Allergenicity, immunogenicity, and safety of immunotherapy with various molecular weight ranges of polymerized ragweed

Allergenicity, immunogenicity, and safety of immunotherapy with various molecular weight ranges of polymerized ragweed Leslie C. Grammer, M.D., Martha A. Shaughnessy, B.S., John J. Shaughnessy, Ph.D.,* Loui Silvestri, Ph.D.,** and ROY Patterson, M.D. Chicago, Ill., Holland. Mich., and Miami, Since

the observation

inversely

with

to determine as part

weight

into

three (50,000

groups

product

pool.

on the basis

ranges

and PRW

B, and C by intradermal or induration

end point ,from

There

were

each

of the molecular-weight

immunogenic, simpllfiing ~MMUNOL

and wfe to administer, of the ,final product pool. the fructionation 76:195-200.

by incidence

reactions

Each

local

>20,000,000

has the adiantuges one chromatogruphic

500

pg PRW

between

reactions,

or

randomized

A X00,000:

that

in clinical

of PRW studied

alone

were

received

PRW

antibody

nor any changes

and thus PRW This

patient

bvas no difference of large

turies this S~ULII~

>20,000,000

pollinosis

ranges:

(PRW)

we conducted

weight

ragbveed

in blocking

ranges

by eliminating

with

molecular-weight

or by rise

euch group.

molecular

There

ragweed

previously,

skin test titration.

titration,

injections,

no systemic

PRW

patients

C >20,000,000.

In summary, as a part

with

Twentyone qf’ rugweed

of polymerized

we hud studied

of one qf the f~~llowing

PNU)

to 20,000,OOO;

erythema

that allergenici&

over

of immunotherapy

the utility

of n final

AgE

200,000

had been mude

moleculur

Fla

PRW

by amount

was significant

laboratory

wus similarly

B

A, of in

parameters.

allergenic,

can be u.sed,for

immunothetqq

of increasing

yields

procexs.

(J ALLERGY

and

of

CLI.V

1985.)

The therapeutic applicability of PRW is the result of its reduced allergenicity and retained immunogenicity.’ In studies comparing PRW and MRW in equivalent doses, similar immunogenicity and symptom reduction were observed.‘, ’ In those studies there were no systemic reactions in 10 patients treated with PRW, but there were seven systemic reactions in nine patients treated with MRW. The overall incidence of systemlc reactions in patients treated with PRW is I .O% per patient course” that is significantly lower than the reported incidence of 7% to 29% per patient courses with conventional immunotherapy. This reduced incidence of systemic reactions is due to the decrease in allergenicity of PRW compared to MRW. ’ With end

From the Section ot Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Chicago. 111..*Hope College. Holland. Mich., and **Key Pharmaceuticals Co., Miam,. Fla. Supported by Umted States Public Health Service Grant AI 11403. the Ernest S. Bazley Trust, and Key Pharmaceuticals Co. Received for publication July 27. 1984. Accepted for publication Nov. 24. 1984. Reprint requests: Leslie C. Crammer. M.D., 303 E. Chicago Ave., Chxago. IL 6061 I.

Abbreviations

used

Polymerized ragweed MRW: Monomeric ragweed CBC: Complete blood count ESR: Erythrocyte sedimentationrate PRW:

point cutaneous titration, allergenicity of PRW has been demonstrated to be inversely proportional to molecular weight.’ The upper limit of molecular weight studied was 20,000,000, the exclusion limit of a Sepharose CL-4B column (Pharmacia Fine Chemlcals, Uppsala, Sweden), which was the initial fractionator of PRW. As PRW MW 20,000,OOOto 200,000 is less allergenic than PRW MW <200,000.h this study was designed to evaluate whether PRW MW >20,000,000 would be even less allergenic. We prepared PRW A as MW >.500,000, PRW B as MW 200,000 to 20,000,000, and PRW C as MW >20,000,000. The details of the chromatographic fractionation are in Table I. The objectives of this study were as follows: (I) to determine if PRW MW >20,000,000 (PRW C) and! or PRW MW >500,000 (PRW A) would be less al195

196 Grammer et al. TABlE

J. ALLERGY

1. Molecular-weight

ranges of ragweed

preparations

MW

PRW A PRW B

2 500,ooo 200,000 to 20,000,000

PRW C PRW P

2 20,000,000 200,000 to 20,000,000

MRW

5,000 to 46,000

TABtE 4. Immunotherapy polymerized ragweed l-._ll__ injection No.

schedule

Dilution

Votume

I:10

0.05

1:lO 0.10 2 3 I:10 0.25 1 1:lO 0.50 Full strength 0.10 5 b Full strength 0.25 -7to 16 Full strength 0.50 -. __--.-.. .--_

for

PNU per injection

50 100 250 500 1000 2500 5000

CLIN. IMMUNOL. AUGUST 1985

Preparation

Fractionation Sephacryl S-300 Fractionation SepharoseCL4B and Sephacryl S-200 Fractionation SepharoseCL4B Fractionation SepharoseCL4B and Sephacryl S-200 as published and used previously4 Before polymerization

TABLE 111.lntradermal polymerized ragweed bg) per injection

end point titration extracts Patients

A&

0.5 1.0 2.5 5 10 25 50

of

I

II

III

0.5* 0.5 0 0.5 0 0.5 0

1.0 1.0 0 1.0 0 1.0 0

3.0 3.0 0 3.0 0 3.0 0

Extracts

PRW P PRW A PRW A and Pt PRW B PRW B and Pt PRW C PRW C and Pt

*Negativelog of the intradermalendpoint titer. t Log differencebetweenPRWP andPRWA, B, or C. Iergenic than PRW MW 200,000 to 20,000,OOO(PRW BI: (2) to determine if PRW A MW >500,000 and/ or PRW C MW >20,000,000 would be equally immunogenic as PRW B; and (3) to determine if PRW A and PRW C could be safely administered. Furthermore. in order to investigate the possibility of the use of PRW C as a part of the final product pool and thus making the fractionation process more efficient by eliminating the Sepharose CL4B column and increasing yield?, we studied allergenicity, immunogenicity, and safety of PRW A. m3-ERfAt Patients

AlrfDlt4imfmS

Twenty-one patients with ragweed pollinosis and 2 + to 4-t prick tests to giant and short ragweed (1: 20, Allergy Laboratories of Ohio [ALO], Columbus, Ohio) were recruited in the spring of 1983. Patients were otherwise healthy as determined by physical examination, history, ESR, CBC, differential leukocyte count, renal function tests. and liver function tests. Patients had not received immunotherapy for at least 5 years before the study. Intradermal end point titrations were performed with weight/volume dilutions of short ragweedand of giant ragweed (lo-‘. 10.“. IO-‘. IO-‘, 10-9, IO-“‘). The end point titration scoresfor patients were ranked, and successivesets of three patients were randomized into three groups.

Polymerization

of ragweed

PRW was prepared at ALO. In brief, short and giant ragweed pollens were defatted with ether, extracted with NH.,HCO,, precipitated by (NH,), SO.,,and fractionated on a SephadexG-15 column, thus resulting in MRW. Protein concentration of MRW was determined by the micro Kjeldahl’s method; AgE content was determined by radialimmunodiffusion. Polymerization with glutaraldehyde was performed at 25” C for 4 hours and was stoppedby addition of glycine. Column chromatographytechniques were then used to preparethree different molecular-weight ranges of polymer. Fractionation

of PRW

One aliquot of PRW was applied to a SepharoseCL4B column, and the fractions from the voided peak containing polymers of 20,000,000 and greatermolecular weight were pooled to produce PRW C. The fractions from the included peak of the SepharoseCL-4B column were applied to a Sephacryl S-200 (PharmaciaFine Chemicals) column, and the voided peak was pooled to produce PRW B containing polymers of 200,000 to 20,000,OOO daltons. Another aliquot of PRW was applied directly to a molecular weight-calibrated Sephacryl S-300 column and was fractionated at MW > 500,000, thus producing PRW A containing polymers of molecular weight mote than 500,000 (Table I).

VOLUME NUMBER

Polymerized ragweed

76 2, PART 1

TABLE IV. Allergic

reactions

to polymerized

ragweed

Total patients Total injections No. of systemic reactions Average centimeter erythema/induration per injection No. of large local reactions requiring repeat injection No. of patients who had large local reactions requiring repeat injections

197

extracts PRW A

PRW B

PRW c

6*(6)t IO0 0 0.1210.2

7*(5)t 89 0 0.11/0.12

7*(5)i 88 0 0.09/0.01

2

1

0

2

f

0

*Total patients who received any injection. “iTotal patients whetcompleted the injection series. No patient dropouts were the result of adversereactions to immunotherapy.

lntradermal

end point titration

of PRW

Three additional ragweed-sensitive subjects were tested by intradermal injection of 0.02 ml of serial 1u05dilutions of PRW A, PRW B, PRW C, and PRW P, a PRW extract previously used in a clinical trial.4 Wheal and erythema were measured at 15 minutes, and the end point was read as the most dilute solution resulting in 5 mm wheal and 10 mm erythema. In the spring of 1983, each of the patients began a series of 16 weekly injections. The schedules. illustrated in Table 11, were identical whether the patient was receiving PRW A, B, or C. Neither patients, investigators, nor treatment nurses were aware of which treatment a given patient was receiving. If a patient had a large focal reaction, the dose was repeated before increasing the next dose.

Immunotherapy Immediate local reactions were measured at 20 minutes and recorded as centimeters of erythema and induration. patients were instructed to measure and record sizes of late local reactions occurring 4 to 48 hours after injections. If a dose was repeated because of a large local reaction, additional injections were administe~d to reach a total of 50,000 PNU and 500 pg AgE.

Laboratory

tests

Each patient had the following Iaboratory tests before receiving injections in order to establish that there were no abnormalities: CBC, differential leukocyte count, ESR. urinalysis, blood urea nitrogen, cteatinine, lactic dehydrogenase, serum glutamy oxytransferase, serum glutamyf phosphotransferase, gamma glutamyl transferase, alkaline phosphatase, and direct and indirect bilirubin. These tests were all repeated after the last injection.

Immunologic

studies

Blood samples were obtained from each patient before the first injection and after the final injection. Serum was separated and stored frozen until immunologic studies were performed.

Total serum AgE binding capacity was determined by a modification’ of the ammonium sulfate precipitation technique of Lidd and Farr.’ Serum aliquots of 0.1 ml were incubated with 10, 100, and 1000 ng of ‘2sI-labeled AgE. Antibody bound to AgE was precipitated with 40% saturated ammonium sulfate, and the amount of AgE bound by I ml of serum in antigen excess was calculated. This is a quantitative estimate of blocking antibody and is expressed as nanogram AgE bound per milliliter of serum. The polystyrene tube radioimmunoassay technique previously described in detail” was used to measure AgE binding by IgE. Polystyrene tubes were coated with myeloma IgE after which anti-IgE was added to form an IgE immunosorbent. The tubes were incubated for 48 hours with 0.1 ml of patient serum and washed. After “‘I-AgE was added in antigen excess, the tubes were incubated for 48 hours, washed, and counted. The amount of specific IgE bound to AgE in 1 ml of serum was calculated.

RESULTS Patients By one-way analysis of variance on pretreatment values of total antibody binding of AgE and IgE against AgE (IgE-a-AgE) and by log rank test on ragweed intradermal end point titration, there were no statisticalIy significant differences between the three groups before treatment.

lntradermal

end point titration

of PRW

The negative logs of the intradermal end point titers of PRW A, PRW B, PRW C, and PRW P in each of three subjects are illustrated in Table III. Also illustrated are the log differences between each test extract and PRW P in each patient. We have previously reported the use of mean log differences for standardization of extract allergenicity.” The mean log difference between PRW P and each test extract was 0, indicating that the atlergenicity of each of the three

198

Grammer

.I. ALLERGY

et al

PRW

A.----

p= 0.0006 PRW

Bp=o.o037

PRW

c&---A p=O.O027

I

I

Pre

significantly different by two-tailed Student’s t-tests. The largest t was 1.37. In the PRW A group, each of two patients required one additional injection for one large local reaction, whereas one patient in the PRW B group required an additional injection for a large local reaction, and no patient in the PRW C group required additional injections for large local reactions. Thus, three patients had large local reactions requiring additional injections. There was no difference between the groups by Fisher’s exact test in terms of numbers of patients or numbers of injections requiring repeat injection for large local reactions. There were no systemic reactions to any PRW extract.

I Rx

Post

Laboratory

Ax

FIG. 1. Change in total serum binding activity of AgE for patients receiving PRW A (v--- 01, PRW B (b-----o), and PRW c (A---- &. The verticalbars are SEM. The rises are highly significant by paired t test in each group: PRW A p = 0.0006; PRW B p = 0.0037; PRW C p = 0.0027.

PRW

A p= 0.05

PRWB

Rx

Post

-

Rx

F1G. 2. Changes in IgE-a-AgE with treatment in groups receiving PRW A (el ), PRW B (o----o), and PRW C (*-.&. The vertical bars are SEM. By paired t test the rise in PRW A (p = 0.05) and PRW B (p = 0.04) are statistically significant, whereas the rise in the PRW C group ip :- 0.31) is not.

test extracts was the same as a PRW extract previously used in a clinical trial.’ Quantitation of aliergic immunotherapy

reactions

to

A summary of allergic reactions to each PRW exis illustrated in Table IV. The total number of patients and injections were similar in the three groups. All possible pairwise comparisons among the three groups for erythema and induration were not tract

tests

All patients entered into the study had values that were within normal limits for their laboratory tests: CBC, differential leukocyte count, ESR, urinalysis, blood urea nitrogen, creatinine, serum glutamyl oxytransferase, serum glutamyl phosphotransferase, gamma glutamyl transferase, alkaline phosphatase, and direct and indirect bilirubin. There were no abnormalities after treatment. Immunologic

Pre

CLIN. IMMUNOL. AUGUST 1985

studies

Log transformed scores were used for all analyses. A one-way analysis of variance was used to compare the three treatment groups. Separate analyses were done on pre- and posttreatment total antibody binding of AgE and IgE-a-A&E. The largest of the four resulting F ratios was F,z.,j, = 1.0, p > 0.4. To assessthe effect of treatment in the three groups, separate paired t-tests were done. The changes in total antibody binding of AgE with injections of PRW A. PRW B, and PRW C are illustrated in Fig. 1. Each treatment caused a significant rise that was at least tenfold in total antibody binding of A&E. (PRW A t = -6.5576, /’ = 0.0006; PRW B t = -5.0331, p = 0.0037: PRW C t = -5.4945, p = 0.0027). The pairwise comparisons of the increase across the three treatment groups demonstrated no significant differences. The largest t obtained was 1.36, p = 0.21. The changes in I&E-a-A&E with injection are illustrated in Fig. 2. Each treatment caused a minor rise in I&E-a-A&E but none more than 2.5-fold (PRW A t = -2.0918, I-’ = 0.05; PRW B t = -2.2475, p = 0.04; PRW C t = -0.5244, p = 0.3139). PRW C was the only preparation that did not result in a significant rise. DlSCUSSlON In preparing PRW, we have previously fractionated on each of two columns. a Sepharose CL4B column

VOLUME NUMBER

76 2, PART 1

and a Sephacryl S-200 column, so that the final product pool was composed only of polymers of 200,000 to 20,000,OOO daltons. We conducted this study because we believed that the use of polymers of more than 20,000,OOO alone or as part of a final product pool might have two advantages: (1) theoretically, higher molecular-weight polymers might be even less allergenic while immunogenicity was retained, and (2) practically. this would make fractionation more efficient by eliminating one chromatographic process. Each of the three molecular-weight ranges of PRW studied was able to be administered to patients safely in an immunotherapy schedule similar to those used in other clinical trials.“. ‘* There were no systemic reactions in any of the 20 patients who received injections, nor were there any changes in the hematologic, renal, or hepatic parameters measured. Moreover, the immunogenicity of each of the three molecular-weight ranges of PRW was not statistically different. PRW C, containing only polymers more than 20,000,OOOdaltons, produced a statistically significant fifteenfold rise in total antibody binding of AgE. The absolute level of that rise was approximately half of that observed with PRW A and B, although when these were tested directly, the rises were not statistically different. Thus, PRW C is comparable in terms of total antibody binding of AgE. It should also be noted, however, that PRW C did not cause a statistically significant rise in IgE-a-AgE, whereas the other two molecular-weight ranges of PRW did. In previous publication@, ‘Owe have demonstrated that allergenicity varies inversely with molecular weight. We have also cited a number of explanations why this occurs. In larger polymers there is intemalization of more antigenic determinants, more steric hindrance of cross-linking IgE on mast cell surfaces, and reduced diffusibility.” By end point intradermal titration, there are no differences in allergenicity of the three PRW molecular-weight ranges studied. Although we are not certain why allergenicity does not appear to decrease with molecular weight in the ranges we tested in this study, we suspect that there is a limiting factor of decreasing allergenicity with polymerization, and that factor may be diffusion; that is, the diffusion constant of a polymer varies inversely and exponentially with its molecular weighV so that polymers, whether 300,000 or 30,000,000, may well be on the asymptotic part of the curve, and thus all have extremely low diffusion coefficients. Also, the average fold difference in molecular weight between MRW and PRW A is several hundred, whereas the mean fold difference in molecular weight between PRW C and PRW A or PRW B is less than tenfold. Thus, the difference in allergenicity between PRW A

Polymerized

ragweed

199

and MRW would be expected to be much greater than the differences between PRW C and PRW A or PRW B. The similarity of allergenicity of the three PRW preparations is also demonstrated clinically in that there were statistically similar numbers of large local reactions requiring repeat injections and statistical] y similar amounts of induration and erythema recorded in each of the three groups. It should be noted that there were no large local reactions requiring repeat injections in the PRW C group and that that group had the lowest average amount of total erythema/induration recorded. At present then, the end point titrations and local reaction data suggest that the allergenicity of PRW C is comparable to that of PRW A and PRW B. This trial of immunotherapy demonstrates safety and immunogenicity of various molecular-weight ranges of PRW. Patients in each group developed significantly increased levels of blocking antibody and tolerated PRW well with no systemic reactions, no changes in laboratory parameters, and in fewer injections than with conventional immunotherapy. PRW C, composed of polymers of MW >20,000,000 daltons, is of comparable allergenicity and is as safe and immunogenic as PRW A and B. Thus, PRW C could be incorporated into the final product pool that would have the advantage of making fractionation more efficient by eliminating one chromatographic process. REFERENCES 1. Patterson R, Suszko IM, Zeiss CR, Ptuzansky JJ, Bacal E: Comparison of immune reactivity to polyvalent monomeric and polymeric ragweed antigens. J ALLERGYCLIN IPIMUNOI 61:28, 1978 2. Bacal E, Zeiss CR, Suszko IM, Levitz D, Patterson R: Polymerized whole ragweed: an improved method of immunotherapy. J ALLERGY CLIN IMMUNOL 62:289, 1978 3. Hendrix SG, Zeiss CR, Levitz D, Suszko IM. Patterson R: Polymerized whole ragweed: a two-year follow-up of patients treated with an improved method of immunotherapy. J Al.LERGYCLIN IMMUNOL 65:57, 1980 4. Grammer LC, Shaughnessy MA, Suszko IM, Shaughnessy JJ. Patterson R: Persistence of efficacy after a brief course of polymerized ragweed allergen: a controlled study. J ALLERGY CLAN IMMLJNOL73:484, 1984 5. Levy DA: Hazards and adverse reactions associated with administration of allergenic extracts. In Brede HD, Going H. editors: Regulatory control and standardization of allergemc extracts. New York, 1980, Gustav Fischer Verlag, p I78 6. Patterson R, Suszko IM, Bacal E, Zeiss CR, Kelly JF, Pruzansky JJ: Reduced allergenicity of high molecular-weight ragweed polymers. J ALLERGY CLIN IMMUNOL 63:47, 1979 7. Zeiss CR, Metzger WJ, Levitz D: Quantitative relationship, between IgE antibody and blocking antibodies specific for antigen E in patients given immunotherapy with ragweed antigen E. Clin Exp Immunol 28:250, 1977

J. ALLERGYCLIN.IMMUNOL. AUGUST1985 8. Lidd D. Farr RS: Primary interaction between “‘l-labeled ragweed pollen and antibodies in the sera of humans and rabbits. J ALLERGY 33:45, 1962 9. Zeiss CR, Levitz D, Suszko IM: Quantification of IgE antibody specific for ragweed and grass allergens: binding of radiolabeled allergens by solid phase bound IgE. J ALWWY CLIN IMMUNOL 61:216. 1978 10. Crammer LC, Silvestri L, Suszko IM, Shaughnessy MA, Patterson R: Evaluation and standardization of polymerized ragweed extracts by chromatography, radioimmunoassay inhibition, and end point cutaneous titration. J ALLERGY CLIN IMUUNOL 72:160, 1983 ! i. Grammer 1 C, Zeiss CR, Suszko IM. Shaughnessy MA, Pat-

Mathea R. Ailartsmith, Kurt J. Bloch, M.D.***

terson R: A double-blind, placebo-controlled trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGYCLIN IMMUNOL 69:494, 1982 12. Hendrix SG, Patterson R, Zeiss CR, Pruzansky JJ, Suszko IM. McQueen RC, Slavin RG, Miller MP, Lieberman PL. Sheffer AL: A multi-institutional trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL66:486, 1980 13. Patterson R: Allergen immunotherapy with modified allergens. J ALLERGY CLIN IMMUNOL 68:89, 1981 14. Brandrup J, Immergut EH, editors: Polymer handbook. New York, 1966, Interscience Publishers, p IV-77

M.D.,**** Robert S. Baird, B.S.,** Boston, Mass.

and

Kiits undergoing ocular unuphylmis induced /J] systemic or 10~1 injection of antigen, topicul ~ippliiation of antigen, or topical upplication qf compound 48180 were evuluated for conjunctivul &let cell changes that might he related to nnnphyluxis. The number of goblet cells in I pm, trlkuline Giemscr-stuined sections nl,eraged SOOlmm” of epithelium in normal ruts; this number WLS not significantly changed in any of the experimental groups. Goblet cells in control ruts c~ccasionuliy demonstrated evacurrtion qf their contents (< 170) or upwurd displacement of the ~ntrucellulur bo1lr.s qf muclls (about 1%); these percentages Mere not increused in anaphFluxi.s. Top&l upplicution qf 2 .O pg of histamine induced an intrucellulur displacement of mucus in i,lr)th c.ontrol animuls und unimuls undergoing unuphylrwis. These ,finding.s suggest that in ocular rrnuph~1a.ui.s the umount of histamine released may be in.sufJicient to produce such intrucellulnr ( hunges. Our results indicate that in ocular anuphyluxis in the rut, there is no light microscopic el?dence of increused mucu.s dischtrrge from conjunctival goblet cells. Increased mucus in .sccretions of patients with ocular cdlergic syndromes muy not be attributable to nnuphyluctic mechanisms ulonc. 1.1ALLERGY CLIN IMMCINOL 76:200-~5~ 1985,)

Excess mucus in the eyes is characteristic of many ocular diseases.‘, .’ particularly vernal conjunctiviQ<;.‘ i 4hhough the mechanism of vernal conjunctivitis has not been delineated, patients have high levels of histamine and IgE antibodies in the tears as well as increased numbers of mast cells in the affected

From the -!-Department of Ophthalmology, Harvard Medical School. ‘Beth lime1 Hospital. the *‘*Department of Cornea Research, I:yc Research Insritute ot Retina Foundation, and the :‘**Departmen! ot Medicine. Harvard Medical School. Clinical IITmunnlogy and .4llergy I’nits of the General Medical Services. Massachusetts General Hospital. Boston. Mass. supported by the National Institutes of Health Grant EY-02099 and hi, a grant from the Artbrtti~ Foundation. Received f& publication June 22, 1984. Accepted for publication Nov. 24. 1984. Reprints not available.

1

PBS:

Phosphate-buffered

sahne

conjunctiva. ‘-’ Thus, it appears that anaphylactic mechanisms may play a role in vernal conjunctivitis. Anaphylaxis, or the mediators of anaphylaxis, have been reported to increase mucus secretions in various models of mucosal anaphylaxis. Goblet cells have been implicated as a major source of this increased mucus. In a model of egg albumin-induced anaphylaxis of the gastrointestinal tract in rats, Lake et al.* obtained indirect evidence of mucus release from duodenal, jejunal, and ileal goblet cells. In these studies goblet cells were implicated as the source of the mucus