Part II Lecture on Immunoglobulins and Fc Receptors 1 st November 2011

1. Part II Lecture on Immunoglobulins and Fc Receptors1st November 2011 by Mike Clark, PhD Reader in Therapeutic and Molecular Immunology Department of Pathology Division of Immunology Cambridge University UK CB2 1QP http://www.antibody.me.uk/

2. IgG schematic

3. Animated Antibody Tutorial • An excellent animated antibody tutorial is available from Eric Martz’s website http://www.umass.edu/molvis/bme3d/materials/jmoltuts/antibody/contents/contents.htm

5. Dynamic model of IgG1

6. Domain structure of IgG Visit the webpage

7. Fc Region of human IgG1 Visit the webpage

8. Fab region structure Visit the webpage

9. Mammalian Antibody Classes • Antibodies are classed according to the type of immunoglobulin heavy chain • In addition each antibody class can have either κ (kappa) or λ (lambda) immunoglobulin light chains • In mammals we recognise the following classes and chains • IgM μ (mu) • IgD δ (delta) • IgG γ (gamma) • IgA α (alpha) • IgE ε (epsilon)

10. Mammalian Antibody Classes

11. Mammalian Antibody subclasses • In many species some of the classes are further subdivided into subclasses dependent upon the existence of multiple sub-types of immunoglobulin heavy chain • In humans the IgG class is divided into four subclasses IgG1, IgG2, IgG3 and IgG4, and the IgA class into IgA1 and IgA2 • In mice the IgG class is divided into IgG1, IgG2a, IgG2b and IgG3. In some laboratory strains of mice a fifth subclass called IgG2c has been identified although historically this was originally classified as an allelic form of the IgG2a subclass. • In rats the IgG class is divided into IgG1, IgG2a, IgG2b and IgG2c • Despite the similarity in nomenclature the subclasses have arisen independently in different species and thus there is no general functional relationship between the subclasses from each species.

12. Crystal structure of whole IgG2a L.J.Harris, S.B.Larson, K.W.Hasel, A.McPherson, "Refined structure of an intact IgG2a monoclonal antibody", Biochemistry 36: 1581, (1997)

13. Schematic view of IgG domains

14. Kabat database variability of VH sequences Mouse VH Human VH

15. Lysozyme CDR regions Antibody Fv

18. Unlike mouse the human IgG subclasses are very similar in sequence but they still have different properties

19. Fc Receptors • Cells of the immune system interact with antibodies using receptors that bind to conserved structures within the constant region domains of the immunoglobulin heavy chains, the so called Fc region • These receptors for antibody are thus called Fc receptors or FcR • Different FcR exist which show specificity for different classes and also for different subclasses of antibody • Of particular importance for therapeutic applications of antibodies are those receptors that recognise IgG, the FcγR

20. Characteristics of human IgG Fc Receptors FcR CD number Protein (Mw) Chromosome Transcripts Affinity Subunits / signaling FcgRI CD64 72 kDa 1q21.1 Ia, Ib, Ic High (108-109 M-1) 3>1>4>>>2 g chain FcgRI AGa motif Low (<107 M-1) IIa-R131: 3>1>>>2,4 IIa-H131: 3>1=2>>>4 FcgRII CD32 40 kDa 1q23-24 IIa, IIb1, IIb2, IIb3, IIc IIa: g chain IIa ITAM IIb ITIM IIc ITAM FcgRIII CD16 50-80 kDa 1q23-24 IIIa, IIIb IIIa: Medium (~2x107 M-1) IIIb: Low (< 107 M-1) 1=3>>>2,4 IIIa: g, x chain b chain in mast cells aAG = antigen presentation

21. Cell distribution and modulation of expression of leukocyte FcR Regulation of expression FcR Cell distribution Up Down FcgRI Monocytes, macrophages, dendritic cellsa, neutrophils (induced) G-CSF, IFN-g, IL-10 IL-4, IL-13 FcgRII Monocytes, macrophages, dendritic cellsa, neutrophils, eosinophils, basophils, B cells, T cellsa, placental endothelial cells, platelets IL-4, IL-13 FcgRIII IIIa: monocytesa, macrophages, NK cells, T cellsa IIIb: neutrophils TGF-b IL-4, IL-13 asubpopulation

22. Human IgG Fc Receptors CD64 CD32 CD16

23. Human Fcg Receptors and their Activities IgG1 IgG3 IgG4>>IgG2 IgG1=IgG3 >>>>>>> IgG4,IgG2 IgG1 IgG2=IgG3 >>IgG4 IgG1=IgG3 >>IgG4 >>>IgG2 FcgRI FcgRIIa FcgRIIb FcgRIIc FcgRIIIa FcgRIIIb (CD64) (CD32) (CD32) (CD32) (CD16) (CD16) a a a a a a-GPI ITAM- ITIM- ITAM- ITAM- ITAM- g2 g2 Inhibitory Affinity High Low-Med Low-Med Low-Med Low Low-Med (108 M-1) (2x106 M-1) (5x105 M-1) Alleles -- IIA-131H -- -- IIIA-158V NA1 IIA-131R IIIA-158F NA2 VV 20% VF 40% FF 40% HH 25% HR 50% RR 25% Slide courtesy of Bill Strohl, Centocor, September 2008

24. Cell signalling by immune complexes (1) Activation

25. Cell signalling by immune complexes (2) Inhibition

29. The Brambell receptor or FcRn • This is a very important receptor with respect to the mammalian IgG class of antibody, first hypothesised by Brambell and thus called by some FcRB • In adult mammals this receptor is widely expressed, particularly on cells of the endothlium and acts by rescuing IgG from degradation within the endocytic pathway • The receptor is also responsible for the transport of maternal IgG to the neonate, hence the alternative name neonatal FcR or FcRn

30. The IgG receptor FcRn Interaction with FcRn and with Protein A through similar region FcRn is important for IgG half-life and transport

31. Effect of half-life on antibody concentration FcRn increases the half-life of IgG to about 21 days as compared to IgM which has a half-life of about 3 days.

32. Pathogen encoded HSV-1 FcR gE (59 kDa + CHO) gI (41 kDa + CHO) Low-affinity FcR 1:1 complex forms high-affinity FcR Binds monomeric IgG in 1:1 ratio FcR function of gE and gI appears to be essential in vivo

33. Binding of human IgG to cells infected with HSV-1 250 G1m(1,17) 200 G2 G4 150 mean fluorescence uninfected control cells: 100 G1m(1,17) 50 G2 0 G4 1 10 100 1000 10000 IgG concentration, nM

34. Binding of human IgG1 allotype variants to cells infected with HSV-1 160 140 120 G1m(1,17) 100 mean fluorescence 80 G1m(3) 60 G1m(null) 40 20 0 0.1 1 10 100 1000 IgG concentration, nM

35. Atherton, A., Armour, K.L., Bell, S., Minson, A.C., & Clark, M.R. Eur J. Immunol. (2000) 30: 2540-547 The Herpes Simplex Virus Type 1 Fc Receptor Discriminates between IgG1 Allotypes