide
`
`TEXT·FIGllRE I.-Preparation of a single
`PLGA.
`
`thio] group·containing
`
`TABLE I.-Chemical data on PLGA-DM
`and Ig-PLGA-DM conjugates·
`
`Data
`
`Average molecular
`weight of PLGA d
`Degree of polymer-
`ization of PLGA
`DM-to-PLGA
`binding ratio'
`Drug-substitution
`rate, %f
`
`PLGA-to-Ig bind-
`ing ratiog
`DM-to-Ig bind-
`ing ratioh
`
`Preparation 1 b
`
`Anti-
`AFP
`
`Normal
`
`Preparation 2'
`Anti-
`AFP
`
`Normal
`
`PLGA-DM
`
`12,800
`
`12,800
`
`15,100
`
`15,100
`
`85
`
`85
`
`100
`
`100
`
`7.0
`
`8.3
`
`7.0
`
`8.3
`
`Ig--PLGA-DM
`
`2.8
`
`19.6
`
`2.1
`
`14.7
`
`10.3
`
`10.3
`
`1.1
`
`11.8
`
`10.3
`
`10.3
`
`1.0
`
`10.3
`
`• The outline of the methods of obtaining the data is described
`to this table. For details, see "Materials and
`in footnotes
`Methods."
`b Details on this preparation are described in "Materials and
`Methods."
`<The conjugates were prepared as in preparation 1. However,
`5MBU was used for introducing the maleimide groups into Ig,
`and a fourfold molar excess of V was used for the reaction with
`VI instead of a tenfold molar excess in the case of preparation 1.
`After dialysis of the final reaction mixture against 15 mM Na
`phosphate-11 mM Na tetraborate buffer (pH 8.4), the conjugates
`were purified by starch-block electrophoresis.
`dDetermined by the end-group (2-pyridyldithio) analysis with
`respect to a lyophilized aliquot of IV.
`'The DM content (mol) was determined spectrophotometrically,
`and the PLGA content (mol) was determined by the end-group
`analysis.
`fThe average percentage of DM-Iinked carboxyl groups among
`total number of carboxyl groups in PLGA.
`g Calculated by the division of the DM-to-Ig binding ratio by
`the DM-to-PLGA binding ratio.
`h This number was obtained from mol of Ig in the conjugate
`preparation (determined by the Bio-Rad protein assay) and mol
`of antibody-linked DM as determined by the multiplication of
`the total DM content (antibody-linked DM plus DM as PLGA-DM)
`of the conjugate preparation (determined spectrophotometrically)
`by pl100 [p=purity (%) of the conjugate shown in table 2].
`
`quantitative end-group analysis by the method described
`in "Materials and Methods." The average molecular
`weight of the isolated II as determined by this method
`is shown in table I.
`
`Preparation of Ig-PLGA-DM Conjugates
`
`First the masked thiol group-containing PLGA de(cid:173)
`rivative II was treated with DM 15% equivalent to the
`carboxyl groups in the presence of the water-soluble
`carbodiimide EDeI followed by the removal of low(cid:173)
`molecular-weight materials to give
`the PLGA-DM
`conjugate (text-fig. 2). The average number of DM
`molecules linked to one molecule of PLGA (DM-to(cid:173)
`PLGA binding ratio) and the ratio (%) of the number
`
`JNCI. VOL. 73, NO.3. SEPTEMBER 1981
`
`SANOFI-AVENTIS Exhibit 1041 - Page 723
`
`IPR for Patent No. 8,951,962
`
`

`
`Downloaded from
`
`http://jnci.oxfordjournals.org/
`
` at University of Chicago on September 10, 2015
`
`724 Tsukada, Kato, Umemoto, et al.
`
`QSS~CO~NHCHCOTp'-fNHCHCOlqOH
`py-SS-PLGA~
`~
`' ;
`II
`CO-OM
`C02H
`Py-SS-PLGA-OM, N
`
`t
`
`, HS-PLGA-OM ________ _
`
`V
`
`o
`Ig~ 19-tNHCO-X-NO] - - - - . .
`o
`
`I
`
`VI
`
`view that this material is a conjugate of OM with
`antibody.
`The average number of the PLGA chains linked to
`one molecule of Ig (PLGA-to-Ig molar binding ratio)
`and the average number of OM molecules conjugated
`with one molecule of Ig through PLGA (OM-to-Ig
`molar binding ratio) are listed in table 1.
`The anti-AFP and normal conjugates prepared in
`preparation 1 were used without further purification in
`biological studies. The conjugates in preparation 2 were
`used after purification by starch-block electrophoresis.
`The purities [percentages of Ig-linked OM in total
`amount of OM (Ig-linked OM plus OM as PLGA-OM)]
`of the conjugates used in the biological studies are
`shown in table 2.
`
`I 9 -fNHCO-X _N)S/'y-CO-f.NH(HCOlp"~NHCHCOlqOH ]
`o 7 ) m
`
`CO-OM
`
`COzH
`
`Ig- PLGA - OM,
`
`vn
`
`o OH
`0
`~CH3
`YVYYOH
`OM-= MeO 0 :~J
`
`H3Cf?:!:J
`HONH_
`
`e, daunomycin-EOCI; t, OTT; g, 5MBE orSMBU
`
`TEXT·FIGllRE 2.-Preparation of Ig-PLGA-OM conjugates.
`
`of drug-substituted carboxyl groups to the total number
`of drug-substituted and drug-unsubstituted carboxyl
`groups (drug-substitution rate) are shown in table 1.
`Next the maleimide group was introduced to aAFP
`with a twentyfold molar excess of 5MBE (14) or 5MBU
`(21). The modified antibody contained, on the average,
`6.7 (SMBE) and 12.1 (SMBU) maleimide groups per
`molecule of antibody as determined by the ONP-Cys
`method. After generation of the free thiol group by
`treatment with OTT, the OM-linked, masked thiol
`derivative IV was conjugated with the maleimide group(cid:173)
`containing antibody by the addition reaction of the
`thiol group to the maleimide group.
`The conjugates were subjected to disk PAGE and the
`Ouchterlony test. Text-figure 3 shows the results of the
`analysis with respect to preparation 1. In disk PAGE
`(fig. IA), negatively charged PLGA-OM migrated very
`fast (gel 5) in contrast to the slow migration of Ig (gels
`3 and 4). In addition to the band of unreacted HS(cid:173)
`PLGA-OM V, the conjugate preparation exhibited two
`new bands that were clearly different from the band of
`Ig (gels I and 2). The material extracted from these
`bands with Tris-HCI buffer (pH 8.2) containing 2 M
`NaCI and O. I % NaN 3 showed a visible absorption
`maximum at 480 nm and formed a precipitin line with
`a rabbit anti-horse IgG antiserum, which supports the
`
`JNCI. VOL. 73, NO.3, SEPTEMBER 1984
`
`10
`
`E
`"-.,
`0
`~ x
`(ji
`...J
`...J
`W
`()
`W
`
`...J co «
`:>
`
`0.1
`
`oL,
`
`o
`rl
`o
`
`I
`I
`i
`3
`0.3
`0.03
`OM CONCENTRATION tug/ml)
`I
`I
`I i i i
`i
`0.4 0.6
`. 46
`4060
`400 600
`Ig CONCENTRATION (,ug/ml)
`
`I
`
`I
`
`..
`30
`
`TEXT-FIGURE 3.-Enhanced cytotoxicity of OM when conjugated with
`aAFP. AH66 cells (5XI0· cells/ml) were cultured in Eagle's
`minimum essential medium (Nissui Seiyaku Co. Ltd., Tokyo; pH
`7.4), containing 10% heat-inactivated calf serum (Flow Labora(cid:173)
`tories, Stanmore, Australia), kanamycin sulfate (6 ~g/rril), and a
`serially diluted test sample (conjugates used were those listed under
`preparation 2 in table I) in a 96-well MicroTest tissue culture plate
`(Falcon Plastics No. 3040) in a humidified atmosphere of 5% C02
`in air at 37°C for 48 hr, and the viable cells were counted by the
`trypan blue dye-exclusion method. 0, nlg; 0, aAFP; X, OM; 0,
`PLGA-OM; ., nlg-PLGA-OM; ., aAFP-PLGA-OM. Points,
`means of triplicate determinations; bars, SE (indicated unless
`smaller than the points as plotted).
`
`SANOFI-AVENTIS Exhibit 1041 - Page 724
`
`IPR for Patent No. 8,951,962
`
`

`
`Downloaded from
`
`http://jnci.oxfordjournals.org/
`
` at University of Chicago on September 10, 2015
`
`An Antibody-Daunorubicin Conjugate 725
`
`MST became 34.9±1.58 and 45.1±2.78 days, respectively.
`The tumor-neutralizing effect of the two nonspecific
`macromolecular DM derivatives PLGA-DM and normal
`conjugate [MST, 39.3±1.45 and 38.3±2.85 days, respec(cid:173)
`tively] was about the same as that of free DM (MST,
`45.l±2.78 days). No significant synergistic effect was
`observed between aAFP and free DM (MST of the
`mixture-of-the-two group, 48.0±3.24 days). The group
`that showed the highest survival rate was the group
`inoculated with the cells preincubated with anti-AFP
`conjugate, and 2 of 8 rats were 70-day survivors, with
`MST of the 6 dead rats being 52.5±3.70 days. The
`tumor-neutralizing effect of the anti-AFP conjugate was
`statistically significant over the effect of either DM
`alone (P<'05) or normal conjugate (P<'OI), even with
`the assumption that two 70-day survivors died on
`day 70.
`
`Therapeutic Effect of Anti-AFP Conjugate
`
`For assessment of the in vivo antitumor activity of
`anti-AFP conjugate on ascites AH66 tumor, DONRYU
`rats, which had been inoculated ip with lXlO4 AH66
`cells (day 0), were treated with an ip injection of test
`materials every other day starting from day 3 for a total
`of 3 . (experiment A, text-fig. 5A) or 5 (experiment B,
`text-fig. 5B) doses. The survival was followed until day
`70 (A) or day 100 (B). The test materials used in
`experiments A and B are those listed under prepara(cid:173)
`tions I and 2 in table I, respectively.
`The aAFP prolonged the lives of AH66 tumor-bearing
`rats (P<'OOI) [MST: A, 31.9±1.91 days; B, 37.0±2.7I
`days (MST of the untreated group: 15.5± 1.22 days)],
`
`100,'----T--.--.-. . --.~
`
`...J
`
`;g;
`~ 50
`:J en
`cf.
`
`o I
`o
`
`10
`
`20
`
`30
`
`"
`40
`
`X "
`50
`
`60
`
`70
`
`DAYS AFTER TUMOR CELL INOCULATION
`
`TEXT-FlGI1RE 4.-Tumor·neutralizing activity of anti-AFP conjugate
`against AH66 tumor. AH66 cells (lXI05 cells/ml of PBS) were
`incubated with PBS (--). nlg (2.500. -0-). aAFP (2.500. -0-).
`OM (500. -X-i. PLGA-OM (500. -0-). mixture of aAFP and OM
`(2.500-500. -A-). nlg-PLGA-OM (2.2\0-560. -.-). or aAFP(cid:173)
`PLGA-OM (2.070-560, -e-) at 37°C for 30 min. The PBS cell
`suspension (0.1 ml) containing IX\04 treated cells was injected ip
`into OONRYU rats. Survival was followed for 70 days. Numbers in
`parentheses in this legend indicate the concentration (}lg/ml) of the
`test materials. For the Ig-PLGA-OM conjugates and the mixture
`of aAFP and OM, two numbers are given for the concentrations of
`Ig and of OM in this order. The conjugates used are those listed
`under preparation I in table I.
`
`JNCI. VOL. 73. NO.3. SEPTEMBER 1984
`
`TABLE 2.-Purity of conjugate preparation used
`in biological studies·
`
`Preparation
`
`1
`2
`
`Purity, %
`
`Anti-AFP
`conjugate
`
`27
`62
`
`Normal
`conjugate
`
`22
`78
`
`a Purity denotes the percentage (p) of Ig-linked DM to total
`amount of DM (Ig-linked DM plus DM as PLGA-DM), and it
`was determined as follows: The conjugate preparations were
`subjected to disk PAGE; in the case of preparation 1, Ig-(cid:173)
`PLGA-DM and PLGA-DM were extracted from the correspond(cid:173)
`ing bands with 0.1 M Tris-HCI buffer (pH 8.2) containing 2 M
`NaCI and 0.1% NaN); in the case of preparation 2, Ig--PLGA-DM
`and PLGA-DM were isolated from the gel by further electro(cid:173)
`phoresis. The contents of DM extracted in the two forms,
`Ig--PLGA-DM and PLGA-DM, were determined spectrophoto(cid:173)
`metrically.
`
`In Vitro Cytotoxicity of Anti-AFP Conjugate
`
`The cytotoxicity of the aAFP-PLGA-DM conjugate
`against hepatoma AH66 cells was assessed by
`the
`determination of the inhibition of the in vitro growth
`of the AH66 cells that were cultured, with the conjugate
`being added to the medium (text-fig. 3). This inhibition
`was compared with the inhibitions obtained with nlg,
`aAFP, unconjugated DM, PLGA-DM, and the nlg(cid:173)
`PLGA-DM conjugate.
`The anti-AFP conjugate exhibited potent concentra(cid:173)
`tion-dependent cytotoxicity and inhibited the cell growth
`or killed the cells at concentrations above 0.3 J.Lg
`(equivalent DM)/ml, whereas normal conjugate showed
`only moderate cytotoxicity. Unconjugated aAFP showed
`even weaker cytotoxicity, though it did show some
`cytotoxicity as compared with the cytotoxicity shown
`by nlg. These results indicate that the potent cyto(cid:173)
`toxicity of anti-AFP conjugate is due to both the
`toxicity of the OM portion and the antigen (AFP)(cid:173)
`binding activity of the aAFP portion.
`The cytotoxicities of PLGA-DM and of nlg(cid:173)
`PLGA-DM were about the same as the cytotoxicity of
`free DM, and the cytotoxicities of these three were
`less than the cytotoxicity of the anti-AFP conjugate;
`i.e., the cytotoxicity of DM was enhanced only when
`conjugated with aAFP via PLGA.
`
`Tumor-Neutralizing Activity of Antl-AFP Conjugate
`
`For the assessment of the tumor-neutralizing activity
`of anti-AFP conjugate by the Winn test (22), groups of
`DONRYU rats were inoculated ip with lXI04 AH66
`cells that had been preincubated with test materials
`(materials tested were those in preparation I, table I) at
`37°C for 30 minutes, and the survival was followed
`(text-fig. 4). MST (±SE) of rats that received the cells
`preincubated with PBS or nlg were 17 .9±0. 72 and
`22.1±0.95 days, respectively. The rats that received the
`cells treated with aAFP or unconjugated DM survived
`considerably longer than the PBS group (P<'OOI), and
`
`SANOFI-AVENTIS Exhibit 1041 - Page 725
`
`IPR for Patent No. 8,951,962
`
`

`
`726 Tsukada, Kato, Umemoto, et al.
`
`100
`
`A
`
`I I
`
`(A, P<'05; B, P<'OI), normal conjugate (P