Toxin-Targeted Design for Anticancer Therapy. II: Preparation
`and Biological Comparison of Different Chemically Linked
`Gelonin-Antibody Conjugates
`
`FRANCO DOSIO, PAOM BRUSA, MAURIZIO CERUTI,
`LAURA DELPRINO, MARIA GIACOMO~I,
`GIORGIO GROSA, AND LUIGI C A n E L X
`Received June 10, 1992, frpm.the lstituto di Chimica Farmaceutica Applicata, University of Turin, C.so Raffaello, 33-10125 Turin,
`Italy. Accepted for publication November 5, 1992.
`
`Abstract 0 To obtain more potent immunotoxins for anticancer therapy
`a gelonin-AR3 antibody immunoconjugate was prepared with different
`new linkers and coupling procedures. The gelonin was derivatized with
`the heterobifunctional thioimidate linkers ethyl-acetyl-3-mercaptopropi-
`onthioimidate (AMPT) and 3-(4-carboxamidophenyldithio)propionthioim-
`idate (CDPT), and with the succinimidyl type reagents Nsuccinimidyl-
`3-(4-carboxamidophenyldithio)propionate (SCDP) and Nsuccinimidyl-
`Sacetyl thiolacetate (SATA). The biological activity of gelonin modified
`with different linkers (AMPT, CDPT, SCDP, SATA) was determined by a
`rabbit reticulocyte assay. We found that AMPT was the molecule of
`choice to derivatize the toxin, confirming the preferability of thioimidate
`linkers. The monoclonal antibody Mab was derivatized with CDPT and
`SCDP. Then the following immunoconjugates were prepared with
`different procedures: Mab-CDPT with gelonin-AMPT; Mab-CDPT with
`gelonin-CDPT; Matj-SCDP with geloninSATA. To verify whether
`selection of the most suitable coupling procedure could affect the
`antitumoral activity of the gelonin-AR3 immunoconjugate, the three
`immunotoxins were tested on target HT-29 human colon carcinoma cells
`versus nontarget MeWo cells. The gelonin immunoconjugate linked via
`the AMPT-CDPT thioimidate reagents showed highest antitumoral
`activity as well as best selectivity for the target cells.
`
`Immunotoxins (ITS) are molecular conjugates formed by
`monoclonal antibodies (Mabs) linked to toxic agents and are
`capable of inactivating the cellular synthesis of proteins.14
`Unlike currently available drugs that usually do not differ-
`entiate between tumor and normal cells, ITS, like the “magic
`bullets” described by Erlich,6 are designed to deliver their
`toxin to the targeted tumor cells.
`ITS are usually obtained with a heterobifunctional linker or
`by gene fusion. The most commonly used toxic agent is the
`ricin A chain, a ribosome inactivating protein (RIP) that
`inactivates protein synthesis in eukaryotic cells by specific
`covalent modification of ribosomal RNA.7 ITS have also been
`synthesized with a large group of RIPS (type 1) of plant origin,
`which resemble the ricin A chain in size and which inactivate
`ribosomes by an identical mechanism.s~g One of these is
`gelonin, a 30 kDa, single-chain glycoprotein obtained from
`the seed of Gelonium multiflorum, which is able to inhibit
`protein synthesis once inside the eukaryotic ceU.10-12 Unlike
`two-chain protein RIPs (type 2), such as abrin and ricin,
`gelonin cannot enter the cells; therefore, it is much less toxic
`to cells in vivo unless it is provided with a mechanism of entry.
`Moreover, gelonin is more stable to chemical and physical
`treatment than ricin A chains and is not toxic at up to 80
`mglkg when injected into mice.13 IT made with gelonin has
`generally displayed similar or better specific toxicity than IT
`prepared with ricin A chain.lPls
`To prepare gelonin-containing IT, heterobifunctional
`agents such as N-succinimidyl-3-(2-pyridyldithio)propionate
`(SPDP, l)19 or 2-iminothiolane20 (2, see structure) were used
`
`N-succinimidyl 3-(2-pyridyldithio)propionate
`
`(SPDP)
`
`3-(4-carboxamidophenyldithio)-propionthoimidate
`
`(CDPT)
`
`N-succinimidyl-acetylthioacetate
`
`(SATA)
`
`(I=a,,
`
`2-iminothiolme
`
`(2-IT)
`
`0 0
`cy-c--s-cy--cy-c~
`
`w*m
`S-Qb-w
`Ethyl S-acetyl-propionthioimidate
`
`(AMPT)
`
`N-succinimidyl 3-(4-corboxamidophenyldithio)propionate
`
`(SCDP)
`
`to introduce a thiol group into the toXin.21.22 The thiolated
`gelonin was then added to an SPDP-derivatized Mabs until a
`stable disulfide bond was formed by a disulfide exchange
`reaction. Some authors have reported that gelonin is not
`afFected by derivatization with 2-iminothiolane in its ability
`to inhibit protein synthesis, while it is inactivated to -90% by
`modification with SPDP.14J6 Recently,23.24 a comparison of
`SPDP and 2-iminothiolane used to derivatize gelonin and
`some other type 1 RIPs has reported analogous results.
`2-Iminothiolane has been considered the reagent of choice to
`covalently link PAP (pokeweed antiviral protein)25 or abrin A
`chain to Mabs26; in this case the 2-iminothiolane-linked ITS
`were found to be much more effective than the SPDP-linked
`ITS as antitumoral agents in mice with murine lymphoma.26
`All these results can be explained by the fact that with SPDP
`the lysine is transformed into an uncharged amido group,
`
`0022-3549/93/0700-0699$02.50/0
`0 1993, American Pharmaceutical Association
`
`Journal of Pharmaceutical Sciences I 699
`Vol. 82, No. 7, July 1993
`
`IMMUNOGEN 2081, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`whereas 24minothiolane (and in general thioimidate re-
`agents) forms an amidinium bond. The maintaining of the
`positive charge of the protein could be an important factor
`that influences the biological activity.
`In a preceding paper27 we reported the preparation of two
`new acyclic thioimidate cross-linking reagents, 344-
`carboxamidopheny1dithio)propionthioimidate (CDPT, 3) and
`ethyl-acetyl-3-mercaptopropionthioimidate (AMPT, 4). Here,
`we report using these reagents to prepare new ITS formed by
`gelonin linked to the AR-3 Mab that recognizes the CAR-3
`antigen widely expressed in stomach and colon adenocarci-
`nomaa.28 To get the optimal coupling procedure, we evaluated
`both the ITS made with AMPT and CDPT, and the immuno-
`conjugates prepared with both a new reagent, N-succinim-
`idyl-3-(4-carboxamidophenyldithio)propionate (SCDP, 6) and
`the commercial N-succinimidyl ligand SATA (5, N-succinim-
`idyl-S-acetylthioacetate). Among those tested, we found that
`ITS prepared with heterobifunctional thioimidate reagents
`showed the best selectivity for the target cells as well as the
`highest antitumoral activity.
`
`Experimental Section
`Mab, Toxin, and Tumor Cell Lines-The Mab AR3 (a mouse IgGl
`that recognizes the CAR-3 antigen widely expressed among human
`adenocarcinomas of the stomach, colon, pancreas, ovary, and U ~ ~ N S )
`was obtained experimentally as described by Prat et al.28 and was
`purified from mouse ascitic fluid by affinity chromatography on
`protein A-Sepharose CL-4B (Sigma, St. Louis, MO). The toxin
`gelonin,' purified as described by Stirpe et al.,9 was from Sigma (St.
`Louis, MO). The tracer used for conjugation and purification was
`12'I-labeled gelonin by the iodogen method29 and then purified by
`high-performance liquid chromatography (HPLC) gel-filtration on
`TSK G2000 SW to a specific activity of 40 mCi/mg. The cell lines used
`were the HT-29 human colon adenocarcinoma, expressing the CAR-3
`antigen, as target line and the MeWo human melanoma as control.
`N-Succinimidyl Esters Preparation-SATA-This
`ligand was
`prepared aceording to the method of Duncan et al.29
`SCDP-The preparation of this ligand is a two-step process. First,
`3-(4-carboxamidophenyldithio)propionic acid (la) was formed as fol-
`lows. 4-Carbo&dobenzensulfenyl
`chloride27 was reacted with
`3-mercaptopropionic acid in glacial acetic acid at reflux for 2 h. After
`cooling to room temperature, the resulting precipitate was removed
`by filtration, and the filtrate was diluted with distilled water. The
`aqueous solution was extracted with ethyl acetate (3 x 50 mL) and
`the organic extracts were dried (Na.$O,)
`and evaporated under
`reduced pressure. The crude product was purified by flash chroma-
`tography (diethyl etherethyl acetate:CH,COOH, 99:1:0.5), giving
`pure la as a colorless crystalline powder (43% yield), mp 170-175 "C;
`IR (KBr): 3400, 3300, 3200, 1700, 1630, 1580, and 830 cm-'.
`Next, SCDP was formed as follows: la (1.27 g, 4.9 mmol) was
`dissolved in 150 mL of refluxing anhydrous tetrahydrofuran (THF).
`After cooling to room temperature, N-hydroxysuccinimide (0.8 g, 7
`mmol) in 5 mL of anhydrous THF was added to the solution. The
`coupling reaction was achieved by the addition of dicycloexylcarbo-
`diimide (1.44 g, 7 mmol) in 5 mL of anhydrous THF. The solution was
`stirred at room temperature for 10 h, and the precipitate dicyclohex-
`ylurea was separated by filtration. The filtrate was then evaporated
`to dryness under reduced pressure, and the residue was crystallized
`from diethyl etherpetroleum ether (50:50) to give SCDP (6) in a 50%
`yield (0.85 g), mp 132 "C; IR (KBr): 3400-3200,1740,1630,1590, and
`810 cm-'; 'H NMR (CD,COCD,): 7.8 (q,4H), 3.1 (m, 4H), 2.9 (s,4H)
`ppm; Mass: 354 (M+).
`Immunoconjugate Preparation-Conjugation of AR3 to Gelonin
`via Thioimidate Esters CDPT (3) and AMPT (4): General Proce-
`dure-To a solution of gelonin (8 mg, 0.267 pmol) in 4.5 mL of
`phosphate-buffered saline (PBS)-EDTA containing 10 pCi of
`['2611ge10nin, was added a solution of AMPT (100 pL, 88 mM) in
`absolute ethanol. After stirring for 40 min at 4 "C, the reaction
`mixture was dialyzed for 16 h at 4 "C. The number of thioacetilated
`groups linked to the protein was calculated by a method described
`elsewhen+; the ge1onin:AMPT molar ratio was 1:1.2. The CDPT
`ligand (43 pL, 16 pM) in dry dimethylformamide (DMF) was added
`to a solution of AR3 Mab in PBS-EDTA (8 mg, 890 pL). The mixture
`
`was stirred for 30 min at room temperature and was dialyzed as
`before. The number of aryldithio groups linked to the protein were
`calculated as described in a preceding pape91; the AR3:CDPT molar
`ratio was 1:1.2. The derivatized proteins were mixed, and a solution
`of hydroxylamine (0.5 M plus 12.5 mM EDTA), neutralized to pH 7.4
`with NaOH, was added (l:lO, v/v). After stirring for 24 h at 4 "C, the
`mixture was treated with N-ethylmaleimide in dry DMF (2 mg, 50
`pL).
`Conjugation of AR3 to Gelonin via CDPT (3)-The CDPT ligand
`(434 pL, 20 mM) in dry DMF was added to a solution of gelonin in
`PBS-EDTA (8 mg, 4.5 mL) containing 10 pCi of ['2'Ilgelonin. After
`stirring for 30 min at 15 "C, the reaction mixture was dialyzed and the
`ge1onin:CDPT molar ratio was determined to be 1:1.3. At the same
`time, a solution of AR3 in PBS-EDTA (8 mg, 890 pL) was derivatized
`with CDPT as previously described. The CDPT-derivatized antibody
`was then mixed with a 10-fold molar excess of dithiothreitol (DTT, 10
`mM) in PBS-EDTA. After 20 min at room temperature, DTT was
`separated from the thiolated antibody by gel filtration on a Bio-Gel
`P6DG column that was pre-equilibrated in PBS-EDTA at 4 "C. The
`protein fraction was directly added in a dropwise manner to the
`CDPT-derivatized gelonin. The reaction mixture was stirred at 4 "C
`for 12 h, and N-ethylmaleimide was then added to block any free thio
`group.
`Conjugation ofAR3 to Gelonin via N-Succinimidyl Esters SATA (5)
`and SCDP (6): General Procedure-The SATA ligand (27 pL, 50 mM)
`in dry DMF was added to a solution of gelonin in PBS-EDTA (8 mg,
`4.5 mL) containing 10 pCi of [1261]gelonin. After stirring for 15 min
`at 20 "C, the reaction mixture was dialyzed and the ge1onin:SATA
`molar ratio was calculated to be 1:1.3. The SCDP ligand (3.8 pL, 28
`mM) in dry DMF was added to a solution of AR3 in PBS-EDTA (8 mg,
`890 pL). The mixture was stirred for 30 min at room temperature and
`was then dialyzed. The Mab:SCDP molar ratio was 1:1.3. The two
`protein solutions were mixed in the presence of hydroxylamine as
`previously described. The conjugation time was reduced with respect
`to the conjugate procedure with CDPT and AMPT to 20 h at 4 "C to
`reduce loss of protein due to flocculation.
`reaction mixtures were
`Purification of Zmmunoconjugates-The
`centrifuged and the supernatants applied to a HPLC gel filtration
`column (TSK G3000 SW, 7.5 x 600 mm) in several steps and eluted
`in a potassium phosphate buffer at pH 7.4 (50 mM phosphate, 300 mM
`NaC1; Figure 1). The elution was monitored spectrophotometrically
`and with a counter on line. The fractions containing the unreacted
`antibody and the conjugate were pooled, concentrated, and loaded
`onto an AfS-Gel Blue column (7 x 100 mm) that was pre-equilibrated
`at 4 "C in 50 mM sodium phosphate buffer (pH 7.4, 50 mM NaCl;
`Figure 1). Unconjugated AR3 did not bind to the solid phase under
`these conditions of ionic strength and was initially eluted from the
`column. The ARSgelonin conjugates were then eluted with the same
`buffer containing 500 mM NaC1. The fractions containing conjugates
`were pooled and finally dialyzed. The purity and the molecular weight
`of the conjugates were monitored by sodium dodecyl sulfate-
`polyacrylamide gel electrophoresis (SDS-PAGE) by the method of
`Laemmli.32 Protein concentrations were determined by measuring
`the absorbance at 280 nm, with extinction coefficients (E:,?)
`of 14.0
`for AR3 and 7.0 for gelonin. The purified conjugates with Mab:toxin
`ratios of 1:l and 1:2 had, respectively, estimated E:Gm values of 13.0
`and 12.0.
`Determination of Antigen Binding Activity of Antibody and
`Conjugates-The binding activity of derivatized AR3 and immuno-
`conjugates 7,8, and 9 was measured by enzyme-linked immunosor-
`bent assay (ELISA). Peroxidase-labeled goat antimouse IgG and
`ELISA kit were from KPL (Kirkegaard & Perry Lab. Inc, Gaithers-
`burg, MD).
`Determination of Toxin Residual Activity after Derivatization
`and Conjugation-Znhibition of Protein Synthesis Assay on a Rabbit
`Reticulocyte Cell-Free System-The Translation Kit Reticulocyte
`Type I1 from Boheringher Mannheim (Germany) was used. The
`reaction mixture contained the following in a final volume of 28
`pL:2.8 pL of translation reaction mixture without leucine, 1.4 pL of
`potassium thioacetate, 2.1 pL of magnesium thioacetate, 0.56 pL of
`RNAse inhibitor (Boheringher Mannheim, Germany), 7.14 pL of
`[3Hlleucine (Amersham International, Buckinghamshire, U.K.), and
`14 pL of a lysate of rabbit reticulocytes. After addition (3.5 pL each)
`of the last samples, water, and mRNA of tobacco mosaic virus
`(Amersham International, Buckinghamshire, U.K.) and rapid mix-
`ing, incubation was performed at 30 "C with and without preincuba-
`
`700 I Journal of Pharmaceutical Sciences
`Vol. 82, No. 7, July 7993
`
`IMMUNOGEN 2081, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`C
`
`t
`
`D
`
`t
`
`Figure 1-Purification of ITS. (A) HPLC gel filtration of CDPT-AMPT IT. The reaction mixture was based on a TSK G 3000 SW column, eluted with
`PBS over several cycles (flow rate, 0.5 mumin). The eluate was monitored spectrophotometrically at 280 nm (-)
`and with an on-line gamma counter
`(- - -). Fraction IT was collected for the next purification step. (B) HPLC gel filtration of CDPT-CDPT IT as described above. (C) Affinity chromatography
`on an Affi-Gel Blue column of CDPT-AMPT gel filtrated mixture, containing immunoconjugate and unreacted AR-3. The AR-3 was eluted with PBS;
`adding NaCl (0.5 mol) in the medium the immunotoxin eluted. (D) Affinity chromatography purification of the CDPT-CDPT IT.
`
`tion with 1% 2-mercaptoethanol. Samples of the reaction mixture (3
`pL) were spotted after 30,60,90, and 120 min on Whatman 3 MM
`paper. Radioactivity incorporated into protein was determined by
`precipitation with trichloroacetic acid and counting in a Beckman
`liquid scintillation spectrometer, with Packard Filter Count Scintil-
`lation liquid (Packard Company).
`Znhibition of Cellular Protein Synthesis-HT-29 and MeWo cell
`lines were tested in the presence of serial dilutions of intact gelonin,
`derivatized gelonin, and ITS 7, 8, and 9, according to the method
`reported by Cattel et (11.31 Results are expressed as percentages of
`['Hlleucine incorporation compared with control cultures (back-
`ground values were subtracted).
`Results and Discussion
`The AR3-gelonin IT was prepared with the thioimidate
`linkers AMPT (4) and CDPT (3)27 (Schemes I and 11) or the
`corresponding N-succinimidyl ester reagents SATA (5) and
`SCDP (6) (Scheme 111). SCDP is a new heterobifunctional
`linker possessing an N-succinimidyl ester function on one side
`and a carboxamidophenyldithio group on the other side of the
`molecule. This reagent was prepared following the known
`procedure used to synthesize SPDP.19
`The thioimidate-type immunoconjugate was prepared by
`two different coupling procedures (A and B, Schemes I and 11,
`respectively). CAI CDPT was fist reacted with the Mabs to
`introduce 1.2 aryldithio groups per protein molecule (Scheme
`I), whereas 1.2 acetylthio residues were introduced into
`gelonin with AMPT at 4 "C. The thioacetylated gelonin was
`coupled to the CDPT-derivatized Mabs with NH,OH
`to
`deprotect the acetylthiogroups. (B) Both the Mab and the
`toxin were derivatized with CDPT by introducing 1.2 and 1.3
`aryldithio groups, respectively (Scheme 11). The modified Mab
`
`was then reduced with DTT and directly coupled to the
`disulfide-containing gelonin by a thiol-disulfide exchange
`reaction.
`The coupling procedure A (CDPT-derivatized Mabs versus
`AMPT-thiolated gelonin) could offer some advantages be-
`cause, during the conjugation reaction, the thiol group intro-
`duced into the toxin, which is proteded as a thioacetyl
`derivative, was liberated in situ. This would prevent sponta-
`neous oxidation of the free thiol group as well as the formation
`of a heteroaggregated polymer.
`The N-succinimidyl ester-type immunoconjugate was pre-
`pared by coupling the SCDP-derivatized AR3 Mab to the
`gelonin previously thioacetylated with SATA (Scheme 111,
`Method C).
`The crude disulfide-linked IT prepared following the pro-
`cedure described in Schemes 1-111 (Methods A, B, and C,
`respectively) was purified by HPLC-gel filtration to remove
`the aggregates and unreacted proteins. By comparing the
`elution and SDS-PAGE electrophoresis profiles under nonre-
`ducing conditions, unreacted gelonin and high molecular
`weight compounds (2300 kDa) most abundantly in the IT 8
`prepared via the CDPT-CDPT procedure (Method B) were
`observed. After AEi-Gel Blue affinity chromatography to
`completely remove the free Mab from the conjugates, the final
`yields of the purified IT were 12% for the AMPT-CDPT
`coupling procedure (Method A), 4.5% for the CDPT-CDPT
`(Method B), and 10% for the SATASCDP (Method C) with
`respect to the molar concentration of Mab used. The relatively
`small yield of the conjugate obtained by Method B was
`probably due to the large amount of intermolecular cross-
`linked products arising from air oxidation of the free sulfydryl
`
`Journal of Phamamutical Sciences I 701
`Vol. 82, No. 7, July 1993
`
`IMMUNOGEN 2081, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Y
`
`w
`
`AR-3
`
`
`1
`
`+
`
`w
`C C W *. s. s -#- CONy
`
`CH$ cy- s'
`
`CDPT
`
`CELONIN
`
`/,
`
`AYFT
`
`Scheme l-Coupling procedure A.
`
`AR-3
`
`I
`
`CDPT
`
`GELONIN
`
`1
`
`CDPT
`
`CELONIN
`
`'0
`
`SATA
`
`Scheme 3-Coupling procedure C.
`
`groups introduced into the Mabs molecule. Because this
`procedure is analogous to that followed for preparing ITS with
`SPDP,22 one could relate the low yield obtained by Method B
`with those observed producing a gelonin-containing IT with
`SPDP.18 In contrast, the very good results obtained with the
`AMPT-CDPT procedure (Method A) were in line with those
`
`already found with mixed 2-iminothiolan4PDP linkers to
`prepare ITS on a large scale.26
`To confirm the supposed superiority of the thioimidate
`ligands versus N-succinimidylesters analogues in making
`highly active ITS, we tested the biological activity of gelonin
`modified with different linkers in the cell-free system. We
`
`702 1 Journal of Pharmaceutical Sciences
`Vol. 62, No. 7, July 1993
`
`IMMUNOGEN 2081, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`found that the thioimidate ligands did not significantly
`reduce gelonin toxicity until 0.6-1.2 groups were introduced
`into the protein. AMPT was the molecule of choice because
`gelonin toxicity was completely maintained (0.8-1.2 mol of
`ligandlmol of protein) whereas SCDP and SATA caused a loss
`of activity (Figure 2). As expected, by further linking the Mab
`to the toxin, the activity of gelonin in a cell-free system was
`strongly reduced for all the tested ITS (Table I). However, by
`addition of 2-mercaptoethanol, gelonin toxicity was com-
`pletely restored only in the case of ITS prepared with thioim-
`idate reagents.
`It is clear that thioimidate heterobifunctional linkers are
`indeed the best of the tested reagents for protein derivatiza-
`tion because they did not affect the biological activities of such
`proteins. This has been already found for 2-iminothio-
`lane.2os33.34 Among them, AMPT was superior to 2-IT (2) in
`maintaining gelonin activity; this also demonstrated the
`superiority of the acetylthio-type reagent over disulfide-type
`for thiolation of proteins.31.36
`Finally, the antitumor activity of the differently linked
`gelonin-AR3 immunoconjugates was compared by incubat-
`ing the IT with the target HT-29 human colon carcinoma cell
`lines and checking the res ective inhibition of protein syn-
`thesis in the presence of [ Hlleucine. Highest activity was
`shown by the IT made with AMPT-CDPT [50% inhibitory
`concentration
`0.2 nM]. This was followed by the IT
`constructed with CDPT-CDPT (IC50, 0.3 nM), and the lowest
`activity was shown by the SATA-SCDP IT (IC50, 13 nM). In
`contrast, free gelonin or gelonin modified with linkers had
`slight toxicity (Figure 3). To test the specificity of the IT, more
`experiments were performed with MeWo melanoma cells that
`do not carry target CAR-3 antigen. All the conjugates were
`much less effective on MeWo cells than on the targeted HT-29
`cell-lines (data not shown). Among the molecules tested, the
`gelonin-IT linked via the AMPT-CDPT thioimidate reagents
`showed the highest antitumoral activity and the best selec-
`tivity, being > 100 times less toxic to control cells compared
`with target cells.
`In conclusion, as can be confirmed by other studies, the
`choice of the most suitable heterobifunctional reagent as well
`as of the best coupling procedure seems of fundamental
`importance for achieving both a good conjugation method and
`for obtaining ITS with the highest activity and specificity.
`Different factors may be involved in the superior protein
`conjugating quality of thioimidate reagents AMPT and
`
`"Ol
`
`Y 0
`
`I
`2
`dsrlvatlzatla7 degree
`
`3
`
`4
`
`Flgure 2-Residual gelonin toxicity after derivatization in cell-free
`system. Key: (0) AMPT (0) SATA; (A) SCDP; (*) CDPT; (x) 2-IT.
`
`Table I-lnhibttlon of Proteln Synthesis in Cell-Free System by
`Dlfferently Llnked ITS'
`Coupling Procedure
`A: CDPT-AMPT
`B: CDPT-CDPT
`C: SCDPSATA
`lCm free gelonin, 20 pM.
`addition of 2-mercaptoethanol.
`
`ICm, PMb
`ICm, pMC
`20 2 9
`220 2 15
`230 2 20
`25 2 5
`120 f 20
`450 2 20
`Tested on intact ITS. Tested after
`
`~
`
`I101
`
`B
`
`c
`
`m
`
`Y
`
`I -12
`
`-10
`-8
`Cexp) Molar concentration
`
`-6
`
`Figure 3-Antitumor activity of gelonin (0), immunotoxin 9 (SCDP-
`SATA, A), immunotoxin 8 (CDPT-CDPT, o), and immunotoxin 7
`(CDPT-AMPT, x) against target cells.
`
`CDPT. One of these is certainly the ability of thioimidate
`linkers to amidinate proteins (instead of creating an amidic
`bond as SPDP does), thus preserving a positive charge on the
`molecule.2033 A second factor could be the highest specificity
`of thioimidates to react with the lysyl groups of proteins.34
`These conditions and the greater polarity connected with the
`positive charge born by the thioimidate ester linkers could
`make the reaction of the linker with the external lysine group
`more selective without causing alteration of the tertiary
`structure of the protein. It is also possible, as recently
`suggested by Thorpe,26 that ITS provided with an amidinium
`bond could be more resistant than those containing an amide
`bond (such as SPDP) to the enzymes capable of splitting the
`linkage. Clearly, a more extensive investigation is needed to
`elucidate the mechanism by which AMPT-CDPT-linked ITS
`possess the highest activity in inhibiting protein synthesis in
`human carcinoma cell lines.
`References and Notes
`1. Vitetta, E. S.; Uhr, J. W . Ann. Rev. Zmmunol. 1985,3, 197-212.
`2. Rybak, S. M.; Youle, R. J. Immunology Allergy Clinics North
`America 1991,11,359-378.
`3. Frankel, A. E. Zmmunotoxins; Ixluwer Academic: Norwell, MA,
`1988.
`4. Fitzgerald, D.; Pastan, I. J. Natl. Cancer Znst. 1989,81,1455-1463.
`5. Blatter, W. A.; Lambert, J. M.; Goldmacher, V. S. Cancer Cells
`1989, 1, 50-55.
`6. Ehrlich, P. In The Collected Papers of Paul Ehrlich; Himmel-
`weite, F.; Marquardt, M.; Dale, H., Eds.; Pergamon: London and
`New York, 1956; pp 596-618.
`7. Blakey, D. C.; Thorpe, P. E. Antibody, Zmmunoconjugates, Ra-
`diopharmaceuticals 1988,1, 1-16.
`8. Barbieri, L.; Stirpe, F. Cancer Suruqs 1982,1,489-520.
`9. Stirpe, F.; Barbieri, L. FEBS Lett. 1986,195, 1-8.
`10. Stirpe, F.; Olsnes, S.; Pihl, A. J. Biol. Chem. 1980, 255, 6947-
`6953.
`
`Journal of Pharmaceutical Sciences I 703
`Vol. 82, No. 7, July 1993
`
`IMMUNOGEN 2081, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`11. Srinivasan, Y.; Ramprasad, M. P.; Surolia, A. FEBS Lett. 1985,
`192,113-118.
`12. Singh, V.; Sairam, M. R. Biochem. J. 1989,263,417-423.
`13. Hirota, N.; Ueda, M.; Ozawa, S.; Abe, 0.; Shimizu, N. Cancer Res.
`1989,49, 7106-7109.
`14. Thorpe, P. E.; Brown, A. N. F.; Ross, W. C. J.; Cumber, A. J.;
`Detre, S. 1.; Edwards, D. C.; Davies, A. J. S.; Stirpe, F. Eur. J.
`Biochem. 1981,116,447454.
`15. Lambert, J. M.; Senter, P. D.; Yau-Young, A.; Blatter, W. A.;
`Goldmacher. V. S. J. Biol. Chem. 1985.260. 12035-12041.
`16. Colombatti, M.; Nabholz, M.; Gros, 0.; Bron, C. J. Zmmunol. 1983,
`131,30914095.
`17. Sivam, G.; Pearson, J. W.; Bohn, W.; Oldham, R. K.; Sadoff, J. C.;
`Morgan, A. C. Cancer Res. 1987,47,3169-3173.
`18. Ozawa, S.; Ueda, M.; Ando, N.; Abe, 0.; Minoshima, S.; Shimizu,
`N. Znt. J. Cancer 1989,43, 152-157.
`19. Carlsson. J.: Drevin. H.: Axen. R. Biochem. J. 1978.173.723-737.
`20. Traut, R.’ R.’; Bollen; A.f Sun, T.; Hershey, J. W. B.’; Sundberg, J.;
`Pierce, L. R. Biochemistry 1973,12,3266-3273.
`21. Ebert, R. F.; Spryn, L. A. Bioconjugate Chem. 1990,1,331336.
`22. Cumber, A. J.; Henry, R. V.; Parnell, G. D.; Wawrzynczak, E. J.
`J. Zmmunol. Meth. 1990, 135, 15-24.
`F.; Barbieri, L.; Tazzari, P. L.; Dinota, A.; Gobbi, M. Eur.
`24. Bolo esi, A.; Barbieri, L.; Abbondanza, A.; Fdasca, A. I.; Car-
`niceeD.; Battelli, M. G.; Stirpe, F. Biochim. Biophys. Acta 1990,
`1087,293302.
`25. Myers, D. E.; Irvin, J. D.; Smith, R. S.; Kuebelbeck, V. M.;
`Uckun, F. M. J. Immunol. Meth. 1991, 136, 221-238.
`
`23- EP ~ m a t o l . Suppl. 1989,43, 173-175.
`
`26.
`Thorpe, P. E.; Blankey, D. C.; Brown, A. N. F.; Knowles, P. P.;
`Kn ba, R. E.; Wallace, P. M.; Watson, G. J.; Wawrzynczak, E. J.
`J. Jatl. Cancer Znst. 1987, 79, 1101-1111.
`27.
`Delprino, L.; Giacomotti, M.; Dosio, F.; Brusa, P.; Ceruti, M.;
`Grosa, G.; Cattel, L. J. Pharm. Sci. 1993,82,506-512.
`Prat, M.; Morra, I.; Bussolati, G.; Comoglio, P. M. Cancer Res.
`28.
`1985,45,579!3-5807.
`Fraker, P. J.; Speck, J. C. Jr. Biochem. Bwphys. Res. Commun.
`29.
`1978,80,849-857.
`Duncan, R. J. S.; Weston, P. D.; Wrigglesworth, R. Anal. Bio-
`30.
`chem. 1983.132. 6%73.
`Cattel, L.; Delpnno, L.; Brusa, P.; Dosio, F.; Comoglio, P. M.;
`31.
`Prat, M. Cancer Zmmunol. Zmmunother. 1988,27,233-240.
`Laemmli, U. K. Nature 1970,227,680-685.
`32.
`Jue, R.; Lambert, J. M.; Pierce, L. R.; Traut, R. R. Biochemistry
`33.
`1978,17,5399-5406.
`34.
`Thumm, M.; Hoenes, J.; Pfleiderer, G. Biochim. Biophys. Acta
`1987,923,263-267.
`Dosio, F.; Cogliati, T.; Canevari, S.; Mezzanzanica, D.; Brusa, P.;
`35.
`Delprino, L:; Colnaghi, M. I.; Cattel, L. Antibody, Zmmumon-
`jugate, Radtopharmaceuticals 1989,2, 101-115.
`
`Acknowledgments
`helpful. We thank Dr. M.
`Dr. P. Cirina’s cooperation was ve
`Mariani for the gift of AR-3 Mab and%. C. Bonino for the protein
`radiolabeling (both from Sorin Biomedica, S a k e , Italy). Thls work
`was supported by MURST 40%-60%, by Assoclazione Italiana per la
`Ricerca sul Cancro, and by CNR (9101677PF70) grants.
`
`704 I Journal of Pharmaceutical Sciences
`Vol. 82, No. 7, July 1993
`
`IMMUNOGEN 2081, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676