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Polymerization of individual species of grass pollen allergens
Original
articles
Polymerization of individual pollen allergens Roy Patterson, M.D., lrena M. Suszko, Martha A. Shaughnessy, B.S. Chicago,
species of grass
B.S., Leslie C. Grammer,
M.D., and
Ill.
Six grass pollen allergens have been individually polymerized. We had previously polymerized mi,red grass pollen allergens. However, since every patient does not react to every grass, we sought to polymerize individual grasses that could subsequently be mixed into a preparation based on a patient’s skin reactivity. As demonstrated by Sepharose 4-B chromatography of individual grass monomers and polymers, all six grasses were polymerized. Polymerized grass preparations as compared with monomer preparations demonstrated a IO”- to I @‘-fold reduction in allergenic@ as determined by cutaneous end point titer. That grass polymer contains the great majority of clinically important allergenic determinants was demonstrated by the abiliv oj polymer to inhibit 78% IgE binding against crude grass in a pool of untreated grass-sensitive patients. Its antigenic similarity to crude grasses is further shown by the abiliv of polymer to inhibit 85% of IgG binding against crude grass in a pool of patients treated with usual crude grass extracts. (.I ALLERGY CWN IMMUNOL 72:129-133, 1983.)
We have reported the polymerization of a mixture of grass pollen allergens’ and initial limited therapeutic trial results with such a preparation.’ Polymerization of mixed grass pollen allergens would produce a mixed polymer that might not be appropriate for immunotherapy of all patients with grass pollinosis because there are patients with no sensitivity to some components of the grass polymer mixture. For this reason we attempted to polymerize individual grass pollen allergens that were subsequently used in a double-blind histamine placebo-controlled therapeutic trial.3
MATERIALS AND METHODS Preparation of grass monomers Perennialrye, orchard,timothy, redtop, Bermuda,and Junegrasspollens(20gmeach)wereobtainedfrom Allergy Labs of Ohio, Columbus. Preparationof each grass monomerwasdonein a similarfashionto that previously described,‘,* exceptthat in this study, preparationof each grassmonomerwasdoneseparately.Briefly, grasspollens weredefattedandextractedovernightat 4” C with 0.125M ammoniumbicarbonate.After centrifugation,proteinsin supernatants were precipitated
at a 90% saturation of am-
monium sulfate. Precipitatesobtained by centrifugation were redissolved in isotonic PBS, pH 7.2, and were exten-
sively dialyzed againstthe samebuffer to remove ammonium sulfate. Grass extracts were then chromatographed on a G-15 Sephadexcolumn(PharmaciaFine Chemicals, From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Chicago. Supported by U.S.P.H.S. Allergy Disease Center Grant AI 11403, the Ernest S. Bazley Grant, and Key Pharmaceutical Co. Received for publication Dec. 6, 1982. Accepted for publication Feb. 4, 1983. Reprint requests: Roy Patterson, M.D., Section of AllergyImmunology, Department of Medicine, Northwestern University Medical School, Chicago, IL 60611.
Piscataway,N. J.), andJnly the excludedportionwasused. For eachgrassa separate G-15 Sephadexcolumnwasused and eachcolumnwascharacterizedfor exclusionvolume with dextran blue 2000 before chromatography of the grass extract. At this stage, monomer grasses were concentrated and dialyzed in order to obtain a monomer grass of 3.12 OD at
280nmasdeterminedon a Gilford 250spectrophotometer. Monomerswerekept at -20” C until use. 129
130
J ALLERGY
Patterson et al
Chromatographic Ahhre~~iufiorzs
PBS: OD: PNU:
TABLE
crass-sensitive Grass
and
end
point
polymeric
titers
grass
of individual
allergens*
in a
patient Monomer
Bermuda June Orchard Redtop
, o--Ii , o- IO
Polymer
Difference
10mX IO-”
, (J-2 ,0-C ,o-” ,o-”
10” IO4 lo” IO4
Rye
1 o-”
1 OF
10:’
Timothy
1o-x
, o-3
lo”
*Dilutions of polymer and monomer were made from preparations having an OD of 3.0 at 280 nm.
Preparation
comparisons of monomers
Each of the six monomers was chromatographed individually on the same Sepharose 4-B column. The applied monomer volume was 4 ml and the column was extensively washed after each use. Wash eluates were tested and found to be free of protein before the column was used again.
mrd
Phosphate-buffered saline Optical density Protein nitrogen units
I. Cutaneous
monomeric
CLIN. IMMUNOL. AUGUST 1983
of polymers
Each grass monomer was polymerized separately in a similar fashion to that previously described. ‘, ’ Polymerization was conducted at room temperature for 4 hr in a total volume of 4 ml. Glutaraldehyde (2.5%), supplied in sealed ampules (Allergy Labs of Ohio), was kept frozen (-20” C) until use. For each individual grass polymerization procedure, a fresh ampule was used. Final molarity of gluteraldehyde was 0.4M in each of six polymerizations. One Sepharose 4-B and one Sephadex G-200 column (both from Pharmacia) were prepared and characterized for exclusion volume by dextran blue 2000. Each polymer was chromatographed on the same Sepharose 4-B column, which was extensively washed after each procedure. Only the included portion of the polymer was kept, and glycine was added to prevent further polymerization. Included polymerized grass was then chromatographed on a G-200 Sephadex column and only the excluded portion was kept. The G-200 Sephadex column was again extensively washed after each procedure. All six excluded polymerized grasses were adjusted to a final OD reading of 3.12 at 280 nm, dialyzed against phenol-PBS, and sterilized via Nalgene micropore filter of 0.22 pm. Determination of PNU per milliliter of each polymerized grass was performed at Allergy Labs of Ohio. With sterile phenol-PBS, dilutions were made so that each polymer contained 10,000 PNUlml. Two mixes were prepared: One containing equal portions of all six polymers and another containing equal portions of five polymers. Bermuda polymer was omitted from the five-polymer mix. In each mixture, the final grass polymer preparation contained 10,000 PNU/ml. Allergy Labs of Ohio performed sterility and safety studies on both polymerized grass mixtures.
Comparison of cutaneous end point titration with monomers and polymers Serial lo-fold dilutions of each polymer and each monomer were tested intradermally in a subject with a prior diagnosis of grass pollinosis. Skin tests results were evaluated by standard criteria’ and the lowest dilution producing a negative reaction was considered to be the end point.
Inhibition
of specific IgG binding by polymer
This polystyrene tube competitive inhibition has been described previously.” Briefly, polystyrene tubes were coated overnight at 4” C with a mixture of five crude grass antigens (redtop, timothy, orchard, June, and Bermuda; Allergy Labs of Ohio) at a concentration of 10 mgiml in 0. IM NaHCO,. Then 0.1 ml aliquots of a I : 100 dilution of serum from a patient treated with usual aqueous grass immunotherapy were incubated overnight at 4” C with various amounts (0, 50, 5000, and 500,000 ng) of the six-grass polymer mixture to inhibit binding. As a control, 0.1 ml of serum diluted 1 : 100 from a nonatopic individual was treated in an identical way. These mixtures were then added to the coated tubes and incubated at 4” C overnight and the tubes were washed. One milliliter of rabbit antihuman IgG (Calbiochem-Behring Corp., La Jolla, Calif.) diluted 1: 5 was added to each tube and incubated for 24 hr at 4” C, at which time the tubes were washed and ““I-labeled IgG was added. The tubes were incubated overnight at 4” C, washed, and counted. Control counts were subtracted and percent inhibition was calculated. The assay was also performed on 0.1 ml of undiluted serum from a serum pool from patients treated with usual grass immunotherapy.
Inhibition
of specific IgE binding by polymer
An assay similar to the above IgG assay was performed to demonstrate inhibition of IgE binding by polymers. Serum from a single untreated patient and from a serum pool of untreated patients were used. Undiluted serum (0.1 ml) and 0, 50, 5000, and 500,000 ng of polymer were used. Anti-IgE (Calbiochem-Behring) at a 1 :40 dilution and IZJIIgE were used instead of anti-IgG and ““I-IgG.
RESULTS Chromatographic patterns of markers and orchard grass monomer and polymer preparations Fig. 1, top,
showsthe profile of dextran blue on a column. Molecules of greater than 20,000,OOOdaltons were in the excluded volume of the Sepharose4-B column and those of less than 20,000,OOOdaltons were in the included volume. On Sepharose
4-B
VOLUME NUMBER
72 2
Polymerization
. ?
..
0.. 0 E
DEXTRAN
of grass
pollen
allergens
131
BLUE
GLUTARALDEHYDE
EXCLUDED
1.6
u az
?-
ORCHARD GRASS: +--.- . POLYMER r MONOMER
,/*““;:,,:, c
;
1.
“,
.*
G ;
‘$;,
* . ..*
FRACTION
NUMBER
FIG. 1. Top, Sepharose 4-B pattern of dextran blue and glurataldehyde. Botrom, Monomer and polymer orchard grass profiles. C-I, Fractions used for final polymerized orchard grass; G, glutaraldehyde peak in polymer.
this standardized column of Sepharose 4-B, free glutaraldehyde in the polymer grass preparation appeared late and was the cause of higher absorbance of the polymer preparations at high elution volumes. Fig. 1, bottom, shows a monomeric orchard grass chromatographic pattern and a polymerized orchard grass. The tailing peak contributed by free glutaraldehyde is shown. Fractions used for the final preparations of polymerized orchard grass are shown and do not include fractions of molecular weights higher than 20,000,000 daltons or fractions containing free glutaraldehyde . Fig. 2 illustrates the Sephadex G-200 chromatographic pattern of dextran blue (top) and of the polymerized orchard grass preparations (bottom). The fractions of polymerized orchard grass used therapeutically are also shown. Preparations of polymers of rye, timothy, redtop, June, and Bermuda grasses Figs. 3 and 4 show the Sepharose 4-B chromatographic profiles of monomeric and polymeric preparations of these grasses. In each case there was a formation of polymers, as demonstrated by the higher
h4 FRACTION FIG. 2. Sephadex tran blue (fop) C-i, Fractions preparation.
NUMBER
G-200 chromatographic and polymer orchard of excluded polymer
patterns of dexgrass Ibortoml. used in the final
OD reading at lower elution volumes of polymer. Formation of high-molecular-weight polymers (more than 20,000,OOO daltons) occurred particularly with Bermuda and June grass polymers. The fractions of each polymer preparation used for pooling and subsequent fractionation on the Sephadex G-200 column are shown. The Sephadex G-200 patterns of rye, timothy, redtop, June, and Bermuda grass polymers were like those for orchard grass and are not shown.
Patterson
10’ 8’
et al.
J ALLERGY
t RED TOP
TABLE
GRASS:
II. Inhibition
of specific
by polymerized
binding
. -‘.-‘. POLYMER ,. 0
87* 6. 5.
RYE GRASS:
.""-4
POLYMER
‘--.-i,
MONOMER
0 58 84 100
0 50 5,000 500,000
0 13 31 04
kG
0 50 5,000 500,000
0 0 38 8.5
IgE”
0 50 5,000 500,000
0 0 27 78
4. 3.
Serum
25 10 61 8. 7.
TIMOTHY
G ..*.*.,
GRASS:
.-- -.-+ ,=OLYMER .. MONOMER
pool
6. 5. 4. 3' 2' OF, 12
AMi.xture of equal parts of all 6 grass polymers. BAssay performed with 0. I ml of serum diluted 1 : 100. “Assay performed with 0.1 ml of serum undiluted.
*a -: \,, A.' , , , 18 20
,
, 24
,
, 28
FRACTION
,
, 32
,
, 36
'
4'0
I
I 44
I 'G..I 48
NUMBER
FG. 3. Sepharose 4-B chromatographic patterns of redtop, rye, and timothy monomer and polymer preparations. G, Free glutaraldehyde in polymer; I--I, fractions of included polymer.
Comparison of cutaneous end point titration with monomers and polymers Results of intradermal end point titrations of monomeric and polymeric grasses are shown in Table I. The monomeric preparations were 1000 to 100,000 times more skin-reactive than the polymeric preparations. inhibition
of specific
IgG binding
by polymer
The ability of polymer grass to inhibit binding of specific IgG to crude grass is shown in Table II. At 500,000 ng of polymer added in the single serum assay, inhibition was 100% complete. The IgG binding of the serum pool was 85% inhibited by adding 500,000 ng of polymer. Inhibition
% inhibition
0 50 5,000 500,000
IgE’
0. 'k.. ' z's ' 2'8 ' 3'2 ' 36 ' 4'0 ' 4'4 ' 49
added* hg)
Single serum IgG”
antibody
grass
Polymer Antibody
grass
CLIN IMMUNOL. AUGUST 1983
of specific
IgE binding
by polymer
The ability of polymer grass to inhibit binding of specific IgE to crude grass is shown in Table II. At
500,000 ng of polymer added to the single serum sample, inhibition was 94% complete. In the serumpool sample 78% inhibition was achieved when 500,000 ng of polymer was added. DISCUSSION In preparing polymerized ragweed, monomersof giant and short ragweedwere mixed and polymerized. This was considered appropriate becausealmost all ragweed-sensitivepatients are reactive to both pollen species. Polymerized ragweed extracts have been shown to have reduced allergenicity (elicitation of immediate-type reactions)6and retained immunogenicity and have been shown to be safe and efficacious in many therapeutic trials.‘-I0 All grass-sensitivepatients do not react to all grasses.Bermuda grassis a prime example of a speciesto which all grass-sensitive patients do not react becauseof its geographic location. These studies were done to show that six grassescould be polymerized individually. In subsequent clinical trials the individual grass polymers were selected and mixed according to patients’ immediate-typereactivity to permit a singleinjection. The change in chromatographic pattern on Sepharose 4-B column from monomeric to polymeric demonstratesthat all grassmonomerswere polymerized. The final molecular sieving on the Sephadex G-200
VOLUME NUMBER
72 2
Polymerization
BERMUDA
GRASS:
,'-'-,
POLYMER
:-..o
MONOMER
20
24
28
JUNE ..,--. 3
32
36
40
44
4S
GRASS:
of grass
pollen
allergens
133
Grass polymers of molecular weights in excess of 20,000,OOO daltons were excluded by Sepharose 4-B chromatography and were not added to the final polymerized grass preparations used in the clinical trial. This finding is analogous to that for the initial polymerized ragweed preparations that were characterized and used clinically. It is probable that the high-molecular-weight polymers (>20,000,000 daltons) are as safe and as immunogenic as the lowermolecular-weight polymers (200,000 to 20,000,OOO daltons) used in the ragweed and grass preparations. Thus the excluded peaks, particularly of Bermuda and June grass (Fig. 4), may be useful when added to final preparations.
POLYMER
REFERENCES
0MONOMER
FRACTION
NUMBER
FIG. 4. Sepharose 4-B chromatographic patterns of Bermuda and June monomer and polymer preparations. G, Location of free glutaraldehyde in polymer; H, fractions of included polymer.
column provided polymerized grasses with molecular weights in excess of 200,000 daltons and analogous to the polymerized ragweed that has already been extensively studied.g The reduction in allergenicity of polymer grasses as compared with monomer grasses further supports our results; we have previously shown that allergenicity of the polymer is less than that of the monomer.” The 3- to 5-log reduction in cutaneous end point titer of polymer as compared with monomer is similar to the differential seen in our polymerization of ragweed,12 mixed grass,’ and mixed tree. I3 This reduced allergenicity affords the safety factor that allows high doses of polymer to be administered over a short time perkI lo That polymer contains a large majority of antigenic determinants to which a pool of grass-sensitive patients develop IgE is well demonstrated by the significant inhibition of binding of IgE against crude grasses by polymer. The antigenic completeness of polymer is further demonstrated by its ability to significantly inhibit binding of IgG in a pool of grasssensitive patients treated with usual aqueous immunotherapy.
1. Patterson R, Suszko IM, Pruzansky JJ, Zeiss CR, Metzger J, Roberts M: Polymerization of mixtures of grass allergens. J ALLERGY CLIN IMMUNOL 59314, 1977. 2. Hendrix SG, Patterson R, Zeiss CR, Suszko IM: The immune responses in humans and rabbits to monomeric and polymeric grass allergens. J Clin Immunol 2:10, 1982. 3. Grammer LC, Shaughnessy MA, Suszko IM, Patterson R: A double-blind histamine placebo-controlled trial of polymerized whole grass for immunotherapy of grass allergy. J ALLERGY CLIN IMMUNOL (In press.) 4. Patterson R, editor: Allergic diseases. Diagnosis and management. Philadelphia, 1972, J. B. Lippincott Co. 5. Patterson R, Roberts M, Zeiss CR, Pmzansky JJ: Human antibodies against trimellityl proteins: comparison of specificities of IgG, IgA and IgE classes. Int Arch Allergy Appl Immunol 66:332, 1981. 6. Patterson R: Allergen immunotherapy with modified allergens (Robert A. Cooke Memorial Lecture). J ALLERGY CLIN IMMUNOL6&85, 1981. 7. Bacal E, Zeiss CR, Suszko IM, Levitz D, Patterson R: Polymerized whole ragweed: an improved method of immunotherapy. J ALLERGY CLINIMMUNOL 62:289, 1978. 8. Kelly JF, Zeiss CR, Patterson R, Levitz D, Suszko IM: Polymerized whole ragweed: human safety and immune response. J ALLERGY CLINIMMUNOL 65~50, 1980. 9. Hendrix SG, Patterson R, Zeiss CR, Pruzansky JJ, Suszko IM, McQueen R, Slavin R, Miller M, Lieberman P, Sheffer A: A multi-institutional trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 66:486, 1980. 10. Grammer LC, Zeiss CR, Suszko IM, Shaughnessy MA, Patterson R: A double-blind, placebo controlled trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 69~494, 1982. 11. Patterson R, Suszko IM, Pruzansky JJ, Zeiss CR Polymerized ragweed antigen E. II. In vivo elimination studies and reactivity with IgE antibody systems. J Immunol 110:1413, 1973. 12. Patterson R, Suszko IM, Zeiss CR, Pruzansky JJ, Bacal E: Comparison of immune reactivity to polyvalent monomeric and polymeric ragweed antigens. J ALLERGY CLIN IMMUNOL 61:28, 1978. 13. Patterson R, Suszko IM, Her&ix SG, Zeiss CR Polymerized treepollenantigens.J ALLERGYCLINIMMUNOL~~: 162.1981.
articles
Polymerization of individual pollen allergens Roy Patterson, M.D., lrena M. Suszko, Martha A. Shaughnessy, B.S. Chicago,
species of grass
B.S., Leslie C. Grammer,
M.D., and
Ill.
Six grass pollen allergens have been individually polymerized. We had previously polymerized mi,red grass pollen allergens. However, since every patient does not react to every grass, we sought to polymerize individual grasses that could subsequently be mixed into a preparation based on a patient’s skin reactivity. As demonstrated by Sepharose 4-B chromatography of individual grass monomers and polymers, all six grasses were polymerized. Polymerized grass preparations as compared with monomer preparations demonstrated a IO”- to I @‘-fold reduction in allergenic@ as determined by cutaneous end point titer. That grass polymer contains the great majority of clinically important allergenic determinants was demonstrated by the abiliv oj polymer to inhibit 78% IgE binding against crude grass in a pool of untreated grass-sensitive patients. Its antigenic similarity to crude grasses is further shown by the abiliv of polymer to inhibit 85% of IgG binding against crude grass in a pool of patients treated with usual crude grass extracts. (.I ALLERGY CWN IMMUNOL 72:129-133, 1983.)
We have reported the polymerization of a mixture of grass pollen allergens’ and initial limited therapeutic trial results with such a preparation.’ Polymerization of mixed grass pollen allergens would produce a mixed polymer that might not be appropriate for immunotherapy of all patients with grass pollinosis because there are patients with no sensitivity to some components of the grass polymer mixture. For this reason we attempted to polymerize individual grass pollen allergens that were subsequently used in a double-blind histamine placebo-controlled therapeutic trial.3
MATERIALS AND METHODS Preparation of grass monomers Perennialrye, orchard,timothy, redtop, Bermuda,and Junegrasspollens(20gmeach)wereobtainedfrom Allergy Labs of Ohio, Columbus. Preparationof each grass monomerwasdonein a similarfashionto that previously described,‘,* exceptthat in this study, preparationof each grassmonomerwasdoneseparately.Briefly, grasspollens weredefattedandextractedovernightat 4” C with 0.125M ammoniumbicarbonate.After centrifugation,proteinsin supernatants were precipitated
at a 90% saturation of am-
monium sulfate. Precipitatesobtained by centrifugation were redissolved in isotonic PBS, pH 7.2, and were exten-
sively dialyzed againstthe samebuffer to remove ammonium sulfate. Grass extracts were then chromatographed on a G-15 Sephadexcolumn(PharmaciaFine Chemicals, From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Chicago. Supported by U.S.P.H.S. Allergy Disease Center Grant AI 11403, the Ernest S. Bazley Grant, and Key Pharmaceutical Co. Received for publication Dec. 6, 1982. Accepted for publication Feb. 4, 1983. Reprint requests: Roy Patterson, M.D., Section of AllergyImmunology, Department of Medicine, Northwestern University Medical School, Chicago, IL 60611.
Piscataway,N. J.), andJnly the excludedportionwasused. For eachgrassa separate G-15 Sephadexcolumnwasused and eachcolumnwascharacterizedfor exclusionvolume with dextran blue 2000 before chromatography of the grass extract. At this stage, monomer grasses were concentrated and dialyzed in order to obtain a monomer grass of 3.12 OD at
280nmasdeterminedon a Gilford 250spectrophotometer. Monomerswerekept at -20” C until use. 129
130
J ALLERGY
Patterson et al
Chromatographic Ahhre~~iufiorzs
PBS: OD: PNU:
TABLE
crass-sensitive Grass
and
end
point
polymeric
titers
grass
of individual
allergens*
in a
patient Monomer
Bermuda June Orchard Redtop
, o--Ii , o- IO
Polymer
Difference
10mX IO-”
, (J-2 ,0-C ,o-” ,o-”
10” IO4 lo” IO4
Rye
1 o-”
1 OF
10:’
Timothy
1o-x
, o-3
lo”
*Dilutions of polymer and monomer were made from preparations having an OD of 3.0 at 280 nm.
Preparation
comparisons of monomers
Each of the six monomers was chromatographed individually on the same Sepharose 4-B column. The applied monomer volume was 4 ml and the column was extensively washed after each use. Wash eluates were tested and found to be free of protein before the column was used again.
mrd
Phosphate-buffered saline Optical density Protein nitrogen units
I. Cutaneous
monomeric
CLIN. IMMUNOL. AUGUST 1983
of polymers
Each grass monomer was polymerized separately in a similar fashion to that previously described. ‘, ’ Polymerization was conducted at room temperature for 4 hr in a total volume of 4 ml. Glutaraldehyde (2.5%), supplied in sealed ampules (Allergy Labs of Ohio), was kept frozen (-20” C) until use. For each individual grass polymerization procedure, a fresh ampule was used. Final molarity of gluteraldehyde was 0.4M in each of six polymerizations. One Sepharose 4-B and one Sephadex G-200 column (both from Pharmacia) were prepared and characterized for exclusion volume by dextran blue 2000. Each polymer was chromatographed on the same Sepharose 4-B column, which was extensively washed after each procedure. Only the included portion of the polymer was kept, and glycine was added to prevent further polymerization. Included polymerized grass was then chromatographed on a G-200 Sephadex column and only the excluded portion was kept. The G-200 Sephadex column was again extensively washed after each procedure. All six excluded polymerized grasses were adjusted to a final OD reading of 3.12 at 280 nm, dialyzed against phenol-PBS, and sterilized via Nalgene micropore filter of 0.22 pm. Determination of PNU per milliliter of each polymerized grass was performed at Allergy Labs of Ohio. With sterile phenol-PBS, dilutions were made so that each polymer contained 10,000 PNUlml. Two mixes were prepared: One containing equal portions of all six polymers and another containing equal portions of five polymers. Bermuda polymer was omitted from the five-polymer mix. In each mixture, the final grass polymer preparation contained 10,000 PNU/ml. Allergy Labs of Ohio performed sterility and safety studies on both polymerized grass mixtures.
Comparison of cutaneous end point titration with monomers and polymers Serial lo-fold dilutions of each polymer and each monomer were tested intradermally in a subject with a prior diagnosis of grass pollinosis. Skin tests results were evaluated by standard criteria’ and the lowest dilution producing a negative reaction was considered to be the end point.
Inhibition
of specific IgG binding by polymer
This polystyrene tube competitive inhibition has been described previously.” Briefly, polystyrene tubes were coated overnight at 4” C with a mixture of five crude grass antigens (redtop, timothy, orchard, June, and Bermuda; Allergy Labs of Ohio) at a concentration of 10 mgiml in 0. IM NaHCO,. Then 0.1 ml aliquots of a I : 100 dilution of serum from a patient treated with usual aqueous grass immunotherapy were incubated overnight at 4” C with various amounts (0, 50, 5000, and 500,000 ng) of the six-grass polymer mixture to inhibit binding. As a control, 0.1 ml of serum diluted 1 : 100 from a nonatopic individual was treated in an identical way. These mixtures were then added to the coated tubes and incubated at 4” C overnight and the tubes were washed. One milliliter of rabbit antihuman IgG (Calbiochem-Behring Corp., La Jolla, Calif.) diluted 1: 5 was added to each tube and incubated for 24 hr at 4” C, at which time the tubes were washed and ““I-labeled IgG was added. The tubes were incubated overnight at 4” C, washed, and counted. Control counts were subtracted and percent inhibition was calculated. The assay was also performed on 0.1 ml of undiluted serum from a serum pool from patients treated with usual grass immunotherapy.
Inhibition
of specific IgE binding by polymer
An assay similar to the above IgG assay was performed to demonstrate inhibition of IgE binding by polymers. Serum from a single untreated patient and from a serum pool of untreated patients were used. Undiluted serum (0.1 ml) and 0, 50, 5000, and 500,000 ng of polymer were used. Anti-IgE (Calbiochem-Behring) at a 1 :40 dilution and IZJIIgE were used instead of anti-IgG and ““I-IgG.
RESULTS Chromatographic patterns of markers and orchard grass monomer and polymer preparations Fig. 1, top,
showsthe profile of dextran blue on a column. Molecules of greater than 20,000,OOOdaltons were in the excluded volume of the Sepharose4-B column and those of less than 20,000,OOOdaltons were in the included volume. On Sepharose
4-B
VOLUME NUMBER
72 2
Polymerization
. ?
..
0.. 0 E
DEXTRAN
of grass
pollen
allergens
131
BLUE
GLUTARALDEHYDE
EXCLUDED
1.6
u az
?-
ORCHARD GRASS: +--.- . POLYMER r MONOMER
,/*““;:,,:, c
;
1.
“,
.*
G ;
‘$;,
* . ..*
FRACTION
NUMBER
FIG. 1. Top, Sepharose 4-B pattern of dextran blue and glurataldehyde. Botrom, Monomer and polymer orchard grass profiles. C-I, Fractions used for final polymerized orchard grass; G, glutaraldehyde peak in polymer.
this standardized column of Sepharose 4-B, free glutaraldehyde in the polymer grass preparation appeared late and was the cause of higher absorbance of the polymer preparations at high elution volumes. Fig. 1, bottom, shows a monomeric orchard grass chromatographic pattern and a polymerized orchard grass. The tailing peak contributed by free glutaraldehyde is shown. Fractions used for the final preparations of polymerized orchard grass are shown and do not include fractions of molecular weights higher than 20,000,000 daltons or fractions containing free glutaraldehyde . Fig. 2 illustrates the Sephadex G-200 chromatographic pattern of dextran blue (top) and of the polymerized orchard grass preparations (bottom). The fractions of polymerized orchard grass used therapeutically are also shown. Preparations of polymers of rye, timothy, redtop, June, and Bermuda grasses Figs. 3 and 4 show the Sepharose 4-B chromatographic profiles of monomeric and polymeric preparations of these grasses. In each case there was a formation of polymers, as demonstrated by the higher
h4 FRACTION FIG. 2. Sephadex tran blue (fop) C-i, Fractions preparation.
NUMBER
G-200 chromatographic and polymer orchard of excluded polymer
patterns of dexgrass Ibortoml. used in the final
OD reading at lower elution volumes of polymer. Formation of high-molecular-weight polymers (more than 20,000,OOO daltons) occurred particularly with Bermuda and June grass polymers. The fractions of each polymer preparation used for pooling and subsequent fractionation on the Sephadex G-200 column are shown. The Sephadex G-200 patterns of rye, timothy, redtop, June, and Bermuda grass polymers were like those for orchard grass and are not shown.
Patterson
10’ 8’
et al.
J ALLERGY
t RED TOP
TABLE
GRASS:
II. Inhibition
of specific
by polymerized
binding
. -‘.-‘. POLYMER ,. 0
87* 6. 5.
RYE GRASS:
.""-4
POLYMER
‘--.-i,
MONOMER
0 58 84 100
0 50 5,000 500,000
0 13 31 04
kG
0 50 5,000 500,000
0 0 38 8.5
IgE”
0 50 5,000 500,000
0 0 27 78
4. 3.
Serum
25 10 61 8. 7.
TIMOTHY
G ..*.*.,
GRASS:
.-- -.-+ ,=OLYMER .. MONOMER
pool
6. 5. 4. 3' 2' OF, 12
AMi.xture of equal parts of all 6 grass polymers. BAssay performed with 0. I ml of serum diluted 1 : 100. “Assay performed with 0.1 ml of serum undiluted.
*a -: \,, A.' , , , 18 20
,
, 24
,
, 28
FRACTION
,
, 32
,
, 36
'
4'0
I
I 44
I 'G..I 48
NUMBER
FG. 3. Sepharose 4-B chromatographic patterns of redtop, rye, and timothy monomer and polymer preparations. G, Free glutaraldehyde in polymer; I--I, fractions of included polymer.
Comparison of cutaneous end point titration with monomers and polymers Results of intradermal end point titrations of monomeric and polymeric grasses are shown in Table I. The monomeric preparations were 1000 to 100,000 times more skin-reactive than the polymeric preparations. inhibition
of specific
IgG binding
by polymer
The ability of polymer grass to inhibit binding of specific IgG to crude grass is shown in Table II. At 500,000 ng of polymer added in the single serum assay, inhibition was 100% complete. The IgG binding of the serum pool was 85% inhibited by adding 500,000 ng of polymer. Inhibition
% inhibition
0 50 5,000 500,000
IgE’
0. 'k.. ' z's ' 2'8 ' 3'2 ' 36 ' 4'0 ' 4'4 ' 49
added* hg)
Single serum IgG”
antibody
grass
Polymer Antibody
grass
CLIN IMMUNOL. AUGUST 1983
of specific
IgE binding
by polymer
The ability of polymer grass to inhibit binding of specific IgE to crude grass is shown in Table II. At
500,000 ng of polymer added to the single serum sample, inhibition was 94% complete. In the serumpool sample 78% inhibition was achieved when 500,000 ng of polymer was added. DISCUSSION In preparing polymerized ragweed, monomersof giant and short ragweedwere mixed and polymerized. This was considered appropriate becausealmost all ragweed-sensitivepatients are reactive to both pollen species. Polymerized ragweed extracts have been shown to have reduced allergenicity (elicitation of immediate-type reactions)6and retained immunogenicity and have been shown to be safe and efficacious in many therapeutic trials.‘-I0 All grass-sensitivepatients do not react to all grasses.Bermuda grassis a prime example of a speciesto which all grass-sensitive patients do not react becauseof its geographic location. These studies were done to show that six grassescould be polymerized individually. In subsequent clinical trials the individual grass polymers were selected and mixed according to patients’ immediate-typereactivity to permit a singleinjection. The change in chromatographic pattern on Sepharose 4-B column from monomeric to polymeric demonstratesthat all grassmonomerswere polymerized. The final molecular sieving on the Sephadex G-200
VOLUME NUMBER
72 2
Polymerization
BERMUDA
GRASS:
,'-'-,
POLYMER
:-..o
MONOMER
20
24
28
JUNE ..,--. 3
32
36
40
44
4S
GRASS:
of grass
pollen
allergens
133
Grass polymers of molecular weights in excess of 20,000,OOO daltons were excluded by Sepharose 4-B chromatography and were not added to the final polymerized grass preparations used in the clinical trial. This finding is analogous to that for the initial polymerized ragweed preparations that were characterized and used clinically. It is probable that the high-molecular-weight polymers (>20,000,000 daltons) are as safe and as immunogenic as the lowermolecular-weight polymers (200,000 to 20,000,OOO daltons) used in the ragweed and grass preparations. Thus the excluded peaks, particularly of Bermuda and June grass (Fig. 4), may be useful when added to final preparations.
POLYMER
REFERENCES
0MONOMER
FRACTION
NUMBER
FIG. 4. Sepharose 4-B chromatographic patterns of Bermuda and June monomer and polymer preparations. G, Location of free glutaraldehyde in polymer; H, fractions of included polymer.
column provided polymerized grasses with molecular weights in excess of 200,000 daltons and analogous to the polymerized ragweed that has already been extensively studied.g The reduction in allergenicity of polymer grasses as compared with monomer grasses further supports our results; we have previously shown that allergenicity of the polymer is less than that of the monomer.” The 3- to 5-log reduction in cutaneous end point titer of polymer as compared with monomer is similar to the differential seen in our polymerization of ragweed,12 mixed grass,’ and mixed tree. I3 This reduced allergenicity affords the safety factor that allows high doses of polymer to be administered over a short time perkI lo That polymer contains a large majority of antigenic determinants to which a pool of grass-sensitive patients develop IgE is well demonstrated by the significant inhibition of binding of IgE against crude grasses by polymer. The antigenic completeness of polymer is further demonstrated by its ability to significantly inhibit binding of IgG in a pool of grasssensitive patients treated with usual aqueous immunotherapy.
1. Patterson R, Suszko IM, Pruzansky JJ, Zeiss CR, Metzger J, Roberts M: Polymerization of mixtures of grass allergens. J ALLERGY CLIN IMMUNOL 59314, 1977. 2. Hendrix SG, Patterson R, Zeiss CR, Suszko IM: The immune responses in humans and rabbits to monomeric and polymeric grass allergens. J Clin Immunol 2:10, 1982. 3. Grammer LC, Shaughnessy MA, Suszko IM, Patterson R: A double-blind histamine placebo-controlled trial of polymerized whole grass for immunotherapy of grass allergy. J ALLERGY CLIN IMMUNOL (In press.) 4. Patterson R, editor: Allergic diseases. Diagnosis and management. Philadelphia, 1972, J. B. Lippincott Co. 5. Patterson R, Roberts M, Zeiss CR, Pmzansky JJ: Human antibodies against trimellityl proteins: comparison of specificities of IgG, IgA and IgE classes. Int Arch Allergy Appl Immunol 66:332, 1981. 6. Patterson R: Allergen immunotherapy with modified allergens (Robert A. Cooke Memorial Lecture). J ALLERGY CLIN IMMUNOL6&85, 1981. 7. Bacal E, Zeiss CR, Suszko IM, Levitz D, Patterson R: Polymerized whole ragweed: an improved method of immunotherapy. J ALLERGY CLINIMMUNOL 62:289, 1978. 8. Kelly JF, Zeiss CR, Patterson R, Levitz D, Suszko IM: Polymerized whole ragweed: human safety and immune response. J ALLERGY CLINIMMUNOL 65~50, 1980. 9. Hendrix SG, Patterson R, Zeiss CR, Pruzansky JJ, Suszko IM, McQueen R, Slavin R, Miller M, Lieberman P, Sheffer A: A multi-institutional trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 66:486, 1980. 10. Grammer LC, Zeiss CR, Suszko IM, Shaughnessy MA, Patterson R: A double-blind, placebo controlled trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 69~494, 1982. 11. Patterson R, Suszko IM, Pruzansky JJ, Zeiss CR Polymerized ragweed antigen E. II. In vivo elimination studies and reactivity with IgE antibody systems. J Immunol 110:1413, 1973. 12. Patterson R, Suszko IM, Zeiss CR, Pruzansky JJ, Bacal E: Comparison of immune reactivity to polyvalent monomeric and polymeric ragweed antigens. J ALLERGY CLIN IMMUNOL 61:28, 1978. 13. Patterson R, Suszko IM, Her&ix SG, Zeiss CR Polymerized treepollenantigens.J ALLERGYCLINIMMUNOL~~: 162.1981.