MOLECULAR BASIS OF ANTIGEN RECOGNITION BY B CELLS AND

ANTIBODIES

The immunoglobulin G (IgG) molecule

COMPLEMENT ACTIVATION

BINDING TO CELLS

DEGRADATION

TRANSPORT

ANTIGEN BINDING

IgG is built from twelve similar shaped immunoglobulin domains

The three-dimensional structure of immunoglobulin C and V domains

There are discrete regions of hypervariability in V domens

•Distinct regions of high variability and conservation led to the concept of a

FRAMEWORK (FR), on which hypervariable regions were

suspended.•Most hypervariable regions coincided with antigen contact points - the

COMPLEMENTARITY DETERMINING REGIONS (CDRs)

The hypervariable regions of antibody V domains lie in discrete loops at one end of the domain structure

CDR: complementarity-determining region

• Within the V domain, sequence variability is localized to

three complementarity-determining regions (CDRs).

• CDRs form three loops that are clustered at one end of the

domain.

• In the antibody molecule the CDRs of the heavy and light

chains form a variable surface that binds antigen.

Mechanisms of epitope recognition

• Linear and discontinuous epitopes

• Multivalent Antigens

• Polymeric Antibodies

• Epitope binding mechanisms

The nature of antigenic determinants

Two kinds of multivalent antigen

IgM is secreted as a pentamer of immunoglobulin monomers

IgA molecules can form dimers

Transcytosis of dimeric IgA antibody across epithelia is mediated by the poly-Ig receptor (pIgR)

IgG is a highly flexible molecule

Different hinge structures distinguish the four subclasses of IgG

Each human immunoglobulin isotype has specializedfunctions correlated with distinctive properties

Maternal IgG is transported by the neonatal Fc receptor (FcRn) across the placenta to the fetus

IgG half-life• FcRn is also present in the adult and involved in protecting IgG from

degradation• Accounts for the long (3 week) half-life of IgG compared to other Ig

isotypes

• Therapeutic agents that are fused to IgG Fc regions take advantage of this property e.g. Enbrel (TNFR-Fc)

Half-life in serumIgG - 21 daysIgA - 6 daysIgM - 5 daysIgE - 2.5 days

The receptor FcRn transports IgG from the bloodstream into the extracellular spaces of tissues

Epitopes can bind to pockets, grooves, extended surfaces, or knobs in antigen-binding sites

The noncovalent forces that hold together the Ag-antibody complex

• Pentameric IgM and dimeric IgA are synthesized in association with the same J chain.

• A poly-Ig receptor (pIgR) is responsible for the transcytosis of dimeric IgA across epithelia.

• FcRn receptors:- transport IgG across the placenta.

- transport IgG from the bloodstream into the extracellular spaces of tissues.

- protect IgG from degradation.

• Antigens are bound to antibodies through noncovalent bonds.

B cell antigen receptor complex

Cross-linking of B-cell receptors by antigens initiates a cascade of intracellular signals

ITAM: immunoreceptor tyrosine-based activation motif

Signal transduction by the BCR complex

Structure and function of the B-cell co-receptor

Signals generated from the B-cell receptor and co-receptor combine to activate B cells in response to surface and soluble antigens

• B cell activation requires

- cross-linking of surface immunoglobulin

- signals from the B-cell co-receptor

(or from PRR-s)

- helper T cells (in the case of protein antigens)

Production of a mouse monoclonal antibody

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

- Therapeutic usage of monoclonal ABs

CD20+ anti-B-cell monoclonals in non-Hodgkin lymphoma

Prevention of organ rejection after transplantation

Anti-inflammatory antibodies (autoimmune diseases)

Monoclonals in tumor therapy

1. „Naked MAb”, unconjugated antibodyAnti-CD20 (rituximab – Mabthera/Rituxan, chimeric): B-cell Non-Hodgkin lymphomaAnti-CD52 (campath – Mabcampath, humanized): chronic lymphoid leukaemiaAnti-ErbB2 (trastuzumab – Herceptin, humanized): breast cancerAnti-VEGF (bevacizumab – Avastin, humanized): colorectalis tu. (+ Lucentis!)Anti-EGFR (cetuximab – Erbitux, chimeric): colorectalis tu. (+ Vectibix, rekomb. human)

2. Conjugated antibodyAnti-CD20 + yttrium-90 isotope (ibritumomab- Zevalin)Anti-CD20 + iodine-131 (tositumomab – Bexxar)

Monoclonal antibodies as treatments for disease

The antibodies made by a hybridoma cell line are all identical and are therefore called monoclonal antibodies.

Monoclonal antibodies are applied in:

- research

- diagnostic tests

- therapeutic treatments