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`Int. Archs Allergy appl. Immun. 48: 505-512 (1975)
`
`Effect of Substitution on Reactivity of B 512 Dextran Fractions
`with Anti-B 512 Dextran in Heterologous Passive Cutaneous
`Anaphylaxis
`
`W olfgang R ichter
`
`Research Division, Pharmacia AB, Uppsala
`
`Abstract. The influence of type and degree of substitution of B 512 dextran frac­
`tions on reactivity with rabbit antibodies against unmodified B 512 dextran was
`studied in heterologous passive cutaneous anaphylaxis in guinea pigs. Low degrees
`of substitution, i.e. 1 substituent group per 7-30 glucose residues, affected reactivity
`with anti-dextran only slightly or not at all. This was shown for the following sub­
`stituents: sulphate, carboxymethyl, phosphate, diethylaminoethyl, hydroxypropyl,
`butyryl, caprylyl, stearoyl, and fluoresceinoyl groups. With high degress of substitu­
`tion, i.e. 1-3 substituents per 2 glucose residues, reactivity with anti-B-512-dextran
`is completely abolished for some substituents, and a new immunological identity
`conferred on the substituted dextran. Strongly charged groups like sulphate, carbox­
`ymethyl and diethylaminoethyl abolish reactivity with anti-B-512-dextran at rela­
`tively lower degrees of substitution than more neutral groups like methyl and acetyl.
`The anti-B-512-dextran represents a specific reagent for a-l,6-linked polyglucose, as
`evidenced by complete cross-reactivity with synthetic linear dextran; its specificity is
`emphasized by non-reactivity with «-1,6-linked synthetic mannan, the monomeric
`residues of the two polymers differing only in position of the C2 hydroxyl groups.
`
`Introduction
`
`Regarding the effect of chemical modification on the immunochemical
`properties of polysaccharides and proteins, the following statement, made
`by Rabat and Mayer [1961], still appears valid: ‘Chemical modification
`of polysaccharides has been used to a much more limited extent in immu-
`nochemistry than chemical modification of proteins.’ In the studies re-
`
`Received: October 15, 1974.
`
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`ported here, one single aspect of chemical modification of polysacchar­
`ides will be considered, namely its influence on the binding to homolo­
`gous antibody, raised against the unmodified polysaccharide.
`Heterologous passive cutaneous anaphylaxis (PCA) was used as a sen­
`sitive in vivo system, to study the reactivity of variously substituted dex-
`tran B 512 fractions with high-titered rabbit anti-dextran antisera, raised
`by immunization with conjugates between B512 dextran and protein.
`Different substituents were introduced, affecting both the charge and the
`balance between hydrophilic and lipophilic properties of the modified
`dextran molecule. The degree of substitution was also varied, in order to
`elucidate minimal requirements for disturbing the complementarity be­
`tween the antigenic determinants of dextran and the anti-dextran combin­
`ing site.
`
`Materials and Methods
`
`Antidextrans. Rabbit anti-dextran antiserum ODX-3 was raised by 4-weekly in­
`tramuscular injections of 0.5 mg of ovalbumin B512 dextran conjugate, emulsified
`in complete Freund’s adjuvant. ODX-3 was harvested 19 weeks after starting immu­
`nization; specific anti-dextran content: 1.3 mg/ml, estimated by reversed single radi­
`al immunodiffusion [Richter, and Kagedal, 1972],
`Unsubstituted polysaccharides. A dextran fraction of average molecular weight
`(mw) 70,000 was obtained by ethanol precipitation from partially hydrolysed native
`dextran produced from sucrose by the Leuconostoc mesenteroides NRRL B 512
`strain. This fraction was used as a reference for comparison with the variously sub­
`stituted B 512 dextran fractions.
`A fraction (D-8) of a completely linear synthetic dextran, with m„ about 50,000,
`was prepared by Uryu and Schuerch [1971].
`A fraction (M-3) of a completely linear synlhetic mannan, with M„- about
`100,000, was prepared by F rechet and Schuerch [1969].
`Substituted dextrans. Syntheses were performed at Pharmacia’s department of
`organic chemistry according to standard procedures. Only B 512 dextran fractions
`of Klw 60,000-261,000 were used for substitution experiments. M„ were kindly de­
`termined by Dr. K. G ranath, using either light scattering or gel chromatography.
`Dextran sulphates [Gronwall et al., 1945]; carboxymethyl dextran (US Pat. 2,
`997, 423, 1961); diethylaminoethyl dextran [McKernan and Ricketts, I960]; meth­
`yl dextran [N orrman, 1968]; acetyl dextran [D e Bf.lder and N orrman, 1968]. hy-
`droxypropyl dextran (Swed. Pat. 221, 926, 1963); stearoyl dextran [Hammerling,
`and W estphal, 1967].
`Butyryl dextran, caprylyl dextran, and benzoyl dextran: these esters of dextran
`were prepared by dropwise addition of the corresponding acid chloride (or anhy­
`dride) to an alkaline solution of the dextran (5-10%>). The reaction mixture was
`thereafter neutralized and the dextran ester recovered by precipitation in ethanol.
`
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`Fluoresceinoyl dextran and dextran phosphate were kindly donated by Dr. A. N. De
`Belder.
`Passive cutaneous anaphylaxis. PCA was used as described by Ovary [1958],
`with minor modifications [Richter, 1970, 1972], Eight intradermal sites of abdomi­
`nal skin were sensitized in each guinea pig, choosing anti-dextran antiserum dilu­
`tions, giving maximal sensitization (0.1 ml, containing 0.3-0.6 /,<g specific anti-dex-
`tran, per site), i.e. PCA lesions of 15-20 mm in diameter on challenge with 0.015
`mg/kg of the reference B 512 dextran fraction, M„ 70,000.
`Reactivity of each substituted dextran was compared to that of the reference
`dextran in two groups of 3-4 equally sensitized guinea pigs. One group was chal­
`lenged with 0.15 mg/kg of the reference dextran, and the other with 0.15 mg/kg of
`the substituted dextran. After sacrificing the animals, the size of the PCA lesions
`was measured on the reflected skin, and the average area of the PCA lesions pro­
`duced by the substituted dextran was expressed as percentage of that produced by
`the reference dextran.
`
`Results
`
`The specificity of the anti-dextran antiserum is demonstrated by com­
`plete cross-reactivity with the synthetic linear dextran fraction D-8, which
`was compared with a dextran B 512 fraction of similar molecular weight
`(table I). The anti-dextran thus represents a specific reagent for «-1,6-
`linked polyglucose. On the other hand, the specificity of the anti-dextran
`is emphasized by the fact that it did not recognize a completely linear
`synthetic «-1,6-linked mannan, the difference between dextran and man-
`nan monomeric residues consisting only in deviating configuration of the
`C, hydroxyl group.
`It is seen from table I that a low degree of substitution (0.03-0.15),
`corresponding to 1 substituent group per 30-7 glucose residues, does
`not affect reactivity with the anti-dextran to a larger extent, as indicated
`by the size of PCA lesions. This is the case for the following substituents:
`sulphate, carboxymethyl, phosphate, diethylaminoethyl, hydroxypropyl,
`butyryl, caprylyl, stearoyl, and fluoresceinoyl.
`When the degree of substitution becomes higher (0.4-1.6), corre­
`sponding to about 1-3 substituents per 2 glucose residues, reactivity with
`the anti-dextran is either completely abolished or inhibited to a varying
`extent, depending on the nature of the substituent. Complete loss of or
`greatly reduced reactivity is illustrated by the examples of highly substi­
`tuted dextran sulphate, carboxymethyl dextran, diethylaminoethyl dex­
`tran, and methyl dextran.
`
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`Table I. Effect of degree and type of substitution on the capability of substituted dextran
`fractions to elicit PCA on intravenous challenge (0.15 mg/kg) in guinea pigs, maximally
`sensitized with rabbit anti-B-512-dextran
`
`Name of substituted
`dextran fraction
`
`m„/1,000 Degree of Number of blue
`substitu- spots
`tion (max.
`= 3.00)
`
`Relative PCA
`reactivity cal-
`culated from
`spot area;
`dextran
`M,v 70,000=100
`
`Dextran sulphate
`Dextran sulphate
`Dextran sulphate
`Carboxymethyl dextran
`Carboxymethyl dextran
`Dextran phosphate
`Diethylaminoethyl dextran
`Diethylamincethyl dextran
`Methyl dextran
`Methyl dextran
`Acetyl dextran
`Hydroxypropyl dextran
`Butyryl dextran
`Butyryl dextran
`Caprylyl dextran
`Benzoyl dextran
`Stearoyl dextran
`Fluoresceinoyl dextran
`Dextran B 512
`Synthetic linear dextranb
`a-l,6-hnked synthetic
`linear mannan
`
`69
`90
`195
`107
`100
`60
`64
`261
`65
`74
`74
`68
`72
`117
`103
`102
`80
`70
`70
`50
`
`circa 100
`
`0.08
`0.51
`1.06
`0.15
`0.50
`0.03
`0.13
`0.52
`0.60
`1.60
`0.60
`0.18
`0.07
`0.41
`0.05
`0.18
`0.09
`0.06
`0.00
`0.00
`
`0.00
`
`24
`24
`24
`32
`24
`24
`32
`32
`32
`32
`32
`24
`32
`24
`32
`56
`32
`24
`616(309 ± 54)“
`32
`
`32
`
`100
`71
`0
`79
`0
`90
`89
`25
`112
`17
`100
`81
`122
`82
`112
`147
`82
`80
`100
`100
`
`0
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`a Figures in parentheses show average spot area (diameter in mm)2, given as mean ±
`standard deviation in 22 groups of 24-32 spots each, from the reference dextran data.
`b Compared with B 512 dextran. mw 42,000 [data from Richter, 1974).
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`Discussion
`
`The extensive hapten inhibition studies of Kabat [1954, 1957, 1960]
`and Mage [1963] indicate that the combining site of anti-dextrans is com­
`plementary to 3, 4, 5, or 6 a-l,6-linked glucose residues. Independent ev­
`idence for this size of the combining site was obtained by PCA experi­
`ments [R ichter, 1972], showing that the smallest dextran fragment en­
`dowed with elicitor action in guinea pigs, maximally sensitized with rabbit
`anti-dextran, was isomaltodecaose, with mw 1,600. In accordance with
`the widely accepted bridging hypothesis for elicitor molecules, requiring
`at least 2 determinants per 1 antigen molecule, the results with isomalto­
`decaose suggest that 1 determinant comprises 3-5 glucose units. From
`these findings one would predict that reactivity with anti-dextran should
`be undisturbed, as long as a certain number of such determinants on a
`large dextran molecule remain unsubstituted. Generally, experimental
`findings are in agreement with this view.
`The question of antigen dosage used for challenge must also be consid­
`ered. As shown earlier [R ichter, 1970], very small antigen doses are re­
`quired for eliciting a minimal PCA response in guinea pigs maximally
`sensitized with rabbit anti-dextran. For example, challenge with B 512
`dextran, mw 70,000, at a dose of 1.5 ng/kg produced PCA lesions in 50%
`of sensitized sites, with mean lesion diameters of about 11 mm; 15 /ugjkg
`and larger doses produced lesions in 100% of sensitized sites, with mean
`diameters of 15-17 mm. Thus, the dose of 150 /ig/kg employed in the ex­
`periments reported here, represents at least 100 times the minimal effec­
`tive dose. A substituted dextran with completely abolished reactivity at
`150 /ug/kg has thus undergone an activity loss of more than 100 times.
`The dextran fractions and the substituted dextran fractions employed
`in this study had mw of 50,000-261,000. B 512 dextran is composed of
`a-l,6-linked glucose units and contains few and short-side chains, about 1
`per 20 glucose residues. As an antigen, a B 512 dextran molecule of Mw
`about 80,000 may be visualized as a chain of about 100 repeating deter­
`minants, each comprising about 5 glucose units [Richter, 1974].
`Upon introduction of a small number of substitutents, sufficient unmo­
`dified determinants are available for normal binding to anti-dextran.
`When the average degree of substitution is increased, so that approxi­
`mately every second glucose residue carries a substituent group, no or
`very few unmodified determinants are accessible and reactivity with the
`anti-dextran is reduced or abolished, depending upon the size and other
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`properties of the substituent group. Because of the large number of deter­
`minants per dextran molecule (of mw about 70,000), it must be consid­
`ered, however, that even with high degrees of substitution one prerequis­
`ite for non-reactivtiy with anti-dextran should be the uniform distribution
`of substituents along the molecular chain. This may be one factor explain­
`ing the differences observed between dextran sulphates and carboxyme-
`thyl dextran at comparable molecular weights and degrees of substitution
`(0.50). Experimental evidence shows that sulphation of dextran, which is
`carried out in pyridine, results in an uneven distribution of sulphate
`groups [De Belder, personal commun.]. In contrast, substitution with
`carboxymethyl groups is performed in aqueous solution and may favour a
`more uniform distribution of substituent groups.
`A factor which may lead to erroneous results in the present experi­
`ments is binding of the substituted dextran to cell surfaces or free ma­
`cromolecules. The strongly basic diethylaminoethyl dextran (DEAE-dex-
`tran) is known to bind to albumin [McKernan and R icketts, 1960] and
`to cell surfaces [Larsen, and O lsen, 1968]. The observed low reactivity
`of DEAE-dextran (degree of substitution 0.52) with anti-dextran could,
`therefore, be due to the fact that only a small fraction of the dose injected
`reached the sensitized skin site.
`In spite of the relatively small number of different substituents studied
`here, some interesting tendencies can be recognized. The strongly charged
`groups sulphate, carboxymethyl and diethylaminoethyl abolish reactivity
`with anti-dextran to a large extent or completely, when at least every sec­
`ond glucose residue carries a substituent. In the case of complete aboli­
`tion of reactivity, a new immunological identity has been conferred to the
`substituted dextran. The more neutral groups methyl and acetyl did not
`interfere with the recognition of the substituted dextran by the anti-dex­
`tran, when one substituent was present on every second glucose residue.
`Much higher degrees of substitution abolished, however, reactivity with
`anti-dextran completely.
`Abolition of precipitability of methylated pneumococcal polysacchar­
`ide type III (Sill) by rabbit antibody against the unmodified polysacchar­
`ide has been reported earlier [Heidelberger and Kendall, 1935], The
`methylated polysaccharide did not inhibit precipitation of rabbit antibody
`by unmodified Sill, showing that it had acquired a new immunological
`identity.
`Five lipophilic substituents: butyryl, caprylyl, benzoyl, stearoyl and
`fluoresceinoyl decreased reactivity of dextran with anti-dextran very little
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`at low degrees of substitution. On the contrary, reactivity appeared to be
`somewhat increased in comparison to that of unmodified dextran. This ef­
`fect may be explained by accessory binding of lipophilic groups to lipo­
`philic areas of the combining site or adjacent parts of the anti-dextran
`molecule.
`The introduction of a small number of stearoyl groups is sufficient to
`confer new properties to the antigen molecule, such as strong binding to
`red cell membranes. Stearoyl derivatives of polysaccharides have, there­
`fore, become useful reagents for detection of anti-polysaccharide antibod­
`ies by passive haemagglutination [H ämmerling and W estphal, 1967].
`
`A cknowledgements
`
`The technical assistance of Mrs. Solveig Tillman is gratefully acknowledged.
`Thanks are due to Prof. Conrad Schuerch, State University of New York, for gen­
`erous gifts of synthetic dextran and mannan; the syntheses of these polymers were
`supported under research grant GM 06168 of the Division of General Medical Sci­
`ences, National Institutes of Health.
`
`References
`
`D e Belder, A. N. and N orrman, B.: The distribution of substituents in partially
`acetylated dextran. Carbohyd. Chem. 8: 1-6 (1968).
`Frechet, J. and Schuerch, C.: Chemical synthesis and structure proof of a stereo­
`regular
`linear mannan, poly-alpha-(l-6)-anhydrc-D-mannopyranose. J. amer.
`chem. Soc. 91: 1161-1164 (1969).
`G rönwall, A.; Ingelman, B., and M osiman, H.: A dextran sulphuric acid ester
`with heparin activity. Upsala LäkFören. Förh. 50: 397-404 (1945).
`H ämmerling, U. and W estphal, O.: Synthesis and use of O-stearoyl polysacchar­
`ides in passive hemagglutination and hemolysis. Europ. J. Biochem. 1: 46-50
`(1967).
`Heidelberger, M. and Kendall, F. E.: Precipitin reaction between type III pneu­
`mococcus polysaccharide and homologous antibody. Quantitative study and
`theory of reaction mechanism. J. exp. Med. 61: 563-591 (1935).
`R abat, E. A.: Some configurational requirements and dimensions of the combining
`site on an antibody to a naturally occurring antigen. J. amer. chem. Soc. 76:
`3709-3713 (1954).
`R abat, E. A.: Size and heterogeneity of the combining sites on an antibody mole­
`cule. J. cell. comp. Physiol. 50: suppl. 1, pp. 79-102 (1957).
`R abat, E. A.: The upper limit for the size of the human antidextran combining site.
`J. Immunol. 84: 82-85 (1960).
`
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`R abat, E. A. and M ayer, M. M. (ed.): Experimental immunochemistry; 2nd ed.,
`chap. 11 (Thomas, Springfield 1961).
`Larsen, B. and Olsen, K.: Inhibitory effect of polycations on the transplantability
`of mouse leukaemia reversed by heparin. Europ. J. Cancer 4: 157-162 (1968).
`M age, R. G.: Immunochemical studies on dextrans. The specificities of rabbit anti-
`dextrans; diss. Columbia University New York (1963).
`M cKernan, W. M. and Ricketts, C. R.: A basic derivative of dextran and its inter­
`action with serum albumin. Biochem. J. 76: 117 (1960).
`N orrman, B.: Partial methylation of dextran. Acta chem. scand. 22: 1381-1385
`(1968).
`Ovary, Z.: Immediate reactions in the skin of experimental animals provoked by
`antibody-antigen interaction. Progr. Allergy, vol. 5, pp. 459-508 (Karger, Basel
`1958).
`Richter, W.: Absence of immunogenic impurities in clinical dextran tested by pas­
`sive cutaneous anaphylaxis. Int. Arch. Allergy 39: 469-478 (1970).
`Richter, W.: Minimal molecular size of dextran required to elicit heterologous pas­
`sive cutaneous anaphylaxis. Int. Arch. Allergy 43: 252-268 (1972).
`Richter, W.: Cross-reactivity of synthetic linear dextran with anti-B512 dextran.
`Int. Arch. Allergy 46: 438-447 (1974).
`Richter, W. and Kagedal, L.: Preparation of dextran-protein conjugates and stud­
`ies of their immunogenicity. Int. Arch. Allergy 42: 887-904 (1972).
`U ryu, T. and Schuerch, C.: Preparation of high molecular weight 2,3,4-tri-O-ben-
`zyl-(l-6)-alpha-D-gluco- and galactopyranan and (l-6)alpha-D-glucopyranan. Ma­
`cromolecules 4: 342-345 (1971).
`
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`Dr. W olfgang R ichter, Research Division, Pharmacia AB, POB 604, S-75125
`Uppsala (Sweden)
`
`PGR2020-00009
`Pharmacosmos A/S v. American Regent, Inc.
`Petitioner Ex. 1071 - Page 8
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