Further studies on the safety of polymerized antigens for immunotherapy

Further studies on the safety of polymerized antigens for immunotherapy Stephen G. Hendrix, M.D., Roy Patterson, lrena M. Suszko, B.S. Chicago, Ill.

M.D., C. Raymond

Zeiss, M.D., and

Six putients receiving immunotherupy with stundurd aqueous extructs for the treatment oj” utopia diseuse were selected from the patient populution oj’ Northwestern Unir,ersig Allergy Clinics ,for continued irnmunotherupy with polymerized untigens. These six patients could not tolerate quuntities of conventional uqueous extracts considered maintenance doses because of immediute-type local or systemic allergic reactions or both to immunotherapy. These six patients were treuted with separute preparutions of’ polymerized ragweed (PRW) und polymerized grass (PC), and euch of the six putients was rupidl! udvanced to previously unobtainable maintenance doses of’ PR W und PC without local or sytemic reactions. Treatment with other aeroullergen stundurd extructs wus continued to muintenunc~e dasuge without further systemic reactions. In patients highly sensitive to rugweed und gruss uyueous extracts, the substitution of these extracts with PRW und PC allows these patients to receive therapeutic immunotherapy injections with decreased risk of immediate-gpe local und systemic reactions.

Recent reviews have supported the effectiveness of immunotherapy in the treatment of atopic disease. ‘3 * Recommended schedules of immunotherapy that are safe are based on long clinical experience.3, 4 However, certain patients who are extremely sensitive to aqueous extract antigens are unable to tolerate the advancement to maintenance doses of immunotherapy according to standard schedules because of the occurrence of very large local and systemic allergic reactions following injections. Such patients require prolonged and expensive therapy with reduced amounts of antigens in the treatment of their atopic disease and are subjected to increased risk of anaphylaxis secondary to immunotherapy. In previous studies at this institution, immunotherapy with polymerized ragweed (PRW) was found to be superior to immunotherapy with standard aqueous extracts because of reduced incidence of local reactions.5, 6 When PRW was compared with standard aqueous ragweed extracts in immunotherapy, reduced

From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School. Supported by U.S. Public Health Service Grant AI 11403 and the Ernest S. Bazley Grant. Received for publication June 19, 1980. Accepted for publication Sept. 26, 1980. Reprint requests to: Roy Patterson, M.D., Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. Vol. 67, No. 2, pp. 124-128

allergenicity was demonstrated. Thus, larger quantities of PRW could be given during the course of immunotherapy with reduced incidence of allergic reactions. The reduced allergenicity of PRW is considered to be the result of coupling of ragweed antigens into large- molecular weight proteins; this effectively conceals some antigenic sites within the protein matrix of the polymer and thus makes them unavailable for interaction with IgE on mast cells.’ Further, high-molecular weight polymers have slower tissue-diffusion rates and the same weight of antigen as a polymer should have less chance to react with the multiple IgE antibodies attached to dermal mast cells as compared with the same weight of aqueous lowmolecular weight monomers. 8 The immune response to PRW has been shown to be similar to that of monomer ragweed.s The polymerization of grass allergens and the chromatographic profiles and retained immunogenicity of the grass polymer have been described. lo To further test the safety of immunotherapy with polymerized antigens, six patients receiving immunotherapy with standard aqueous extracts were selected from the clinic population of Northwestern University to receive polymerized antigen immunotherapy. These patients had in common severe reactivity to their standard allergen extracts. They required weekly injections of reduced amounts of aqueous extracts because attempts to advance these patients to higher

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0 1981 The C. V. Mosby

Co.

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Safety of polymerized antigens 125

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doses or to monthly maintenance injections resulted in reoccurring systemic or very large local reactions. Their standard extract mixtures contained equal parts on a weight-per-volume basis of ragweed, grass, mold, and tree pollens and house dust and these were injected at a single site. In this study, grass and ragweed pollen extracts were eliminated from the standard mixtures and the remaining extracts were injected in one arm at a single site. PRW and polymerized grass (PG) were injected in the opposite arm. Each patient was observed for the appearance of local or systemic reactions and was advanced to maintenance immunotherapy at monthly intervals. METHODS Selection of patients Six patients receiving standard immunotherapy extracts for the treatment of atopic disease who could not be advanced to maintenancedosagesbecauseof immediate local and systemic reactions were selected for treatment with polymerized antigens. Patient characteristics are described in Table I. Skin-testing

antigens

Cutaneous testing was performed with commercially prepared aqueousglycerinated 1: 10 (w/v) giant and short ragweed (RW), orchard grassand Kentucky bluegrass(G), oak and elm trees (T), Aspergillus and Penicillium molds (M), and house dust (HD) antigen extracts purchasedfrom Hollister-Stier Laboratories (Spokane, Wash.). Intradermal skin testing was performed with commercially prepared aqueous glycerinated 1: 1,000 (w/v) extracts. Results of skin testing are shown in Table I. Standard aqueous immunotherapy

extracts

six patients to standardextract injections and the maximum dosagetoleratedwithout systemicor large local reactionsby eachpatient at time of PG and PRW substitution aredetailed in Table 1. Polymerized

antigens

The preparation of PRW and PG have been described.“‘1 ‘I Briefly, thesematerials are aqueousextractsof ether-defatted giant and short ragweed pollens obtained from Greer Laboratories (Lenoir, N. C.) and grass pollen mixture from Greer containing equal amounts of redtop, Bermuda, orchard, June, timothy, and perennial rye. These were polymerized by glutaraldehydetreatmentand collected after molecular fractionation on Sepharose 4B and SephadexG-200 columns, Sterility was assuredby appropriate cultures in the bacteriology laboratories of Northwestern Memorial Hospital. The final preparationsof PRW and PG antigenshad molecular weight rangesof 200,000 to 20,000,OOOdaltons. The PRW and PG solutions were standardizedby optical density analysis at 280 nm and by determining nitrogen with the Kjeldahl method. Stock solutions of PRW were adjusted to contain 17,000 PNU/ml. Analysis of ragweed antigen E (AgE) content of monomer ragweed (MRW), which is the starting material for PRW, demonstratedthat 17,000 PNU of MRW contains 100 pg of AgE and this is entirely incorporated into PRW. Stock solutions of PG were adjustedto contain 10,000 PNUlml. Schedule for immunotherapy polymerized antigens

with

Table III shows the schedule for injections of PRW and PG given each of the six patients. When maintenancedosage was obtained, each patient was gradually advancedto monthly injections.

for

These were commercial extracts purchasedfrom Hollister-Stier. The grass mixture (G) consisted of equal parts June, orchard, redtop, timothy, and Bermuda grasses.The tree mixture (T) consisted of ash, beech, birch, walnut, cottonwood, elm, hickory, maple, oak, sycamore, and willow. The mold mixture (M) consistedof 40% Alrernaria, 20% Aspergillus, 20% Hormodendrum, and 20% Penicilhum. House dust (HD) was supplied by Hollister-Stier as an extract in 50% glycerin. Extracts were diluted on a weight-per-volume basis as shown in Table II. Standard schedule for immunotherapy with aqueous extracts A typical schedule used for immunotherapy for atopic diseaseat Northwestern University is shown in Table 1I.a.1 Weekly injections are begun with dilutions of extracts of 1: 10,000 (w/v) in a single injection site. The initial dose, 0.05 ml, contains approximately 5 protein nitrogen units (PNU) of eachpollen extract. Patientsable to tolerate injections of 0.5 ml of 1: 100 standard mixtures are gradually advancedto monthly injections. Immediate reactions in the

RESULTS Immediate reactions immunotherapy

to polymerized

antigen

No systemic reactions to injections of PRW or PG were noted. All six patients tolerated the schedule of PG and PRW immunotherapy outlined in Table III. Reactions at the sites of PG and PRW injections were judged to be minimal by patients and health personnel. In addition, all six patients were advanced to maintenance dosage of the remaining standard aqueous extracts as shown in Table I. All patients were then gradually advanced to monthly maintenance injections of the polymerized antigens and standard extract antigens without difficulty. DISCUSSION In this study, six atopic patients who displayed marked sensitivity to standard aqueous immunotherapy extracts were selected for treatment with polymerized antigens in an effort to safely increase aller-

126 Hendrix et al.

TABLE Age (yrll Sex

I. Patient characteristics, Diagnosis

J. ALLERGY

diagnosis,

CLIN. IMMUNOL. FEBRUARY 1981

and treatment Standard

Skin tests

28/F

Perennial allergic rhinitis symptoms uncontrolled with antihistamines

Prick: G, 3+; RW, 4+ Intradermal: M, 3-l; T, 3+

36/F

Perennial allergic rhinitis symptoms uncontrolled with antihistamines

RW, 4+; T, 4+ Prick: Intradermal: M, 3 +; HD, 3 +; G, 3 +

50/M

Perennial allergic rhinitis with side effects from antihistamines unacceptable to patient

Prick: G, 3+; RW, 4+ Intradermal: M, 3+; HD, 3+

23/F

Perennial allergic rhinitis with side effects from antihistamines unacceptable to patient

Prick: RW, 4+; G, 3+ Intradermal: M, 3+; T, 3+

24/F

Allergic rhinitis with symptoms uncontrolled with antihistamines

Prick: G, 2+; RW, 4+ Intradermal: T, 2+

32/F

Seasonal asthma with perennial allergic rhinitis

Prick: G, 3+; RW, 4+ Intradermal: M, 4f; HD, 3 +; T, 3 -t

immunotherapy

Type: Equal parts (w/v) T, G, RW, M Immunotherapy begun: March, 1979 Documented immediate reactions: 3/79: Large local; 0.05 ml 1: 10,000 5/79: Bronchospasm; 0.25 ml 1: 1,000 6/79: Bronchospasm; 0.15 ml 1: 1,000 10/79: Bronchospasm; 0.05 ml 1: 100 Highest dose: 0.20 ml 1: 100 (2,000 PNU weekly) Type: Equal parts (w/v) T, G, RW, M, HD Immunotherapy begun: January, 1977 Documented immediate reactions: 4/77: Large local; 0.5 ml 1: 100 5/77: Diffuse urticaria; 0.4 ml 1: 100 7/77: Bronchospasm; 0.3 ml 1: 100 Highest dose: 0.15 ml 1: 100 (1,500 PNU weekly) Type: Equal parts (w/v) T, G, RW, HD Immunotherapy begun: July, 1978 Documented immediate reactions: 7/78 to 1 l/79: Approximately 20 large local reactions Highest dose: 0.025 ml 1: 100 (250 PNU weekly) Type: Equal parts (w/v) T, G, RW, Immunotherapy begun: January, 1972 Documented immediate reactions: l/72: Large local; 0.05 ml 1:50,000 l/72 to 5/79: Approximately 12 episodes of bronchospasms noted Highest dose: 0.025 ml 1: 100 (250 PNU weekly) Type: Equal parts (w/v) G, RW Immunotherapy begun: January, 1978 Documented immediate reactions: l/78: Large local; 0.05 ml 1: 10,000 6/78: Bronchospasm; 0.20 ml 1: 1,000 8/78: Large local; 0.20 ml 1: 1,000 Highest dose: 0.25 ml 1: 1,000 (250 PNU weekly) Type: Equal parts (w/v) G, RW, M. HD, T Immunotherapy begun: November, 1977 Documented immediate reactions: 5/78: Bronchospasm; 0.35 ml 1: 1,000 5/79: Urticaria; 0.25 ml 1: 100 6/79: Bronchospasm; 0.075 ml 1: 100 Highest dose: 0.10 ml 1: 100 (1,000 PNU weekly)

PNU = protein nitrogen units; G = grass; RW = ragweed; M = mold; HD = house dust; T = tree; PRW = polymerized ragweed;

PC = polymerized grass. gen (protein nitrogen units per injection) and decrease the frequency of injections. None of the six patients had been able to achieve the usual dose of maintenance immunotherapy with standard aqueous extracts

because of recurring local or systemic reactions or both following injections. When PG and PRW were substituted for the standard grass and ragweed extracts each patient was advanced to maintenance im-

munotherapy without the occurrence of significant local reactions and without systemic reactions. Maintenance immunotherapy at this institution is reached when 5,000 PNU of each allergen extract can be injected at one site at monthly intervals without development of significant local reaction. For this study a maintenance dosage of 8,500 PNU of PRW was chosen because experience with multiple clinical trials

VOLL ’ IE 67 NUMt< IH 2

Safety of polymerized

antigens

127

TABLE II. Northwestern University allergy service for standard extract immunotherapy

-. Substituted

immunothempy Dilution

Kight arm: PC and PRW SC, edule per Table Ill Left rm: Aqueous T. M AC Janced IO 0.5 ml I : IO0 each (5,000 weekly injections

PNU)

after

PNU)

after

Righi. .trrn: PC and PRW Schedule per Table III

775 2.7.50 ‘7 5(H) _.._ 5.O(HJ PNU pel ir!icctilm

TABLE III. Schedule for polymerized immunotherapy following attempted extract immunotherapy Injection number

Left ;Irm: Aqueous T, HD advanced to 0.5 ml I : iO0 each (5.000 PNU) after 6 weekly injections Right arm: PRW Schedule per Table III Left ;I-m: PG Schedule per Table 111

I 2 3 4 5 6

PNU PG lfebemd 500 I .OOO 1.ooo 3,000 -l.ow 5.ooo

antigen standard

WPRW dSW I .ooo 3 .WO S.000 0.5ol) 8.500

Injections are given weekly until maintenance dosage I\ achieved and then gradually advanced to monthly injecticln\ PNU = protein nitrogen units.

Right x-m: PRW SC+ :dule per Table III Left ,3 .m: PC Set :dule per Table III

Right xrn: PG and PRW Schl:dule per Table 111 Left alrn: aqueous T, HD, M Advanced to 0.5 ml I : 100 each (5,000 8 weekly injections

IO IO IO

Total PNU of each extract deibvemd

Injecttons are given weekly until maintenance dosage i\ achieved and then gradually advanced KI monthly injection\ PNU -T protein nitrogen units.

Righ. arm: PRW SC, edule per Table III Left xm: Aqueous T, G, M, HD Ad lanced to 0.5 ml I : IO0 each (5,000 I- weekly injections

I : I0.000 (w/v) 1 : 1,000 I : l(K) Weekly injection of I : IO0

Number of injectiins

PNU) after

using PRW had shown that we could achieve this dose with safety. Less experience with PG resulted in greater caution in selecting the maintenance dose in these patients. Based on our experience with these six patients, we believe that a dosage of 8,500 PNU of PG could be safely administered. Each patient tolerated a schedule in which only six injections of PG and PRW were required to reach maintenance immunotherapy and only six to eight injections of the remainder of the former aqueous extract were required. Each

patient was then advanced gradually from weekly to monthly injections. This suggests that the antigens responsible for these patients’ previous intolerance were contained in aqueous ragweed or grass. One patient, a 36-yr-old woman (Table I) with minimal skin sensitivity to grass (positive to intradetmal tests tolerated advancement to maintenance only), immunotherapy with the substitution of only PRW for aqueous ragweed in the immunotherapy mixture. In a large multi-institutional study of PRW immunotherapy, 15 injections of PRW were given to achieve similar maintenance doses.” A more accelerated schedule was used here because these six patients had previously received a large number of injections containing ragweed and grass antigens. Further, careful observation of the patients following each dose of polymerized allergens showed the absence of local or systemic reactions and permitted the use of the schedule in Table III in all patients. Over the past four yr, approximately 3.000 injections of PRW have been administed to 200 patients in a series of studies. Three patients have had mild systemic reactions following PRW in these combined studies. This is an incidence of one systemic reaction

128

Hendrix

et al.

per 1,000 injections. These reactions consisted of mild urticaria and bronchospasm and were successfully treated with a subcutaneous injection of 1: 1,000 epinephrine; each patient was subsequently advanced to maintenance immunotherapy without further reactions. In the past year, 22 patients have received immunotherapy with PG and no systemic reactions have occurred. This study has reported the successful use of PRW and PG in patients highly sensitive to standard extracts of ragweed and grass. The use of PRW and PG allows these patients to achieve monthly maintenance immunotherapy without increased risk of painful local reactions or anaphylaxis. REFERENCES 1. Norman PS: An overview of immunotherapy: Implications for the future. J ALLERGY CLIN IMMUNOL 6587, 1980. 2. Patterson R: Clinical efficacy of allergen immunotherapy. J ALLERGY CLIN IMMUNOL 64:155, 1979. 3. Patterson R: Immunotherapy, in Middleton E, Reed C, Ellis E, editors: Allergy, principles and practice. St. Louis, 1978, The C. V. Mosby Co., p. 877. 4. Melam H: Principles of immunologic management of allergic diseases due to extrinsic antigens, in Patterson R, editor: Allergic diseases. Philadelphia, 1972, J. B. Lippincott Co., p. 293.

J. ALLERGY

CLIN. IMMUNOL FEBRUARY 1981

5. 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. 6. Hendrix Xi, 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 AILERGYCLIN IMMUNOL 65:57, 1980. 7. Patterson R, Suszko lM, Metzger J, Zeiss CR, Pruzansky JJ: Immunotherapy with polymerized pollen antigens. Proceedings of Ninth International Congress of Allergology, Buenos Aires, 1976. Excerpta Medica International Congress Series No. 414. 8. Patterson R, Suszko IM, Pruzansky JJ, Zeiss CR: Polymerized ragweed antigen E: In viva elimination studies and reactivity with IgE antibody systems. J Immunol 110:1413, 1973. 9. 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. 10. Patterson R, Suszko IM, Pruzansky JJ, Zeiss CR, Metzger WJ, Roberts M: Polymerization of mixtures of grass allergens. J ALLERGY CLIN IMMUNOL 59:314, 1977. 11. Patterson R, Suszko IM, McIntire FC: Polymerized ragweed antigen E: Preparation and immunologic studies. J Immunol 110:1402, 1973. 12. Hendrix SG, Patterson R, Zeiss CR, et al: A multi-institutional trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 66:486, 1980.