Erika Jensen-Jarolim

Medizinisches Propädeutikum, 18. November 2009

AllergoOncology:

Novel biological functions of IgE antibodies

Clemens Freiherr von Pirquet

Definition of „Allergy“In: Münchner Med. Wochenschrift 1906

IgE

Allergen

Triggering allergy effector cells causes symptoms

• Rhinoconjunctivitis

• Asthma

• Food allergy

• Urticaria

• Anaphylaxis

FceRI

mast cell

basophil

eosinophil

• Sensitization phase: Production of IgE

• Effector phase: Symptoms

Mediator release

Oral, intestinal Respiratory

Asthma, Asthma, AtopicAtopic TriadTriadFood allergyFood allergy

IgE incidence tripled during last decades.

Predicted 40% in New Zealand.

What makes an allergen an allergen?

From the following article:Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine dataStephan C. Bischoff. Nature Reviews Immunology 7, 93-104 (February 2007)

How is the specificity of antibodies guaranteed ?

multimericantigens/allergens

monovalentantigen/allergen ?

flexible, multivalent antigens ?

B-cell crosslinking by allergens

multivalent, multiepitope antigens ?

No secreted IgE

multimericantigens/allergens

monovalentantigen/allergen

flexible, multivalent antigens

B-cell crosslinking by allergensnon-productive productive

No secreted IgE Secreted IgE

multivalent, multiepitope antigens

Table 1: Examples of allergens, which are able to form dimers or multimers (sorted by year of publication).

(Suck et al., 2000)2000Ves v 5 (wasp venom)

(Lascombe et al., 2000)2000Equ c 1 (horse dander)

(Maleki et al., 2000)2000Ara h 1 (peanut)

(Xia et al., 2000)2000ABA-1 (Ascaris, nematodes)

(Reese et al., 1999)1999Tropomyosin (seafood)

(Gregoire et al., 1999)1999Equ c 1 (horse dander)

(Eriksson et al., 1999)1999Aca s 13 (Acarus siro, dust mite)

(Petersen et al., 1998)1998Phl p 1 (grass pollen)

(Shin et al., 1998)1998Ara h 1 (peanut)

(Rautiainen et al., 1996)1996Bos d 2 (BDA20) and Bos d 5 (bovine dander allergens)

(Wellhausen et al., 1996)1996Bet v 1 (birch pollen)

(De Vouge et al., 1996)1996Alt a 1 (Alternaria, fungi)

(Gimona et al., 1995)1995Tropomyosin (e.g. fish)

(Hoffman, 1993)1993Sol i II (fire ant)

(Ogawa et al., 1993)1993Gly m Bd 30K (soy bean)

(Bocskei et al., 1992)1992Mus m 1 (MUP; mouse urinary protein)

(Bocskei et al., 1992)1992A2U (rat urinary allergen)

(McGibbon et al., 1990)1990ABA 1 (Ascaris, nematodes)

(Terwilliger et al., 1982)1982Api m 4 (bee venom)

(Huecas et al., 2001)2001Ole e 1 (olive pollen)

(Aas and Jebsen, 1967)1967Gad c 1 (Baltic codfish)

ReferenceYear of publicationAllergen (source)

(Tan et al., 2009)2009Per a 4 (cockroach)

(Bellinghausen et al., 2008)2008Per a 3 (cockroach)

(Weghofer et al., 2008)2008Der p 21 (house dust mite)

(Li et al., 2008)2008Bla g 2 (cockroach)

(Kamata et al., 2007)2007Can f 1 and 2 (dog dander)

(de Halleux et al., 2006)2006Der p 1 (house dust mite)

(van Boxtel et al., 2006)2006Ara h 1 (peanut)

(van Oort et al., 2005)2005Dac g 5 (grass pollen)

(Schöll et al., 2005)2005Bet v 1 (birch pollen)

(Gronlund et al., 2003)2003Fel d 1 (cat dander)

(Fahlbusch et al., 2003)2003Cav p 1 (guinea pig dander)

(Wopfner et al., 2002)2002Profilin (panallergen)

(Verdino et al., 2002)2002Phl p 7 (grass pollen)

(Rajashankar et al., 2002)2002Phl p 5b (grass pollen)

(Das Dores et al., 2002)2002Gad m 1 (Atlantic codfish)

(Sen et al., 2002)2002Ara h 2 (peanut)

(Sakurai et al., 2001)2001Beta-lactoglobulin (bovine milk)

(Suck et al., 2000)2000Ves v 5 (wasp venom)

ReferenceYear of publicationAllergen (source)

…The immediate phase reaction occursas a result of cross-linking with multivalent allergen…..Current Opinion in Allergy and Clinical Immunology 2002, by R. Pawankar

Multivalent antigen crosslinks bound IgE…. Immunobiology, by Janeway & Travers 1997.

Aggregation of FcRI, typically by the binding of di- or multivalent allergen recognized by the IgE, induces mast cell and basophil activation. In: Nature Medicine 11, 381 – 382, 2005,by J. Kalesnikoff & SJ Galli.

What makes a protein an effector allergen, i.e. triggering symptoms?

Multivalency ?

mediatorschemokines

cytokines

multimericantigens/allergens

monovalentantigen/allergen

Flexible, multivalent antigens

IgE-effectorcells ?

enhancedcell survival &

FcRI expressionmonomericIgE

INTERPHASES

• BETWEEN ALLERGEN AND IgE

• BETWEEN IgE and FcRI, CD23

Effector cell crosslinking by allergensnon-productive productive

+ Ko

- Ko

Monomer

Dimer

Bet v 1 monomer does not trigger HR

CD spectrum

SAXS

Type I skin test in BALB/c

From: Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice. Schöll I et al. J Immunol. 2005 Nov 15;175(10):6645-50.

Isabella Pali-Schoell

From: Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1.Vrtala et al, FASEB J. 2001 Sep;15(11):2045-7. Epub 2001 Jul 24.

A Bet v 1 trimer does not trigger HR

Disagreement with hypothesis?

From the following article:Integrated signalling pathways for mast-cell activation

Alasdair M. Gilfillan and Christine TkaczykNature Reviews Immunology 6, 218-230 (March 2006)

Epitope spacing is critical for Crosslinking

From: Cancer and IgE: Introducing the concept of AllergoOncology.(Eds. Penichet and Jensen-Jarolim) Springer, in press.

Does IgE against innocuous antigens make sense?

The function of IgE: Protection against parasites ?

„…a causal association between geohelminth infection and atopic disorders has not been established.“Reddy A, Fried B. Adv Parasitol. 2008;66:149-91.

„…evidence from experimental animal models infected with helminth parasites and treated with anti-IgE antibodies do not support a critical role for IgE in mediating protection against helminths.“Cooper PJ et al, Allergy. 2008 Apr;63(4):409-17

Read more in: Survival of the fittest: allergology or parasitology?Fitzsimmons CM, Dunne DW. Trends Parasitol. 2009 Oct;25(10):447-51.

Parasite-antigens

• Highly elecated IgE levels in sera• Eosinophils elevated to 50% of leucocyte counts

FcRI

IgE

Schistosoma mansoni

IgE: exclusively cooperation with cells

With DCs and other APCs, IgE supportsAntigen transport and immunity

• Antigen focussing: IgE and FcRI, CD23

With eosinophils, mast cells, basophils, macrophages:Effector functions

• ADCC - Cytotoxicity: IgE and FcRI• ADCP - Phagocytosis: IgE and CD23

From: Taking our breath away: dendritic cells in the pathogenesis of asthmaBart N. Lambrecht & Hamida Hammad. Nature Reviews Immunology 3, 994-1003 (December 2003)

Immune cells present in tumor tissue (Red and Green)

„Chronic infiltration of innate immune cells belonging to the myeloid cell lineage,including mast cells, macrophages, and neutrophils, correlates with poor disease prognosis. “

Cancer inflammation: IgE-cooperating cells on-site

Source: Nature review in Cancer 2006

Cancer mortality (n= 1,102,247) and allergies.„…..significant inverse associations between asthma

and hay fever and cancer mortality ….“

Turner et al. Am J Epidemiol. 2005 Aug 1;162(3):212-21.

Anti-IgE therapy and Cancer.„…Xolair patients had a 0.5% malignancy rate

compared to 0.2% of control patients.“

(FDA Advisory commitee)

IgE - Protection against tumors?

• IgE is more effective in cytotoxicity than IgG Kershaw MH et al. Oncol. Res. 1998.

Reali E. et al. Cancer Res. 2001.

Evidence for IgE combating tumors

IgE inhibits tumor growth in SCID.

From: Gould HJ et al. Eur. J. Immunol. 1999

Dense infiltration by macrophages tumor transplanted SCID mice treated with IgE.

From: Activity of human monocytes in IgE antibody-dependent surveillance and killing of ovarian tumor cells. Karagiannis et al. Eur. J. Immunol. 33, 2003.

Table I. IgE in allergy and oncology

Clin Exp Immunol.153(3): 401-9. 2008.Immunoglobulin E antibodies from pancreatic cancer patients…Fu SL, et al.

Cancer Imm. Imm.. 57(2): 247-263. 2008.Characterisation of an engineered trastuzumab IgE antibody ….Karagiannis P and Singer J, et al.

J. Immunol Meth 323(2): 160-71. 2007.Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis.

Bracher M, et al.

J Immunol. 179(5): 2832-43. 2007IgE-antibody-dependent immunotherapy of solid tumors: cytotoxic and phagocytic..Karagiannis SN, et al.

Allergy 63 (10): 1255-66. 2008.AllergoOncology: The role of IgE-mediated allergy in cancerJensen-Jarolim E, et al.

2006 Definition of AllergoOncology

Cancer Res. 67(7): 3406-11. 2007.Active induction of tumor-specific IgE antibodies by oral mimotope vaccination.Riemer AB, et al.

Am J Epidemiol. 162(3): 212-21. 2005.Cancer mortality among US men and women with asthma and hay fever.Turner MC, et al.

Eur J Immunol.33(4): 1030-40. 2003.Activity of human monocytes in IgE antibody-dependent surveillance and killing of ovarian tumor cells.

Karagiannis SN, et al.

Cancer Res 61: 5517-22. 2001.IgEs targeted on tumor cells: therapeutic activity and potential in the design of vaccines.Reali E, et al.

J Immunol. 29: 3527-37. 1999.Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.

Gould HJ et al.

Oncol Res 10: 133-42. 1998Tumor-specific IgE-mediated inhibition of human colorectal carcinoma xenograft growthKershaw MH et al.

Cancer Immunol Immunother 34: 63-9. 1991.

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus.

Nagy E et al.

Int Arch Allergy Appl Imm.. 41: 141-3. 1971.

IgE levels and allergic skin reactions in cancer and non-cancer patients.Augustin R and Chandradasa KD.

Cancer 27: 93-9. 1971.A study of allergy in patients with malignant lymphoma and chronic lymph. leukemia.McCormick DP et al.

Scott Med J 14: 51-4. 1969.Negative association between allergy and cancer.Ure DM.

1967 Identification of IgE

Strahlentherapie 114: 203-24. 1961.Is there an allergy against malignant tumor tissue and what can it signify in regard to the defense of the body against cancer?

Schlitter HE.

Int Arch Allergy Appl Immunol 9: 200-21. 1956.

Mechanism of tumor allergy and its importance in tumor pathogenesis.Berdel W, et al.

Cancer Res 15: 181-3. 1955.The effect of an allergic inflammatory response in the tumor bed on the fate of transplanted tumors in mice.

Molomut N et al.

Zentralbl Chir 77: 1873-81. 1952.Tumor metastasis in the light of allergology.Bienengraber A.

Ann Surg 102: 56-61. 1935.Predisposing Factors and Diagnosis of Rectal Cancer: A Discussion of Allergy.Martin EG.

1906Definition of Allergy & Era of Reagins

ReferenceTitleAuthors

• Occurrence of natural IgE in head and neck cancerNeuchrist et al, Int. Archs Allergy Clin Immun. 1994

• Occurrence of natural IgE in pancreatic cancer: tumoricidic

From: Immunoglobulin E antibodies from pancreatic cancer patients mediate antibody-dependent cell-mediated cytotoxicity against pancreatic cancer cells.

Fu SL et al. Clin Exp Immunol. 2008 Sep;153(3):401-9.

Natural IgE in melanoma ?

IgE

IgG

Melanomasample1774/08/1

Green: anti human IgE/IgG-FITCBlue: DAPI to stain nuclei

IgE120 / 83.674positive

IgG2.233 / 84.386positive

IgE

IgG

Natural IgE in melanoma by TissueFAXs

Passive immunotherapy with IgE

Passive Antibody-immunotherapy• ADC,CDC

• Radioimmunoconjugates

• Immune cytokines

• Immunotoxins

• Direct Apoptosis

• Internalisation and downregulationEGFR

HE

R-2

Tumor cell

Cetuxi

mab (E

rbitux

© )

Trastu

zumab

(Herce

ptin© )

Growth arrest -

Colon cancer

Breast cancer

Herceptin-IgE construction

• cDNA encoding heavy and light chains of Herceptin® (source: www.pdb.org; 1n8z)• Cloning into vectors with epsilon heavy chain, and human kappa light chain• Transfection into HEK293 cells• Antibodies purified by affinity chromatography

Karagiannis and Singer et al, Cancer Immunol. Immunother, 2008

Panos Karagiannis

Josef Singer

Herceptin-IgE: affinity to HER-2 and FcRI

Precondition for effector function: Herceptin-IgE binds to IgE receptors FcRI and CD23

100 101 102 103 104FL1-Height

M1

62.8%

FcεRI expressing RBL-SX38

62.8%

CD23 expressing A8866 cells

Visualization of effector functions

M. Bracher et al., Journal of Imm. Meths 2007

Macrophages

Tumor cell

Phagocytosis

Control IgEHerceptin-like IgE

Phagocytosis in 3-color assay

100 101 102 103 104FL1-H

Tauf1.1.010

R1

R2

αCD89 PE stained cells-> Monocytes U937

CFSE-stained cells->Tumor cells

Double stained cells->phagocyted tumor cells

Propidium Iodide positive cells->Cytotoxicity

100 101 102 103 104FL1-H

Tauf1.1.010

R3

M. Bracher et al., Journal of Imm. Meths 2007

Quantification of effector functions

Karagiannis and Singer et al, Cancer Immunol. Immunother, 2008

IgG IgEControl IgG Control IgE

Phagocytosis

Cytotoxicity

IgG IgE

Herceptin-IgG or –IgE in 3-color assay

MTT cell viability assay with EGFR+ A431 cells

Cetuximab (Erbitux ®) targets EGFR

Construction of a Cetuximab-IgE

Cooperation withProf. R. Bredehorst,and Dr. E. Spillner

tumor cells effector cells

After 16 hours two separate populations

IgG

Mixed populations

IgE

Co-culture EGFR positive A-431 cells and RBL-SX38 effector cells with cetuximab IgG or IgE

Diana Mechtcheriakova

CoCo--culture culture AA--431/RBL431/RBL--SX38 andSX38 and IgEIgE (3 h)(3 h)

Active immunotherapy with IgE

subcutaneous mimotope-vaccine induces Herceptin® -like IgG antibodies

Riemer AB et al, J Immunol. 2004, JNCI 2006, and ms. in preparationKnittelfelder et al. Expert Opin. Biol. Ther.9 (4):493-506. 2009.

Mimotope-serum TT-serum Herceptin

• receptor internalisation

• ADCC, CDC

• in vivo tumor arrestin transgenic model

Breast cancer cells express HER-2

% tu

mor

-free

m

ice

time until tumor (weeks)50403020

1,0

0,8

0,6

0,4

0,2

0,0

Kum

. Übe

rlebe

n

QMW-KLH-zensiertPBS-zensiertKLH-zensiertQMW-KLHPBSKLH

Group

HerceptinHerceptin

HER-2 Epitope

Mimotope

Angelika Riemer Silke Gruber

How to induce IgE: Lessons from Food Allergy

Untersmayr & Jensen-Jarolim. JACI 2008

Formation of IgE-antibodiesagainst non-digested proteins

Protein and acid inhibitor

Eva Eva UntersmayrUntersmayr

Herceptin-mimotopeoral / acid suppression

oral mimotope vaccineinduces Herceptin® -like IgE antibodies

Riemer AB et al., Cancer Research 2007 Untersmayr and Jensen-Jarolim, J. Allergy Clin Immunol 2008

mediator release

MastzelleIgE

tumor cell

IgE

mediators

cytotoxicity

Regina Knittelfelder Regina Knittelfelder

Commons of allergens and tumor antigens

Mast cell

IgE

Allergen PrototypeHomoMultimersAggregation and Clustering of epitopes

Mast cell

IgE

Tumor PrototypeHomodimersClustering of overexpressed TAAsAra h 1

Shin et al. JBC 1998

AAMPs:Allergen-Associated Molecular Patterns

TAMPs: Tumor-Associated Molecular Patterns

mediatorschemokines

cytokines

overexpressingtumor cell

multimericantigens/allergens

monovalentantigen/allergen

Flexible, multivalent antigens

IgE-effectorcells

enhancedcell survival &

FcRI expressionmonomericIgE

Effector cell crosslinking by tumor antigens: ADCC, ADCPnon-productive productive

• Natural anti-tumor IgE

• Recombinant IgE antibodies

• IgE-based oral vaccine

Erika Jensen-Jarolim

Medizinisches Propädeutikum, 18. November 2009

AllergoOncology:

Novel biological functions of IgE antibodies