272
`
`BBA 21171
`
`Biochimica et Biophysica Acta, 717 (1982) 272w277
`Elsevier Biomedical Press
`
`A COIVIPARISON OF THE IN VITRO AND IN VIVO ACTIVITIES OF CONJUGATES OF
`ANTI-MOUSE LYMPHOCYTE GLOBULIN AND ABRIN
`
`D.C. EDWARDS ‘, W.C.J. ROSS, AJ. CUMBER, D. MCINTOSH, A. SMITH, P.E. Tl-IORPE, A. BROWN,
`R.I-I. WILLIAMS and A.J.S. DAVIES
`
`Divisions of Biology and Chemistry, Institute of Cancer Research, Chester Beatty Research Institute, Fulham Road, London SW3 6JB
`(UK)
`
`(Received October 28th, 1981)
`(Revised manuscript received March 29th, 1982)
`
`Key words: Antibody-toxin conjugate; Abrin; Immunoglobulin; Immunosuppression; (Mouse lymphocyte)
`
`immunoglobulin have been conjugated to ahrin using two
`Anti-mouse lymphocyte globulin and normal
`procedures, one involving linkage through an amide bond and a piperazine ring and the other the introduction
`of two amide bonds flanking a disulphide bridge. The four conjugates produced were equipotent as inhibitors
`of protein synthesis in rabbit reticulocyte lysates. Each antibody-containing conjugate was a more effective
`inhibitor of protein synthesis in cultured cells than the equivalent normal
`iinmunoglohulin-containing
`conjugate. In addition the conjugates with disulphide linkage groups were ten times more potent than their
`counterparts. The disulphide conjugates were also twice as toxic to mice in an acute toxicity test but when
`used to suppress their immune responses to sheep red blood cells it was the non-disulphide-liriked conjugates
`that were superior. In all instances antibody-containing conjugates were more powerful imm m
`than those containing normal IgG. The results are taken to indicate a relative lack of stability of the
`disulphide conjugates in the tissues.
`
`Introduction
`
`Recently, chemical conjugates of antibodies and
`certain toxins or their component A-chains have
`been employed in attempts to produce a new
`generation of putative chemotherapeutic agents
`[I-6]. For the most part the emphasis has been
`upon the introduction of a disulphide bridge be-
`tween the component parts and for this various
`methods have been devised [7—9]. Studies from our
`laboratory [2,lO] have described the preparation of
`
`* D.C.E. is an external member of the staff of the Wellcomc
`Foundation Ltd.
`ixmnunopurificd horse anti-mouse
`Abbreviations: AMLG,
`lymphocyte globulin; nIgG, normal horse immunoglobulin;
`SPDP, N-suocinimidyl 3-(2-pyridyl-dithio)-propauate; PFC,
`plaque-forming cells.
`
`0304-4165 /82/0000-—O000/$02.75 © 1982 Elscvier Biomedical Press
`
`conjugates by the use of a derivative of chlo-
`rambucil which results in conjugation through a
`bond not susceptible to reduction or to cleavage
`by sulphydryl exchange.
`In the present work the properties of conjugates
`formed by the use of a disulphide bridge have
`been compared with those of conjugates not in-
`volving such a linkage group. Effects on cell-free
`extracts, cells in tissue culture and living animals
`have been measured and susceptibility to cleavage
`by dithiothreitol studied.
`
`Materials and Methods
`
`Immunopurified horse anti-mouse lymphocyte
`globulin (AMLG), normal horse immunoglobulin
`(nIgG) and abrin were obtained as described pre-
`viously [11,12].
`
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`Disulphide bridge conjugates. N-Succinimidyl 3-
`(2-pyridyl-dithio)-propionate (SPDP) was ob-
`tained from Pharmacia (U.K.) Ltd., Hounslow,
`Middlesex and used in accordance with the makers
`
`to prepare AMLG-S-S-abrin and
`instructions
`nIgG-S-S-abrin which were isolated by chromatog-
`raphy on Sephacryl 300 and analyzed as previ-
`ously described [10]. The general structure and
`abrin to globulin ratios are given in Table I.
`Chlorambucil
`(CB 1348) bridge
`conjugates.
`AMLG-1348-abrin and nIgG-1348-abrin were pre-
`pared, isolated and analyzed as described previ-
`ously [10]. The structures and abrin to globulin
`ratios are given in Table I.
`Electrophoresis in sodium dodecyl sulphate-com
`raining gel. Samples of protein were made up in a
`solution of 2% (w/v) SDS, 80 mM Tris-HCl, pH
`6.8, 10% (w/v) glycerol and 0.002% (w/v) brorno—
`phenol blue. A second set of samples were pre-
`pared in the same solvent with the addition of 2.5
`mM dithiothreitol. All samples were heated at
`60°C for 15 min prior to electrophoresis which
`was carried out as described by Laemmli [13]. A
`3% stacking gel and a 10% running gel were used
`and the gel was stained with Coomassie brilliant
`blue and destained in an aqueous mixture of 10%
`(w/v) methanol and 10% (v/v) acetic acid.
`Tests in vitro and in viva. Details of the cell—free
`protein synthesis inhibition assay, of [3H]leucine
`uptake inhibition in tissue culture, acute toxicity
`(LD50) in mice and of suppression of the immune
`response of the mouse to an injection of sheep red
`blood cells have all been given elsewhere [10,12,14].
`Serum haernagglutinating titres were measured
`by the method described by Takatsky [15]. The
`
`TABLE I
`
`273
`
`values shown are the means of the reciprocals of
`the dilutions. of serum used.
`
`Results
`
`Analysis of SDS-polyacrylamide gel electrophore-
`sis before and after treatment with dithiothreitol.
`The abrin: IgG ratios shown in Tablel support the
`view that the major constituent molecular species
`is a conjugate with immunoglobulin and abrin in a
`1:1 molecular combination and the presumptive
`structures of the conjugates are shown.
`The different sensitivities of the two types of
`conjugate to reduction by 2.5 mM dithiothreitol
`are illustrated in Fig. 1. Lanes 1_——4 show the con-
`jugates, their constituent globulin and abrin to be
`relatively stable to SDS in the absence of reducing
`agent. Lanes 5-8 show the materials run on the
`same gel but after pretreatment with the reducing
`agent. Comparison of lanes 1 and 5 shows nIgG-S-
`S-abrin to be largely dissociated by dithiothreitol
`giving products corresponding to those given by
`reduced abrin and globulin (lanes 7 and 8). nIgG-
`1348-abrin (lanes 2 and 6) is relatively unaffected
`giving only small amounts of breakdown products
`which correspond to those from free abrin (lane 7).
`Comparison with lanes 4 and 8 which demonstrate
`the breakdown of immunoglobulin by the reducing
`agent suggests that in the nIgG-1348-abrin con-
`jugate the immunoglobulin is stabilized presuma-
`bly by the formation of internal cross-links formed
`when the chloro—ethy1 groups are activated.
`Inhibition of protein synthesis in rabbit reticula-
`cyte lysate. The four conjugates were used as in-
`hibitors of protein synthesis in a lysate of rabbit
`
`STRUCTURES AND ANALYSES OF CONJUGATES OF IMMUNOGLOBULINS AND TOXINS PRODUCED BY TWO
`DIFFERENT METHODS
`
`
`Conjugation
`method
`
`
`Structural formula
`
`Conjugate
`
`Ratio ab rin/IgG
`
`Chlorambucil
`[10]
`
`/j
`IgG-NHCO(Cl-I2)3 N-abrin
`
`AMLG-1348-abrin
`
`nIgG-1348-abrin
`AMLG- S-S-abrin
`
`0.81
`
`0.83
`0.84
`
`SPDP [8]
`
`IgG-NHC0(Cl-12)2—S-S-(CH2):-CONH-abrin
`
`0.78
`n1gG-S-S-abrin
`
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`
`274
`
`Fig. 1. Elecrophoresis of abrin, nlgG, nIgG-S-S-abrin and
`nIgG—l348~abrin in gels containing SDS with and without
`dithiothreitol. Lanes 1 and 5, 7.7- 10'” mols nIgG-S-S-abrin;
`2 and 6, 7.6- 10-“ mols nIgG-1348-abrin; 3 and 7, 6.5-10'”
`mols abrin; 4 and 8, 5.6- 10' “ mols nIgG. Lanes 5-8 were run
`in the presence of 2.5 mM dithiothreitol.
`
`reticulocytes and the results shown in Fig. 2 were
`obtained. From the figure it is clear that the levels
`of inhibition are indistinguishable from each other
`indicating A-chain activity to be identical in each
`preparation. Thus, differences in A-chain activity
`can be eliminated as a source of any other varia-
`tion in biological effect of the conjugates.
`Cytotoxicity in tissue culture. Both AMLG—S-S-
`abrin and AMLG-1348-abrin were about ten times
`more effective at inhibiting protein synthesis in
`mouse spleen lymphocytes in tissue culture than
`the corresponding conjugates with normal
`im-
`munoglobulin (Fig. 3). The superiority of the anti-
`body—based conjugates on this occasion exceeded
`
`(cmx1033
`[3911-teucineincorporationintoprotein
`
`
`
`1.4
`0.14
`Concentration of eonjugnu (M x 1040)
`
`14
`
`Fig.2. The effect of antibody-abrin conjugates on cel1~free
`protein synthesis. 0, AMLG-l348—abrin; E1, nIgG-1348-abrin;
`O. AMLG—S—S-abrin; I, nIgG-S-Snabrin.
`
`that reported previously [12]. The two disulphide
`linked conjugates were each about twice as cyto-
`toxic as
`the corresponding non-reducible con-
`jugate.
`
`113)
`
`50
`
`o
`
`
`
`
`
`t3H1—Iuucineuptdza(%o¢eormoi)
`
`1.sx1o"3
`
`1.5x1o“'
`
`1.ax1o'l°
`Cononnuttion (M)
`
`1.sx1e°
`
`1.5x10‘3
`
`Fig. 3. The effect of antibody-abrin conjugates on protein
`synthesis of mouse spleen cells in tissue culture stimulated with
`
`concanavalin A. O '
`O, AMLG-1348-abrin; D
`D,
`
`nIgG-1348-abxin; O
`O, AMLG-S-S-abrin; I
`I,
`nIgG-S-S-abrin.
`
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`
`TABLE I1
`
`EFFECT OF CONJUGATES ON THE ABILITY OF MICE TO RESPOND IMMUNOLOGICALLY TO AN INJECTION
`(INTRAPERITONEAL) OF SHEEP RED BLOOD CELLS
`
`A total of 5108 sheep red blood cells were injected in all treatments except for the blank control.
`
`
`Treatment
`(intraperitoneal)
`
`Dose
`(mol X
`10' ”)
`
`PFC/ 105 spleen cells
`
`PFC/spleen
`
`Geo-
`metric
`mean
`
`x S.D.
`+
`
`Geo-
`metric
`mean
`
`X S.D.
`-I~
`
`P
`
`Serum
`haemag-
`glutinating
`titre.
`
`275
`
`(Control) none
`None
`AMLG-1348-abrin
`
`nlgG-1348-abrin
`
`AMLG-S-Svabrin
`
`nIgG-S-S-abrin
`
`(x 102)
`
`66
`1.29
`O
`l 905
`1.17
`2.23
`<0.001
`646
`1.26
`74
`<0.001
`<0.001
`468
`1.44
`23
`<0.001
`<0.001
`398
`1.48
`18
`<0.001
`N.S.
`1 148
`1.58
`223
`N.S.
`<0.001
`617
`1.55
`97
`<0.0l
`<0.001
`851
`1.20
`74
`<0.001
`<0.05
`1380
`1.23
`111
`n.s.
`<0.001
`912
`1.05
`111
`n.s.
`<0.001
`759
`1.45
`97
`n.s.
`n.s.
`1288
`1.86
`169
`n.s.
`1.36
`1 279
`3.8
`n.s.
`1 380
`1.45
`215
`<0.05
`1.19
`1 186
`7.5
`n.s.
`1047
`1.99
`128
`n.s.
`1.33
`1685
`15.0
`
`
`0
`0
`3.8
`7.5
`15.0
`
`3.8
`7.5
`15.0
`3.8
`7.5
`15.0
`
`79
`1 561
`669
`617
`449
`
`1 158
`681
`819
`1 429
`1 479
`1 549
`
`1.39
`1.07
`1.18
`1.39
`1.46
`
`1.35
`1.47
`1.10
`1.23
`1.29
`1.12
`
`Acute toxicity to mice. AMLG—l348-abrin and
`nlgG-1348-abrin were equipotent, the LD5(, values
`being 77 - 10"” and 80 « 10"” mol, respectively.
`AMLG-S-S-abrin and nIgG—S-S-abrin were also
`equipotent but with LDSO values of 35 - IO‘‘3 and
`40 - 10"?’ mol, respectively, were twice as toxic to
`mice as the chlorambucil-linked materials.
`
`Immunosuppressioe activities. Table II shows the
`effects of the conjugates on the immune response
`of the mouse to an injection of sheep erythrocytes.
`It is clear from the number of plaque-forming cells
`(PFC) produced per 10“ spleen cells that AMLG-
`1348-abrin has reduced the immune response and
`that it is approximately twice as effective as nIgG-
`1348-abrin. AMLG—S-S-abrin and NIgG-S-S-abrin
`on the other hand are without discernable effect.
`
`To take into account changes in the sizes of the
`spleens of the mice results are also expressed on a
`PFC per total spleen cells number basis. Again the
`superiority of AMLG-1348—abrin over nIgG-
`1348-abrin is demonstrated but now in addition
`
`AMLG-S-S-abrin but not nIgG-S-S—abrin is seen
`to be immunosuppressive. The potency of AMLG-
`S-S-abrin appears to be of similar magnitude to
`
`that of nlgG-1348-abrin and is clearly less than
`that of AMLG-1348-abrin. Confirmation of these
`
`findings is given by the circulating anti-sheep
`erythrocyte antibody titres.
`
`Discussion
`
`linkages between immunoglobulins
`Covalent
`and the toxic protein, abrin, have been introduced
`by two methods. In the first the two proteins were
`joined through the formation of an amide and
`probably a piperazine ring (Table I). The second
`method involved the introduction of a 3-3’-di-
`
`thiobis(propionyl) group linking an amino group
`on each protein. In both procedures the attach-
`ment
`to the protein is thought
`to be through
`c-amino groups of lysine residues. The main dis-
`tinguishing feature between the two types of lin-
`kage is the susceptibility of the disulphide bridge
`to reduction and sulphydryl exchange. This is
`clearly illustrated by the break-down of the con-
`jugate occasioned by the use of dithiothreitol.
`Toxins like abrin owe their ability to kill cells to
`an inhibitory effect of their constituent A—cha.ins
`
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`
`276
`
`[16]. In the
`on protein synthesis in the cytosol
`present experiments, A-chain activities were in-
`distuinguishable in all four conjugates, suggesting
`that
`the linkages between the immunoglobulins
`and the toxin are similarly distributed between the
`A- and B-chains. The superior cytotoxicity of the
`disulphide-linked conjugates in vitro could be ex-
`plained by the fact
`that both antibody-S-S-A-
`chain ~ B-chain and antibody-S-S-B-chain - A-chain
`would be able to release A-chain or a derivative
`will only a small
`increase in molecular weight
`within the cell, whereas only the antibody-1348-
`B-chain-A chain derivatives would do so. This
`would also probably account
`for the increased
`acute toxicity of the disulphide~linked conjugates
`in vivo.
`
`The relative failure of AMLG-S-S—abrin as an
`immunosuppressive agent may reflect a suscept-
`ibility to reduction or sulphydryl exchange in the
`tissues. Albumin in particular is known to exist
`with a proportion of sulphydryl groups and would
`be available for exchange, as could other sulphydryl
`containing molecules [17]. The possibility of un-
`protected disulphide bridges exchanging with other
`—S-S- compounds such as the widely distributed
`glutathione must also be recognised. nIgG-S-S-
`abrin may likewise be subject to dissociation in the
`body fluids. This may explain its inefficacy rela-
`tive to nIgG—1348-abrin which would retain its
`binding capacity for cells via its abrin B-chain.
`In previous studies abrin has been shown to be
`a potent immunosuppressive agent in its own right
`[10,18] and it is important to note that any abrin
`released by reduction of the —S-S- bond would be a
`derivative of the form abrin-(HN-CO-(CH2);
`SH)” where n = l or 2. This substitution number
`may be critical since in a study in which abrin was
`reacted with succinic anhydride [19] to give three
`succinyl groups per abrin molecule, the product,
`although retaining 80% of the A-chain activity of
`the original toxin, was 13% as toxic for mice. As
`with SPDP the reaction involved the formation of
`amide bonds.
`Since abrin itself comprises two polypeptide
`chains linked by a single disulphide bridge we
`must also enquire why it too does not dissociate in
`plasma. In a study on the recombination of puri-
`fied A- and B—chains it has been claimed that the
`preferrred combination is A-S-S-B and that the
`
`toxicity of the product suggests that virtually all
`the chains reassociate in this fashion [20]. If this is
`so it argues strongly for a preferrred orientation
`probably independent of the sulphydryl groups.
`This view is reinforced by a study on a closely
`related plant toxin, ricin in which it was shown
`that the molecule remained intact even when the
`disulphide bridge was fully reduced [21]. Also,
`native protein required
`50-fold more
`mercaptoethanol for reduction of the bond than
`did denatured ricin. Thus, the evidence seems to
`favour the view that the disulphide bridge in ricin
`is protected within the molecule and from the
`‘postulated homologies, abrin may be presumed to
`be similar. Artificially introduced reducible bonds
`would be unlikely to benefit from such stabiliza-
`tion, either through favourable orientation or fold-
`ing within the molecule, or both, and this is be-
`lieved to be reflected in the results presented in
`this paper. It
`is concluded that
`in all cases in
`which disulphide bridges are used to form new
`molecular pairings it will be important to establish
`their stability in vivo.
`
`References
`
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`
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