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IMMUNGLOBULINS STRUCTURE and FUNCTION

Immunoglobulin structure Valency, affinity, avidity Direct and indirect functions Immunoglobulin types Fc receptors Immunoglobulin transport Monoclonal and polyclonal antibodies Passive immunization Therapeutic antibodies Chimera and humanized monoclonal Ab

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IMMUNGLOBULINS STRUCTURE and FUNCTION

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  1. Immunoglobulin structure Valency, affinity, avidity Direct and indirect functions Immunoglobulin types Fc receptors Immunoglobulin transport Monoclonal and polyclonal antibodies Passive immunization Therapeutic antibodies Chimera and humanized monoclonal Ab Tumortherapy, Immunsuppression Case study (mieloma multiplex) B cell malignancies More and more therapeutic monoclonal antibodies CARs and TRUCKs IMMUNGLOBULINSSTRUCTURE and FUNCTION

  2. IMMUNOGLOBULINS Definition: Glycoprotein molecules that are present on B cells (BCR) or produced by plasma cells (antibodies) in response to an immunogen immunoglobulin ~ antibody

  3. STRUCTURE • heavy andlight chains • disulfide bonds • inter-chain • intra-chain disulfide bond carbohydrate CL VL CH2 CH3 CH1 hinge region VH

  4. disulfide bond carbohydrate CL CH2 CH3 CH1 hinge region VH STRUCTURE • variable andconstant regions • hinge region immunoglobulin domen • domains • VL & CL • VH & CH1 - CH3 (or CH4) • oligosaccharides VL

  5. Ribbon structure of IgG

  6. mIg = BCR

  7. Hypervariableregion - ComplementaryDeterminingRegion (CDR) 3 CDR regions in a V domain VH & VL domains 3+3 CDR (the circled part from the previous slide)

  8. IMMUNOGLOBULIN FRAGMENTS STRUCTURE/FUNCTION RELATIONSHIPS antigenbinding complement binding site placental transfer binding to Fc receptors

  9. papain VH VL IMMUNOGLOBULIN FRAGMENTS STRUCTURE/FUNCTION RELATIONSHIPS • Fab • antigenbinding (valence=1, monovalent) • specificity determined by VHand VL domains • some effector function is impaired Fc • Fc (effector functions) Fab

  10. IMMUNOGLOBULIN FRAGMENTS STRUCTURE/FUNCTION RELATIONSHIPS • F(ab’)2 • bivalent antigen binding (valency=2) pepsin Fc peptides + F(ab’)2 Some effektor functin is impaired. It can mediate agglutination/precipitation (other seminar)

  11. MODEL OF ANTIGENBINDING

  12. EFFECTORFUNCTION:NEUTRALIZATION neutralized pathogen / toxin toxin effect or pathogen adhesion is blocked by steric hindrance

  13. the (Fab)2 fragment can • detect antigen • precipitate antigen • block the active sites of toxins or pathogen-associated molecules • block interactions between host and pathogen-associated molecules but can not activate (role of Fc region) • inflammatory and effector functions associated with cells • inflammatory and effector functions associated with complement system • the trafficking of antigens into the antigen processing pathways (opsonized phagocytosis) Why do antibodies need an Fc region? NEUTRALIZATION

  14. EFFECTORFUNCTIONS OF ANTIBODIES • Neutralization (variable domain associated) • Marking theantigensforeffectorfunctions • (variábledomain and Fcdependent) • phagocytosis (oppsonization) • ADCC and IgEmediatedmastcellactivation • activation of thecomplementsystem(nextseminar) Fc part is recognizedbyFcreceptors DifferentantibodieshavedifferentFc part

  15. VARIABILITY IN DIFFERENT REGIONS OF THE IG DETERMINES IG CLASSES OR SPECIFICITY isotype allotype idiotype (Classes/subclasses) Sequence variability of H/L-chain constant regions Sequence variability of H and L-chain variable regions (individual, clone- specific) Allelic variants

  16. HUMAN IMMUNOGLOBULIN CLASSESencodedbydifferentstructuralgenesegments (isotypes) • IgG - gamma (γ) heavy chains • IgM - mu (μ) heavy chains • IgA - alpha (α) heavy chains • IgD - delta (δ) heavy chains • IgE - epsilon (ε) heavy chains light chain types • kappa (κ) • lambda (λ)

  17. Az Fcreceptors FcγR : γ IgG binding FcεR : ε IgE binding FcαR : α IgA binding

  18. OPSONIZATION Phagocytosis is ineffective/slowwithoutopsonization

  19. complex antigen antibodies complexed to complex antigen (A) High-affinity FcRs on the surface of the cell bind monomeric Ig before it binds to antigen. (B) Low-affinity FcRs bind multiple Igs that have already bound to a multivalent antigen.

  20. ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY (ADCC) lowaffinityFcRmediatedNKcell / Basophylgranulocytedegranulation

  21. MASTCELLDEGRANULATION Cross-linking of IgE via bound pathogen/allergen Mast cell pre-armed with antigen specific IgE degranulation pathogen expulsion (allergy symptoms)

  22. FEATURES OF ANTIBODY-ANTIGENINTERACTION - valency and avidity Valency: numbers of bonds between antigen and antibody Affinity: the strength of interaction between a specific antigen and one binding site of the antibody Avidity: „sum” of affinities of the binding sites of a given antibody

  23. MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 1. valence free IgM pentamer (star shape) 2 2 2 4 Antigen bound IgM(crab shape) 10

  24. MAIN CHARACTERISTICS OF ANTIBODY ISOTYPES 2.

  25. THE ROLE OF THE HINGEREGION

  26. 100-1000x erősebb az első válasznál antigénfüggő magas alacsony ANTIBODYPRODUCTIONDURING THE PRIMARYAND THE SECONDARYIMMUNERESPONSE

  27. ANTIBODY PRODUCTION DURING THE PRIMARY AND THE SECONDARY IMMUNE RESPONSE level of antibodies secondary response against antigen A primary response against antigen A primary response against antigen B napok Antigen A Antigen A and B

  28. ANTIBODY ISOTYPE AND FUNCTION IgG Isotype distribution in the body

  29. BEFORE BIRTH AFTER BIRTH breast milk IgA IgM 100% (adult) maternal IgG IgG IgA 0 3 6 9 1 2 3 4 5 adult month year PRODUCTION OF IMMUNOGLOBULINS

  30. Fcreceptors and transportingFcreceptors

  31. human FcgRn Az IgG placental transport: neonatal Fcg receptor (FcRn) humán MHCClass I (the structure of FcRn and MHC I is related)

  32. FcRn, FcγRn FcRn can transport IgG and albumin • Locatedindifferentcelltypes: • endothelialcells • epithelialcells (digestive and respiratorysystem) • differentimmunecellseg. professional APC (crosspresentation)

  33. FcRn (FcγRn) function pinocytosis IgG is bound with high affinity in acidic conditions IgG can be released at the opposite side of the epithelia: transcytosis IgG can be transported to various sites of the body circulation  tissues tissues  epithelial surfaces(IgG is more abundant in the rectum and in some upper respiratory tract than IgA) maternal circulation  foetal circulation

  34. Az FcRn salvages IgG from lysosomal degradation

  35. S S S S S S S S S S S S S S S S S S S S S S S S S S S S s s s s s s s s s s s s s s C C C C C C C J J J J J J J C C C C C C C S S S S S S S S S S S S S S C C C C C C C C C C C C C C pIgR and IgA are internalised S S S S S S S S S S S S S S Polymeric Ig receptors are expressed on the basolateral surface of epithelial cells to capture IgA produced in the mucosa C C C C C C C C C C C C C C B SECRETORY IgA AND TRANSCYTOSIS ‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR (the secretory component) MUC US IgA and pIgR are transported to the apical surface in vesicles Epithelial cell B cells located in the submucosa produce dimeric IgA

  36. The cargo of pIgR(poliimmunoglobulin receptor) Immunoglobulins with ”J-chain”: IgA, IgM (Do not confuse this J chain with the VJ/VDJ rearrangement’s J region!) multimeric IgM can exist without J chain, but not transported The destination epithelial surfaces, sercretums (saliva, breast milk)

  37. SUMMARY OF THE EFFECTORFUNCTIONS OF ANTIBODIES (next seminar)

  38. MONOCLONAL ANTIBODIES and POLYCLONAL ANTIBODIES

  39. Ag Ag Ag POLYCLONAL ANTIBODY RESPONSE Polyclonal antibody Immunserum Set of B-cells Activated B-cells Antibody-producing plasma-cells Antigen-specific antibodies

  40. PRUDUCTION OF MONOCLONAL ANTIBODIES

  41. MONOCLONAL ANTIBODIES • products of one B lymphocyte clone • homogeneous in antigen specificity, affinity, and isotype • can be found in pathologic condition in humans(the product of a malignant cell clone) • advantages against polyclonal antibodies: • antibodies of a given specificity and isotype • can be produced in high quantity and assured quality • therapeutic usage of monoclonal ABs: • anti-inflammatory antibodies (autoimmune diseases) • tumor therapy

  42. FEATURES OF POLYCLONAL AND MONOCLONAL ANTIBODIES

  43. POSSIBLE USE OF MONOCLONAL ANTIBODIES • Identifying cell types Immunohistochemistry Characterization of lymphomas with CD (cluster of differentiation) markers • Isolation of cells Isolation of CD34+ stem cells for autologous/allogeneic transplantation (from peripheral blood!) • Blood group determination (with anti-A, anti-B, and anti-D monoclonals) • Identification of cell surface and intracellular antigens Cell activation state • Targeted chemotherapy CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma Prevention of organ rejection after transplantation

  44. Monoclonal antibodies as drugs? Mouse monoclonal antibodies may elicit an immune response upon administration in human subjects. (see immunogenicity-determining factors!) How can we solve this problem?

  45. Human Mouse Humanized EVOLUTION OF MONOCLONAL ANTIBODIES Chimeric Humanized antibodies are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.

  46. mouse monoclonal antibodies immunization humanized mouse monoclonal antibodies immunization Human immunoglobulin transgenic mouse human monoclonal antibodies ENDANGERED SUBJECT PASSZÍV IMMUNIZÁLÁS PROTECTED SUBJECT serum antibody This is a case of PASSIVE IMMUNIZATION Immune system is not activated prompt effect temporary protection/effect Immunoglobulin degradation

  47. Passive immunization II. Natural passive immunization: maternal IgG placental transfer maternal IgA in breast milk

  48. PassiveimmunizationIII. ”Antisera” / Policlonalantibodies Antivenom snakes, scorpions, spiders, dangerous toxic sea creatures Slow, gradial hyperimmunization of large animals (horses) Purification of the gamma globulin fraction from the sera of the hyperimmunized animals (Purified animal proteins (Ig) are milder immunogens than raw serum in the patients body. Repeated therapy could be possible)

  49. MONOCLONALS AS DRUGS - Tumor therapy Monoclonals can be used for targeted chemotherapy of tumors. It is cell-type specific, but not specific to malignant cells! • Immunsuppressive monoclonals Cell-type specific immunsuppression

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