Intra-lymphatic Immunotherapy ( ILIT)

Theerapan Songnuy M.D.

July 26, 2013

Outlines

• Introduction• Mechanism of ILIT• Animal model of ILIT• Outcomes of ILIT in human study• Conclusion & take home message

Introduction• Allergic rhinitis is a health problem that causes worldwide

disability : social life, sleep, school, and work 1

• Immunotherapy is the only treatment that diminishes

symptoms, improves quality of life, prevents new

sensitizations, and reduces the development of asthma in

patients suffering from allergic rhinitis 2,3

1Bousquet J, Khaltaev N, Cruz AA, Denburg J, Fokkens WJ, Togias A, et al. Aller-gic Rhinitis and its Impact on Asthma (ARIA) 2008 update (in collaboration with the World Health Organization, GA(2)LEN and AllerGen). Allergy 2008;63:8-160

2 Frew AJ. 25. Immunotherapy of allergic disease. J Allergy Clin Immunol 2003; 111:S712-93 Larche M, Akdis CA, Valenta R. Immunological mechanisms of allergen-specific

immunotherapy. Nat Rev Immunol 2006;6:761-71

Introduction

• Subcutaneous immunotherapy ( SCIT) requires numerous

injections and take several years to complete 4

• Adverse events

• Physician-oriented

• High Cost

• Only 5% of allergic patients with insufficient symptom control undergo SCIT 5

4Cox L, Nelson H, Lockey R, Calabria C, Chacko T, Finegold I, et al. Allergen im-munotherapy: a practice parameter third update. J Allergy Clin Immunol 2011;127: S1-55 5 Frew AJ. Allergen immunotherapy. J Allergy Clin Immunol 2010;125:S306-13.

Introduction• Sublingual Immunotherapy (SLIT)

- Efficacy in AR, prevent asthma in AR,

prevent new sensitization

- Better safety profile

- Convenience & ease of administration at home

- Increase adherence

- Cost-efficacy if compared to pharmacological

treatment alone

But

- Not approved in the US

- Long duration of treatmentImmunol Allergy Clin North Am.2011; 31(3): 561-599

Introduction

• When antigens are administered subcutaneously, just only

small fractions of the antigenic epitopes reach the lymph nodes 8

• As immune responses are initiated in secondary lymphoid organs, seems reasonable that the direct administration of antigens into the highly immuno-competent environment of the lymph nodes induces greater immunogenicity 9

8 Martinez-Gomez JM, Johansen P, Erdmann I, Senti G, Crameri R, Kundig TM.Intralymphatic injections as a new administration route for allergen-specific immu-notherapy. Int Arch Allergy Immunol 2009;150:59-65

9 Zinkernagel RM. Localization dose and time of antigens determine immune reac-tivity. Semin Immunol 2000;12:163-71; discussion 257-344

New Direction of Immunotherapy

Intra-Lymphatic Immunotherapy ( ILIT)

Micro-architecture of the lymph node

• Lymph nodes are organized into 3 zones

1. An antigen- sampling zone

- Subcapsular area & medulla

- Rich in APCs

2. B-cell activation zone

- In the cortex, containing B-cell follicles

3. T-cell activation zone

- In the paracortex

Junt T, Scandella E, Ludewig B. Form follows function: lymphoid tissue microarchitecture in

antimicrobial immune defence . Nat Rev Immunol 2008; 8( 10): 764-75.

Picture

Nat Rev Immunol 2008; 8( 10): 764-75.

Nat Rev Immunol 2008; 8( 10): 764-75.

Pathologic mechanism• Animal studies : vaccines can be given in lower

concentrations & reduced number of injections

when administered directly into the lymph node11

• In mice, intra-lymphatic immunization

- Enhanced allergen-specific IgG &T-cell

responses

- Stimulated the production of the TH1-dependent

subclass IgG2a ( improved protection against

allergen-induced anaphylaxis) 8

11 Eur J Immunol 2005;35:568-74

8 Int Arch Allergy Immunol 2009;150:59-65

Aims

• To compare subcutaneous with direct intra-lymphatic administration of allergens in mice as a means for improving efficacy

Methods• Immunization Protocols

- Female CBA mice ( 6-8 wk old; Harlan, Horst, The Netherland)

- Immunized 3 times with 2-wk intervals

- Injection with major bee venom allergen phospholipase A2 ( PLA2; n=5;

Sigma- Aldrich, Buchs, Switzerland)

- Another group use recombinant Fel d1 ( n=4), Fel d1 was cloned into

the Pqe30 expression vector as an N-terminal [ His]6- tag fusion

protein, produced in E. coli & then purified

- Lipopolysaccharide content of Fel d1 was 1.27 ng/mg of protein

- Allergens were mixed with aluminum hydroxide ( Alhydrogel 3%) &

saline 1 hr before use ( 90 ug for ILIT, 450 ug for SCIT)

- Injection volume were 10 ul for ILIT, 50 ul for SCIT

Methods

Mice

ILIT gr

SCIT gr

Poscontrol

Neg control

Cat fur allergen extract intra-

peritoneal6-wkly ( 1 ug)

Sensiti-zed

Mice

Desensitizedwith1 ug Feld1

2 wk later ( 3 doses)

Adapted from

Methods

• Induction of Anaphylaxis

- All 4 groups were challenged 3 wk after last

desensitization

- Using 30 ug of cat fur allergen extract in saline

( 50 ul intra-peritoneally)

- Body temperature was measured before &

30 min after challenge

Methods

• Antibody Measurements

- Process of incubation:

- Plates coated with cat fur allergen extract, PLA2

- Serial dilution of sera

- Biotinylated goat anti-mouse IgG1, IgG2a

- Absorption was read at 405 nm on Model 550

microplate reader

Methods

• Detection of PLA 2-sIgE

- Using 2 ug/ml anti-mouse IgE capture Ab

- An in-house biotinylated PLA 2 at 3 ug/ml,

for binding to mouse serum

• Detection of Fel d1- sIgE

- Using 2 ug/ml anti-mouse IgE capture Ab

- Incubated with serum from immunized mice

- Then rFel d1 0.6 uM was added

- Finally use anti-Fel d1-biotin Ab at 1:1,000

Methods

• Flow Cytometry & Cytokine Secretion Assay

- One wk after injection, spleens were isolated

then single-cell suspension were prepared

- Measure CD4,CD8, CD 44 etc.

- Analysis of cytokines by ELISA ; IL-2, IL-4,

IL-10, IFN- gamma

Methods

• Bio-distribution

- Compare the relative bio-distribution of ILIT & SCIT

- CBA mice were injected with 99MTc pertechnetate-

labelled human Ig ( Technescan R HIG)

- Using 3 MBq /dose IL or SC at inguinal area

- Four animals each were euthanised at 90 min & 17 hr

- Lungs, spleens, livers, inguinal lymph nodes were

dissected , then analyzed in a Cobra II gamma counter

( radioactive count /min)

In Conclusion

• ILIT may represent a promising method to

- Increase the efficacy of SIT

- Reduce the required allergen doses

- Reduce number of injections

- Reduce the risk of adverse events

as compared to SCIT

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Aims

• To evaluate the clinical feasibility & safety of

allergen injection directly into lymph nodes

• To compare its efficacy with subcutaneous

immunotherapy

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Methods• RCT study• Time : June 2001-March 2005• Place : The University Hopital Zurich, Switzerland• Participants : seasonal rhino-conjunctivitis, allergic asthma

• Inclusion criteria:

- History of rhino-conjunctivitis

- Aged 18-65 y

- Positive SPT to grass pollen

- Springtime AR with SPT positive to tree pollen

( birch, hazel, alder)

- No clinical symptom but SPT positive to cat dander or HDM

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Methods

• Exclusion criteria:

- Blood donation or surgery within 30 d

- Use of investigational drug within 90 d

- Pregnancy or nursing

- Mastocytosis

- Significant cardiovascular, hepatic, renal

hematologic, autoimmune or infectious diseases

- History of HT, malignancy, using B blocker, ACE-I

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Methods

• Treatment & F/U Procedure:

Group ILIT

- Three 0.1-ml injection with 1,000 standardized

quality unit of aluminum hydroxide-adsorbed

grass pollen extract (Alutard SQ; ALK-Abello)

- Timing : at day 0, 4 wk, 8 wk

- Route: superficial inguinal lymph node under

US guidanceProc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Superficial

Inguinal

Lymph

Node

Clinical Radiology 2003; 58(5): 367-371

Methods Group SCIT

- A total of 54 SC injections over 3 y

( cumulative doses of 4,031,540 SQ-U)

- The allergen doses were increased weekly

for 16 wk

- After reaching maintenance dose(100,000 SQ-U)

injections were given monthly

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Methods• NPTs

- Adaptation to room temperature for 10 min

- Anterior rhino-scopy was done at base line

- The 50 ul of isotonic test solution was pushed to middle

nose conch using a pipette

- Allergen diluent : 100- 1,000-10,000-100,000 SQ-U aqueous

grass pollen extract ( Aquagen SQ ) in 10-min intervals

- A symptom score ranging from 0-6 points

- Nasal secretion: none = 0, mild =1, severe = 2

- Sneezing : 0-2 = 0, 3-5 =1, >5 = 2

- Remote symptoms : none = 0, lacrimation or pruritus = 1, chemosis,

urticaria, shortness of breath = 2Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Methods• NPTs

- Scoring greater than 4 defined as the maximal tolerated pollen conc

- If scoring < 4 with conc of 100,000 SQ-U, the maximum tolerated

dose was 1 Million SQ-U

• SPTs

- A 30 ul serially diluted of grass pollen allergens ( Soluprick Alutard-

SQ;ALK-Abello)

- Recording of severity of hay fever, asthma, nasal congestion

nasal itching, sneezing, red eye & dry cough

* Serum sIgE

- By UniCAP for grass-pollen mix & timothy grass

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

Proc Natl Acad Sci USA 2008; 105 ( 46): 17908-17912

In Conclusion

• Intra-lymphatic Immunotherapy

- Reduce both number & dose of allergen

- Reduce duration of treatment

- Increase safety

- Less painful

- More compliance

Allergy Clin Immunol 2012;129:1290-6.

Aims

• The phase I/IIa trial• To evaluate the safety, immunogenicity & efficacy

of of ILIT with MAT-Fel d1

• Primary efficacy objective :

- Tolerance to cat dander extract in a titrated

NPT• Secondary efficacy objectives:

- Allergen tolerance in IDTs & SPTs &

immunologic parameters Allergy Clin Immunol 2012;129:1290-6.

Methods

• Patients with cat dander allergy• Enrolled during Aug-Sep 2008• At University Hospital Zurich, Switzerland

• Inclusion criteria :

- History of cat dander allergy ( AR, AC)

- Age of 18-65 y

- Positive at least one concentration of cat

dander extract in SPT

- Positive NPTs to Fel d1

- Detection of serum sIgE to Fel d1

Allergy Clin Immunol 2012;129:1290-6

Methods

• Exclusion criteria:

- Serious immunologic diseases

- Serious cardiovascular diseases

- Malignancies

- Chronic infections

- Use of B-blockers

- Uncontrolled & severe asthma

- Pregnancy or nursing

- Severe psychiatric disorders

Allergy Clin Immunol 2012;129:1290-6

Methods

Allergy Clin Immunol 2012;129:1290-6

Methods• Allergen & placebo preparation

- MAT-Fel d1: a recombinant nonglycosylated fusion protein consisting of 291 amino acid

( Strathmann Biotec GmbH & Co KG ; Hamburg, Germany)

- Stored at -80 C

- Both MAT-Fel d1 & placebo were incubated at room temperature with aluminum hydroxide for > 60 min before injection

- Cat dander allergen extract from Stallergenes ( Antony, France)

- Placebo ( saline in alum )

Allergy Clin Immunol 2012;129:1290-6

Methods

• Intra-nodal injection under US guidance

- Superficial inguinal lymph node

- Administration of 100 ul of either MAT-Fel d1

or placebo

- Using a 1-ml hypodermic syringe with

a 25-gauge needle

Allergy Clin Immunol 2012;129:1290-6

Methods

• NPTs

- Baseline evaluation by rhinoscopy &

rhinomanometry

- A 100 ul of allergen solution ( Alyostal;

Stallergenes, Antony, France) was sprayed

into the wider side of the nose

- Symptoms were scored

- The end point : nasal flow reduction > 40 %Allergy Clin Immunol 2012;129:1290-6

Rhino-manometer

From : www.nagelnetwork.com/rhino.htm

Methods

• SPTs

- Serially diluted allergen solutions

- Prick on the volar forearm

- A wheal size of > 7 mm2 defined as positive

• IDTs

- A 1000-fold dilution of SPTs

- A 100 ul was injected by using 0.5 ml syringe

with 26-gauge needle

- Wheal reaction > 6 mm in diameter as positive Allergy Clin Immunol 2012;129:1290-6

Methods• Determination of Ab

- IgE analyzed by ImmunoCAP

- ELISAs to detect cat dander-specific IgG ,IgG1, IgG4

• Cellular antigen stimulation test

- Leukocytes were isolated then mixed in stimulation

buffer containing IL-3

- Then stimulate with rFel d1, MAT-Fel d1, nFel d1

at 37 C for 40 min

- Allergens were tested at 5, 0.5, 0.05 & 0.005 nmol/l Fel d1

- Supernatants were assayed for soluble leukotrieneAllergy Clin Immunol 2012;129:1290-6

Methods

• T-cell analyses

- Allergen-specific T-cell were determined

- By stimulation of 200,000 PBMCs with LPS-free rFel d1

- Supernatants were analyzed for IL-2, IL-4,IL-5, IL10,

IL17 & IFN gamma

• Mini-rhino-conjunctivitis quality-of-life Q

- Assess symptoms during the last week

Clin Exp Allergy 2000; 30: 132-40

Clin Exp Allergy 2000; 30: 132-40

Clin Exp Allergy 2000; 30: 132-40

Allergy Clin Immunol 2012;129:1290-6

Allergy Clin Immunol 2012;129:1290-6

Allergy Clin Immunol 2012;129:1290-6

Allergy Clin Immunol 2012;129:1290-6

Allergy Clin Immunol 2012;129:1290-6

Allergy Clin Immunol 2012;129:1290-6

In Conclusion

• ILIT with MAT-Fel d1 was safe & induced tolerance

to NPT after 3 injections• This yields over SCIT or SLIT in duration of treatment & less

side effects• Limitations :

- Low pt number

- Symptoms were not assessed at baseline• Future challenges:

- Compare ILIT with MAT-Fel d1 VS rFel d1

- Compare SCIT VS ILIT with MAT-Fel d1

Take Home Messages

• Allergic rhinitis, asthma, & prevention of a new sensitization would be successfully treated by AIT

• Intra-lymphatic immunotherapy yields in less side effect, less time, less dose required but more convenient & adherence than SCIT

• But ILIT needs US-guided & physician-administrated• RCT is needed to confirm the cost-effectiveness of

ILIT in the real-world situation before applying in daily practice

Thank You Very Much