Intra-lymphatic immunotherapy (ILIT) directly administers allergens into lymph nodes to improve efficacy of immunotherapy. A human study on ILIT for cat allergy found: 1) ILIT with recombinant Fel d 1 was safe and increased tolerance to cat allergen in nasal provocation tests. 2) Secondary outcomes like skin prick tests, intradermal tests and immunological parameters also improved with ILIT. 3) This phase I/IIa trial demonstrated safety and preliminary efficacy of ILIT for cat allergy, justifying further studies.

1. Intra-lymphatic
Immunotherapy ( ILIT)
Theerapan Songnuy M.D.
July 26, 2013

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

3. 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:S712-9
3 Larche M, Akdis CA, Valenta R. Immunological mechanisms of allergen-specific
immunotherapy. Nat Rev Immunol 2006;6:761-71

4. 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.

5. 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 treatment
Immunol Allergy Clin North Am.2011; 31(3): 561-599

6. 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

7. New Direction of Immunotherapy
Intra-Lymphatic Immunotherapy ( ILIT)

8. 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.

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

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

11. 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

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

14. 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

15. Methods
Mice
ILIT
gr
SCIT
gr
Pos
control
Neg
control
Cat fur allergen
extract intra-
peritoneal
6-wkly ( 1 ug)
Sensiti-
zed
Mice
Desensitized
with1 ug Feld1
2 wk later
( 3 doses)
Adapted from

16. 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

17. 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

18. 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

19. 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

20. 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)

26. 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

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

28. 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

29. 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

30. 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

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

32. Superficial
Inguinal
Lymph
Node
Clinical Radiology 2003; 58(5): 367-371

33. 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

34. 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 = 2

35. 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

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

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

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

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

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

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

42. In Conclusion
• Intra-lymphatic Immunotherapy
- Reduce both number & dose of allergen
- Reduce duration of treatment
- Increase safety
- Less painful
- More compliance

43. Allergy Clin Immunol 2012;129:1290-6.

44. 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.

45. 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

46. 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

47. Methods
Allergy Clin Immunol 2012;129:1290-6

48. 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

49. 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

50. 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

51. Rhino-manometer
From : www.nagelnetwork.com/rhino.htm

52. 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

53. 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 leukotriene
Allergy Clin Immunol 2012;129:1290-6

54. 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

55. Clin Exp Allergy 2000; 30:
132-40
Clin Exp Allergy 2000; 30: 132-40

56. Allergy Clin Immunol 2012;129:1290-6

57. Allergy Clin Immunol 2012;129:1290-6

58. Allergy Clin Immunol 2012;129:1290-6

59. Allergy Clin Immunol 2012;129:1290-6

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61. Allergy Clin Immunol 2012;129:1290-6

62. 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

63. 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

64. Thank You Very Much