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`
`/. Biochem. 91, 1583-1591 (1982)
`
`Importance of the Antigen-Binding Valency and the Nature of the
`
`Cross-Linking Bond in Ricin A-Chain Conjugates with Antibody
`
`Yasuhiko MASUHO, Kazuo KISHIDA, Masahiko SAITO,
`Naoji UMEMOTO, and Takeshi HARA
`Teijin Institute for Bio-medical Research, Asahigaoka, Hino, Tokyo 191
`
`Received for publication, October 20, 1981
`
`As a continuation of our work on toxin A-chain conjugates with antitumor anti-
`bodies for selective delivery of the toxin to the target cells, four ricin A-chain conju-
`gates were prepared by linking A-chain to Fab' or F(ab')2 of rabbit IgG against
`L1210 with or without employing a cross-linking agent, iVjiV'-o-phenylenedimaleimide
`(PDM), iV-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or N-succinimidyl
`m-(7V-maleimido)benzoate (SMB), and the effects of antigen-binding valency and of
`the nature of the cross-linking bond on their in vitro cytotoxicity were studied.
`The relative potencies of the conjugates in terms of IC84's were as follows: F(ab')2-
`SPDP-A-chain, 100; Fab'-S-S-A-chain, 21; F(ab')2-SMB-A-chain, 1.3; Fab'-
`PDM-A-chain 0.38. Among the four conjugates, F(ab')2-SPDP-A-chain and Fab'-
`S-S-A-chain can be cleaved into the homing and the cytotoxic components with
`2mM 2-mercaptoethanol. These results suggest that divalency in antigen-binding
`and susceptibility of the cross-linking bond to cleavage by mercapto reagent are
`desirable for high potency. Protein synthesis in a cell-free system of rabbit reticu-
`locyte lysate was inhibited by Fab'-S-S-A-chain and by Fab'-PDM-A-chain as
`effectively as by free A-chain, indicating that the liberation of A-chain is not im-
`portant, at least on ribosomes, but it is important for the A-chain to reach a ribo-
`some after binding of the conjugates to the cell-surface.
`
`approach to such agents there has been a con-
`Since most of the present cytotoxic antitumor
`siderable amount of research on conjugates of
`agents show unwanted side-effects due to their
`cytotoxic agents with antitumor antibodies (1).
`detrimental action on normal cells, it is highly
`desirable in cancer chemotherapy to develop anti- The importance of this type of approach has been
`increased owing to the recent rapid advancement
`tumor agents with improved selectivity. As an
`of the hybridoma method as a means of preparing
`Abbreviations: 2-ME, 2-mercaptoethanol; EDTA,
`a n t l b o d i es «•
`ethylenediaminetetraacetate; PDM, iV.JV'-o-phenylene-
`Previously we prepared a conjugate by coupl-
`dimaleimide; PBS, 10mM phosphate buffer-0.14M NaCI,
`i n8 ^ ^^ of r i c i n. t he intracellularly active en-
`PH7.2; SDS-PAGE, sodium dodecyl sulfate-polyacryl-
`amide gel electrophoresis; SMB, iV-succinimidyl m-(N- zymc p o r t i on of t he p i a nt toxin, with the Fab'
`maleimido)benzoate; SPDP, iV-succinimidyl 3-(2-pyridyl-
`fragment of immunoglobulin. The conjugate thus
`dithio)propionate.
`prepared exhibited a potent in vitro cytotoxicity
`
`Vol. 91, No. 5, 1982
`
`1583
`
`IMMUNOGEN 2091 pg. 1
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`1584
`
`Y. MASUHO, K. KISHIDA, M. SAITO, N. UMEMOTO, and T. HARA
`
`against the target cells through its binding to the
`cell-surface antigens via the Fab' portion of the
`conjugate (3). Several other research groups have
`also studied the ricin A-chain conjugates with
`several antibodies including monoclonal antibodies
`prepared by the hybridoma method' (4-7). The
`cytotoxicity of these conjugates against the target
`cells, however, are in general much smaller than
`that of ricin.
`It is extremely desirable to make a
`more potent antitumor conjugate because of the
`general paucity of the target antigens on the tumor
`cell surface. Therefore, in the present study, we
`have prepared four types of ricin A-chain con-
`jugates with antibody having different linkages for
`combining the two components of the conjugate
`and different antigen-binding valencies, and we
`examined the effect of these variables on the cyto-
`toxic activity.
`
`MATERIALS AND METHODS
`
`Materials—Ricinus communis beans were pur-
`chased from Kinokuniya Pharmacy, Tokyo; N,N'-
`o-phenylenedimaleimide
`(PDM)
`from Nakarai
`Chemicals, Ltd. Kyoto; iV-succinimidyl 3-(2-pyri-
`dyldithio)propionate (SPDP) from Pharmacia Fine
`Chemicals AB, Uppsala, Sweden; 2-mercapto-
`ethanol (2-ME) from Nakarai Chemicals, Ltd.;
`culture medium RPMI1640 from Nissui Seiyaku
`Co., Tokyo; kanamycin sulfate from Banyu Phar-
`maceutical Co., Tokyo; fetal calf serum from
`Grand Island Biological Co., Grand Island, N.Y.;
`ATP, GTP, creatine phosphate, and creatine ki-
`nase from Boehringer Mannheim GmbH, Mann-
`heim; hemin from Sigma Chemical Co., Saint
`Louis, Mo.; and L-[4,5- 3H]leucine (60Ci/mmol)
`from the Radiochemical Centre, Amersham. N-
`Succinimidyl /w-(;V~-maleimido)benzoate (SMB) was
`synthesized by employing a published method (8)
`with some modification.
`LI210 Cells— Murine leukemia LI210 cells
`were kindly provided by Dr. T. Kataoka, Cancer
`Chemotherapy Center, Japanese Foundation for
`Cancer Research, Tokyo, and maintained serially
`by intraperitoneal passage of 1 x 10s cells in a
`DBA/2 mouse (purchased from Charles River
`Japan, Atsugi) at weekly intervals. For the cyto-
`toxicity tests, five days after inoculation, ascites
`L1210 cells were collected in 10 mM phosphate
`buffer-0.14M NaCl, pH 7.2 (PBS) and washed
`
`three times with the culture medium before use.
`The F(ab')% and Fab' Fragments and Ricin A-
`Chain—These components of the conjugates were
`prepared as previously described (3). Briefly, anti-
`serum against L1210 cells was produced in rabbits
`by four weekly subcutaneous injections of 5 x 106
`cells emulsified
`in complete Freund's adjuvant.
`The F(ab')2 fragment obtained by pepsin digestion
`of the IgG fraction of the antiserum was reduced
`with the minimal amount (2 mM) of 2-ME to give
`Fab' resulting from the cleavage of only the inter-
`heavy-chain disulfide bond. Ricin was extracted
`from Ricinus communis beans, and its A-chain was
`isolated after treatment with 2-ME (9).
`.
`The Fab'-S-S-A-Chain Conjugate—-This type
`of conjugate was prepared as previously described
`(3) by the activation of the sulfhydryl group of
`Fab'-SH with Ellman's reagent followed by the
`reaction of ricin A-chain with Fab' having the
`3-carboxy-4-nitrophenylthio group introduced.
`The Fab'-PDM-A-Chain Conjugate—A mix-
`ture of 2.7 mg of PDM and 5 ml of 5 mM Na
`acetate buffer-0.14M NaCl-1 mM ethylenediamine-
`tetraacetate (EDTA), pH 5.5 (buffer A) was vigor-
`ously stirred for 5 min and centrifuged. A 1.75 ml
`aliquot of the supernatant (saturated PDM solution)
`was added to 1.75 ml of Fab'-SH in buffer A (3.9 mg
`protein/ml), and the reaction was allowed to proceed
`at room temperature for 30 min. The excess PDM
`was removed by gel filtration on Sephadex G-25
`(0.8 x 43 cm) in buffer A to give Fab'-PDM. To
`2.3 ml of a Fab'-PDM solution (2.1 mg protein/ml)
`thus obtained were added 0.7 ml of ricin A-chain in
`buffer A (7.0 mg protein/ml) and 0.3 ml of 0.3 M Na
`phosphate buffer, pH 6.5, and the reaction was
`allowed to proceed at 4°C overnight. The mixture
`was chromatographed on Sephadex G-150 super-
`fine (1.6x93 cm) in 0.9% NaCl to give the pure
`conjugate Fab'-PDM-A-chain.
`The F(ab')2-SPDP-A-Chain Conjugate—Fifty
`/A of 12 mM SPDP (70) in ethanol was added to
`2.0 ml of F(ab')2 in 0.1 M Na phosphate buffer-
`0.1 M NaCl, pH 7.5 (5.0 mg protein/ml), and the
`mixture was incubated at 26°C for 30 min. The
`excess SPDP and TV-hydroxysuccinimide generated
`were removed by gel filtration on Sephadex G-25
`(0.8 x 43 cm) in buffer A to give F(abO2-SPDP.
`The average number of 3-(2-pyridyldithio)propionyl
`groups introduced into one molecule of F(ab')2 was
`determined to be 3.0 by reducing the product with
`
`IMMUNOGEN 2091 pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
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`
`RICIN A-CHAIN CONJUGATES WITH ANTIBODY
`
`1585
`
`2-ME, followed by measurement of the absorbance
`at 343 nm due to free pyridine-2-thione generated
`(11). A mixture of 1.9 ml of F(ab')2-SPDP in buffer
`A (3.1 mg protein/ml), 2.3 ml of ricin A-chain in
`buffer A (1.8 mg protein/ml), and 0.41 ml of 0.4 M
`Na phosphate buffer-lOmM EDTA, pH7.5 was
`incubated at 26°C for 4 h and chromatographed on
`Sephadex G-150 superfine (1.6x93 cm) in 0.9%
`NaCl to give the desired conjugate.
`
`The F(ab')2-SMB-A-Chain Conjugate—To 1.0
`ml of F(ab')2 in 0.1 M Na phosphate buffer, pH
`7.0 (5.2 mg protein/ml) was added 50/*l of a
`solution of SMB in TV.iV-dimethylformamide (7.0
`mg/ml), and the mixture was incubated at 26°C
`for 30min. The excess SMB and A^-hydroxy-
`succinimide generated were removed by gel filtra-
`tion on Sephadex G-25 (0.8 x 43 cm) in buffer A
`to give F(ab')2-SMB. Two ml of F(ab')2-SMB in
`buffer A (2.2 mg protein/ml) and 2.3 ml of ricin
`A-chain
`in buffer A (1.8 mg protein/ml) were
`mixed, and the reaction was allowed to proceed
`at room temperature overnight. After removal
`of a small amount of precipitate by centrifugation,
`the mixture was chromatographed on Sephadex
`G-150 superfine (1.6 x 93 cm) in 0.9% NaCl to give
`the desired conjugate.
`
`Sodium Dodecyl Sulfate-Polyacrylamide Gel
`(SDS-PAGE)—The
`conjugates
`Electrophoresis
`were analyzed by SDS-PAGE carried out by the
`method of Weber and Osborn (12) before and
`after reduction with 2-ME.
`In the latter case,
`0.1 ml of a conjugate in 0.9% NaCl (0.5-1.0 mg
`protein/ml) was treated with 10 n\ of 0.3 M Tris-HCl
`buffer, pH8.3, containing 20 mM 2-ME at 37°C for
`1 h and then with 10 fi\ of 0.2 M iodoacetamide in
`water at room temperature for 30 min.
`
`Cytotoxicity Assay—Serially diluted conju-
`gates were added to RPMI1640 medium (0.2 ml)
`containing L1210 cells (2xlOVml), 10% fetal
`calf serum, 20 /IM 2-ME, and kanamycin sulfate
`(0.1 mg/ml), and after incubation in a humidified
`atmosphere of 5% CO2 in air at 37°C for 48 h,
`the number of viable cells was determined by
`counting undyed cells after the addition of one-
`tenth volume of 3% trypan blue in PBS. The
`cytotoxicity of the conjugates is expressed in terms
`of the molar concentration necessary to decrease
`the number of viable cells after 48-h incubation
`from 1.6xlO5/ml (control) to 1 x 104/ml, desig-
`nated as IC94.
`
`Inhibition of Protein Synthesis in a Cell-Free
`System by Ricin A-Chain—Rabbit reticulocyte ly-
`sate used for the test was prepared according to
`the method of Pelham and Jackson (13) and stored
`at -80°C. Serially diluted conjugates and other
`test samples were added to 50 pi of the assay
`mixture containing the above lysate (20 /<!), A^-(2-
`hydroxyethyl)piperazine-iV'-2-ethanesulfonic
`acid
`(15 mM, pH7.6), CH3COOK (80 mM), Mg(CH3-
`COO)2 (1 mM), ATP (1 mM), GTP (0.4 mM), cre-
`atine phosphate (8 mM), creatine kinase from rabbit
`L-[4,5-3H]-
`muscle (0.15 mg/ml), hemin (20 m),
`leucine (0.25 fid) and cold amino acid mixture
`without leucine (3 /UM). After incubation at 37°C
`for 10 min, 40 /A of each assay mixture was placed
`on a paper disk (Whatman 3 MM, 2.5 cm), and
`the radioactivity of the fraction insoluble in hot
`trichloroacetic acid was determined on a liquid
`scintillation counter (Packard 3255) according to
`the method of Igarashi et al. (14).
`
`RESULTS
`
`Preparation of Conjugates—Two conjugates
`which are both monovalent
`in antigen-binding
`valency but differ from each other in the cross-
`linking bond were prepared (Fig. la, b). The
`ricin A-chain conjugate with Fab' coupled by a
`disulfide bond, Fab'-S-S-A-chain, was prepared
`by employing Ellman's reagent for the activation
`of the sulfhydryl group of Fab'-SH.
`In order to
`prepare the conjugate cross-linked by a sulfide
`bond, Fab' was allowed to react with excess PDM
`to give Fab'-PDM resulting from the addition of
`the sulfhydryl group of Fab' to only one of the two
`maleimide groups of the cross-linking agent. Then,
`Fab'-PDM was treated with ricin A-chain. Fig-
`ure 2a shows the elution pattern on Sephadex
`G-150 superfine chromatography of the resulting
`reaction mixture. The pure conjugate
`(Fab'-
`PDM-A-chain) was prepared efficiently by this
`method, without formation of the homodimer.
`
`Two other conjugates having the divalent
`antigen-binding site were prepared employing
`F(ab')2 as the homing component to be coupled
`with ricin A-chain with SPDP or SMB. The
`N-hydroxysuccinimide ester group, an active ester,
`of the coupling agents reacts efficiently with the
`lysine residue of F(abO2 to form the amide bond.
`The sulfhydryl group of ricin A-chain reacts with
`
`IMMUNOGEN 2091 pg. 3
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`IPR2014-00676
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`
`Y. MASUHO, K. KISHIDA, M. SAITO, N. UMEMOTO, and T. HARA
`
`MONOVALENT CONJUGATES
`
`DIVALENT CONJUGATES
`
`a
`
`Fab'—S-S-A-CHAIN
`
`Flab'lz-SPDP-A-CHAIN
`
`I A-CH/
`
`IH-CO-IChU-S-S-t
`
`Fab^PDM-A-CHAIN
`
`F(ab'l2—SMB-A-CHAIN
`
`In the cases of the divalent
`Fig. 1. Structures of ricin A-chain conjugates with antibody.
`conjugates, they contain F(ab')2-(A-chain), («=2 or 3) in addition to the major product
`FCab'MA-chain)!.
`In the figure, however, only the structure of FCab'MA-chain), is
`shown cis Si representative in C3.cn csse.
`
`30
`
`40
`
`50
`
`60
`
`30
`
`40
`
`A-CHAIN
`
`, /\
`50
`
`,
`60
`
`Flab'1,
`
`A . C H A ,
`
`30
`
`40
`
`50
`
`60
`
`FRACTION NO.
`
`Fig. 2. Elution profile on Sephadex G-150 superfine chromatography of coupling reaction mixtures
`of Fab' or F(ab'), and ricin A-chain. a, Fab'-PDM-A-chain; b, F(ab') 2-SPDP-A-chain; c, F(ab')2-
`SMB-A-chain. Arrows indicate the elution positions of uncoupled components. The fractions indicated
`by the shaded area were pooled and employed as the conjugates.
`
`the second functional group of the coupling agents
`remaining in F(ab')2-SPDP (the active disulfide
`group) and F(ab')2-SMB (the maleimide group) to
`form the disulfide bond (Fig. 1c) and the sulfide
`bond (Fig. Id), respectively. These reaction mix-
`tures gave products whose molecular weights were
`larger than that of F(ab')2 (Fig. 2b, c). The pro-
`teins of the shaded area in Fig. 2a-c were used for
`the subsequent studies.
`Analysis by SDS-PAGE—The molecular
`weights of the conjugates and the susceptibility of
`the cross-linking bond to reductive cleavage were
`determined by SDS-PAGE. The conjugates Fab'-
`
`PDM-A-chain and Fab'-S-S-A-chain both mi-
`grated to the position of molecular weight 76,000
`daltons, which is close to the sum of the molec-
`ular weights of Fab' (46,000 daltons) and A-chain
`(32,000 daltons), supporting the view that the
`conjugates were composed of one molecule of
`Fab' and one molecule of A-chain (Fig. 3, disks
`3 and 4). These conjugates and intact ricin were
`treated with 2-ME (final 2 and 20 HIM). Although
`Fab'-S-S-A-chain was completely cleaved to Fab'
`and A-chain even at the lower (2 ITIM) concentra-
`tion of 2-ME (Fig. 3, disk 7), Fab'-PDM-A-chain
`and ricin remained intact at this concentration
`
`IMMUNOGEN 2091 pg. 4
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`RICIN A-CHAIN CONJUGATES WITH ANTIBODY
`
`Fig. 3. Analysis of monovalent conjugates by SDS-
`PAGE. Disk 1, Fab'; disk 2, ricin A-chain; disk 3,
`Fab'-PDM-A-chain; disk 4, Fab'-S-S-A-chain; disk
`5, ricin; disk 6, Fab'-PDM-A-chain treated succes-
`sively with 2mM 2-ME and iodoacetamide; disk 7,
`Fab'-S-S-A-chain treated successively with 2 niM 2-ME
`and iodoacetamide; disk 8, ricin treated successively
`
`PDM-A-chain treated successively with 20 mM 2-ME
`and iodoacetamide; disk 10, Fab'-S-S-A-chain treated
`successively with 20 mM 2-ME and iodoacetamide;
`disk 11, ricin treated successively with 20 mM 2-ME and
`iodoacetamide.
`
`12 3 4 5 6 7
`
`The conjugates F(ab')2-SPDP-A-chain and
`F(ab'),-SMB-A-chain gave four bands (Fig. 4,
`disks 4 and 5). Their molecular weights as deter-
`mined from the respective migration positions sug-
`gested that the proteins detected separately were
`the F(ab'), molecule having zero, one, two, and
`three molecules of A-chain; F(ab')2, F(ab')2-(A-
`chain)1; F(ab')2-(A-chain)2, and F(ab')2-(A-chain)3.
`The major conjugate produced was F(ab')2-(A-
`chain)!, whichever of the two coupling agents,
`SPDP and SMB, was employed. When these
`conjugates were treated with 2-ME (final 2 mM),
`F(ab')2-SPDP-A-chain was cleaved to Fab' and
`A-chain (Fig. 4, disk 6), but F(ab')2-SMB-A-chain
`was cleaved to Fab' and Fab'-SMB-A-chain, and
`no free A-chain was detected (Fig. 4, disk 7).
`Cytotoxicity of the Conjugates—The target
`cell cytotoxicity was examined by determining the
`cell viability of L1210 cells cultured with the
`conjugates. Figure 5a shows the cytotoxicity of
`the conjugates having the monovalent carrier moi-
`ety, Fab'. Although an equimolar mixture of
`Fab' and A-chain did not affect the cell growth,
`Fab'-PDM-A-chain decreased the viability. How-
`ever, the concentration required to suppress the
`number of the viable cells to 107ml at the assay
`time (IC94) was 3.9X10- 7M, considerably larger
`(7.2X10" 9M) of Fab'-S-S-A-chain.
`than
`that
`Therefore, for the manifestation of cytotoxicity,
`the disulfide bond is better than the sulfide bond
`for coupling the homing and toxic components of
`the conjugates. The 1C94 of ricin was found to
`be 3.3 x 10-11 M, and the cytotoxicity of Fab'-S-S-
`A-chain was 1/200 of the highly potent cytotoxicity
`
`Fig. 4. Analysis of divalent conjugates by SDS-PAGE.
`Disk 1, F(ab')2; disk 2, Fab'; disk 3, ricin A-chain;
`disk 4, F(ab')2-SPDP-A-chain; disk 5, F(ab')2-SMB-A-
`chain; disk 6, F(ab')2-SPDP-A-chain treated succes-
`sively with 2ir>M 2-ME and iodoacetamide; disk 7,
`F(ab')2-SMB-A-chain treated successively with 2mM
`2-ME and iodoacetamide.
`
`(Fig. 3, disks 6 and 8). Fab'-PDM-A-chain did
`not give free A-chain even at the higher (20 mM)
`concentration of 2-ME, though the disulfide bond
`between the L and H chains was cleaved to give a
`considerable amount of H-chain fragment-PDM-
`A-chain (Fig. 3, disk 9). Ricin, at this concentra-
`tion, was considerably cleaved at its disulfide bond
`giving rise to free A-chain (Fig. 3, disk 11).
`
`Next, the cytotoxicities of the divalent con-
`jugates were compared with each other (Fig. 5b).
`The ICM's of F(ab')2-SPDP-A-chain, F(ab')2-
`SMB-A-chain, and an equimolar mixture of
`F(ab')2 and A-chain were 1.5x10-', 1.2 xlO"7,
`and > 10-«M, respectively. Here again, the SPDP
`conjugate from which A-chain could be reductively
`liberated with 2-ME exhibited a greater cytotoxic-
`ity than the SMB conjugate. The divalent conju-
`gates F(ab')2-SPDP-A-chain and F(ab')2-SMB-A-
`chain were five and three times more cytotoxic than
`the corresponding monovalent conjugates Fab'-S-
`S-A-chain and Fab'-PDM-A-chain, respectively.
`Protein Synthesis Inhibitory Activity in a Cell-
`Free System—Fab'-S-S-A-chain and Fab'-PDM-
`
`Vol. 91, No. 5, 1982
`
`IMMUNOGEN 2091 pg. 5
`Phigenix v. Immunogen
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`
`Y. MASUHO, K. KISHIDA, M. SAITO, N. UMEMOTO, and T. HARA
`
`PROTEIN (Ml
`Fig. 5. Cytotoxicity of the conjugates. L1210 cells (2x10* cells/ml) were incubated at 37°C for
`48 h with ricin, serially diluted conjugates, or an equimolar mixture of Fab' or F(ab')2 and A-chain,
`c,
`y
`d h
`b
`f ibl
`ll
`re determined by the trypan blue dye exclusion method, a, monova-
`and the numbers of viable cells were
`(a) D, ricin; O, Fab'-S-S-A-chain; •, Fab'-PDM-A-chain;
`lent conjugates; b, divalent conjugate
`xture of Fab and Achain. (b) A, F(ab')2-SPDP-A-chain; A , F(ab')2-SMB-A-chain;
`X, equimolar mixture of Fab' and A-c
`ixture of F(ab')2 and A-chain.
`X, equimolar
`
`A-chain were as active as free A-chain in inhibit-
`ing the protein synthesis of rabbit reticulocyte
`lysate (Fig. 6). Therefore, A-chain can inhibit
`protein synthesis even when it is bound to the Four types of conjugates were prepared by coupl-
`carrier antibody moiety.
`ing the A-chain of ricin with the Fab' or F(ab')2
`fragment of anti-murine
`leukemia LI210 IgG
`with or without a cross-linking agent PDM,
`SPDP, or SMB. The bonds which couple the
`
`IMMUNOGEN 2091 pg. 6
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`RICIN A-CHAIN CONJUGATES WITH ANTIBODY
`
`S 5
`
`10°
`
`103
`I EQUIVALENT Ipg)
`RICIN A-CHAIN
`Fig. 6. Inhibition of protein synthesis in a cell-free system. Rabbit reticulocyte lysate
`synthesis
`was used as the cell-free protein synthesis system. The lysate in an assay mixture con-
`taining L-[4,5-3H]leucine was incubated at 37°C for 10 min with serially diluted conjugates
`or ricin A-chain, and the incorporated L-[4,5-3H]leucine was determined. O, Fab'-S-S-
`A-chain ; •, Fab'-PDM-A-chain; •, ricin A-chain.
`
`104
`
`homing and the toxic components of the conju-
`gates are disulfide, sulfide, and/or amide bond(s).
`Among these bonds, only the disulfide bond can
`be reductively cleaved with 2-ME. Therefore, the
`characteristics which differentiate the conjugates
`from each other are the antigen-binding valency
`and the susceptibility to reductive liberation of
`ricin A-chain from the conjugate; Fab'-S-S-A-
`chain (monovalent, susceptible), Fab'-PDM-A-
`chain (monovalent, insusceptible), F(ab')2-SPDP-
`A-chain (divalent, susceptible), F(ab')2-SMB-A-
`chain (divalent, insusceptible).
`The cytotoxicities of Fab'-PDM-A-chain and
`F(ab')2-SMB-A-chain were 1/54 and 1/80 of those
`of Fab'-S-S-A-chain and F(ab')2-SPDP-A-chain,
`respectively. This result, together with the sus-
`ceptibility of the respective cross-linking bond to
`reductive cleavage with 2-ME, suggests the im-
`portance of the liberation of A-chain from the
`conjugate for the manifestation of cytotoxicity.
`Oda and Funatsu (75) observed that the cytotox-
`icity of ricin decreased to 1/10 of the original upon
`cleaving the disulfide bond connecting the A- and
`B-chains followed by cross-linking of the two
`chains with PDM. This is compatible with our
`results summarized above.
`
`Gill and Pappenheimer (16) showed that the
`enzyme activity of the A moiety responsible for
`the cytotoxicity of diphtheria toxin was, when
`linked to the B moiety, less than 1/1,000 of that
`in the free state. In the case of ricin, whose A-
`chain is known to terminate protein synthesis by
`catalytically inactivating ribosomes (77), Olsnes
`and Pihl (18) showed that the activity of the A
`moiety, when bound to the B moiety, was lowered
`to several tenths of its activity in the free state.
`These results led us to examine the possibility that
`the activity of ricin A-chain is blocked in Fab'-
`PDM-A-chain, a conjugate insusceptible to the
`liberation of A-chain with a mercapto reagent, by
`being coupled with Fab'. Measurement of the
`enzyme activity of ricin A-chain employing a cell-
`free, protein synthesis system of a lysate from
`rabbit reticulocytes indicated that Fab'-PDM-A-
`chain exhibited almost the same magnitude of
`enzyme activity as that of free A-chain. There-
`fore, the lower cytotoxicity of Fab'-PDM-A-chain
`compared to that of Fab'-S-S-A-chain is not due
`to any blocking effect of the bound Fab' moiety
`on the enzyme activity of A-chain.
`In addition,
`Fab'-S-S-A-chain was as active as free A-chain.
`These observations that A-chain is active even
`
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`Y. MASUHO, K. KISHIDA, M. SAITO, N. UMEMOTO, and T. HARA
`
`when bound to Fab', though it is almost inactive
`when bound to B-chain in native ricin may be
`because in the conjugates, due to the flexibility of
`the cross-linking bonds, the active site of A-chain
`is available for enzyme action to a much greater
`extent than
`in ricin in which B-chain is more
`tightly coupled with A-chain not only by a disulfide
`bond but also by non-covalent bonds. Ricin A-
`chain may have to be liberated from the conjugate
`to reach ribosomes after binding of the conjugate
`to the cell surface, and the liberation of A-chain
`can take place more easily from Fab'-S-S-A-chain
`than from Fab'-PDM-A-chain.
`
`The divalent conjugates F(ab') 2-SPDP-A-
`chain and F(ab') 2-SMB-A-chain were five and
`three times more active than the corresponding
`monovalent
`conjugates Fab'-S-S-A-chain
`and
`Fab'-PDM-A-chain, respectively. Therefore, di-
`valency in antigen-binding is more advantageous
`than monovalency for the conjugate
`to exhibit
`cytotoxicity. This is probably not only because
`the divalent antibody has a stronger affinity for
`the target cell but also because divalent binding
`effects a more efficient internalization of the con-
`jugate into the cell. According to the electron
`microscopic observations of De Petris (19), F(ab') 2
`can be taken into pinocytotic vesicles more effec-
`tively than Fab'.
`
`Among the four conjugates, F(ab') 2-SPDP-A-
`chain showed the highest cytotoxicity. However,
`ricin was forty-five
`times more cytotoxic
`than
`F(ab')2-SPDP-A-chain
`in
`terms of
`the IC M's.
`Ricin B-chain may permit more efficient internali-
`zation of its A-chain than F(ab') 2 of immunoglob-
`ulin. Secondly, most of the cell-surface antigens
`which the anti-L1210 IgG employed for the pre-
`sent study recognizes may be inferior as regards
`endocytic capability.
`It has been shown that there
`is a difference in endocytic potential among cell-
`surface determinants (20). Studies on ricin A-
`chain conjugates prepared from monoclonal anti-
`bodies may clarify the importance of this different
`endocytic potential and afford a more potent con-
`jugate.
`In the present study, the IgG fraction of
`an antiserum was used, and only a small portion
`of this is the antibody. This is another factor
`which diminishes the potency of the conjugate.
`Again, the use of a highly pure monoclonal anti-
`body for the preparation of the conjugate should
`be advantageous. The present study showed that
`
`the bond connecting A-chain with B-chain in ricin
`is considerably more resistant to reductive cleavage
`than the cross-linking bonds in F(abO 2-SPDP-A-
`chain and Fab'-S-S-A-chain. This suggests that
`although ricin is cleaved to liberate its A-chain
`only after its binding to the cell surface or there-
`after, some of
`the F(ab') 2-SPDP-A-chain and
`Fab'-S-S-A-chain molecules might be cleaved to
`the two components outside the cell, decreasing
`the efficiency of entry of A-chain into the cell.
`This is the first report on the effects of anti-
`gen-binding valency of ricin A-chain conjugates
`with an antibody and of the nature of the cross-
`linking bond on their in vitro cytotoxicity.
`In
`summary, the present study has demonstrated an
`advantage of employing F(ab') 2 rather than Fab'
`as the homing component of the ricin A-chain
`conjugates; the conjugates with F(ab') 2 were more
`potent than the corresponding Fab' conjugates.
`Comparison of the cytotoxicities of the differently
`linked conjugates revealed that the disulfide-linked
`conjugates from which A-chain could be liberated
`by a mercapto reagent showed potent cytotoxicity,
`suggesting the importance of the liberation of A-
`chain for the manifestation of cytotoxicity. How-
`ever, since Fab'-PDM-A-chain inhibited cell-free
`protein synthesis as effectively as free A-chain, the
`liberation of A-chain is not important, at least on
`ribosomes, but it is important for the A-chain to
`reach a ribosome after binding of the conjugates
`to the cell-surface.
`It should be noted that al-
`though in ricin, which is much more cytotoxic
`than the disulfide-linked conjugates, the receptor-
`binding portion is also linked to A-chain via a
`disulfide bond, the bond is less easily cleaved by
`a mercapto reagent. Further studies on
`ricin
`A-chain conjugates employing hybridoma-prepared
`monoclonal antitumor antibodies should be useful
`to explore the potential of the strategy of selectively
`homing toxin A-chain to the target cancer cells.
`
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`IMMUNOGEN 2091 pg. 9
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