Monoclonal Antibodies

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• Monoclonal Antibodies
• Antibodies (Abs). Also known as immunoglobulins
  (Ig).
• Comprised of 2 heavy chains and 2 light chains
• Monoclonal Abs bind specifically to a single
  site (epitope) on a particular antigen
• - Abs are produced by B lymphocytes
• Because of their specificity and ease of
  generation, they are extensively used as
  therapeutics (passive immunotherapy) and as
  diagnostic and research tools
• -They can be generated in large (unlimited)
  amounts in vitro

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Antibodies are made by B-cells
B cells develop in the bone marrow ?
hematopoietic stem cells and
lymphoid stem cells
? T-cells and B-cells progenitor pro-B
cell (B220) precursor pre-B cells
heavy-chain rearranged immature B cell
IgM light-chain rearranged mature B cell IgM
IgD an antigen encounter in spleen or lymph
nodes goes to periphery Terminally
differentiated cell plasma cell, periphery, Ig
secretor
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Domain structure of an immunoglobulin molecule
disulfide bonds
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Laboratory fragmentation of antibodies
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Ig molecule showing polarity, disulfides,
carbohydrate
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Fc functions
Opsonization Complement activation Antibody-depe
ndent cell-mediated cytotoxicity
(ADCC) Transcytosis
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Fc functions
Opsonization Direct uptake of bacteria coated
with antibody molecules Complement activation
Activated complement proteins lyse cells by
making holes in their mebranes(e.g.
bacteria) Antibody-dependent cell-mediated
cytotoxicity (ADCC) Killer T-cells use
antibodies on their surface to target cells with
an antigen and kill them. Transcytosis Antibody
-antigen complexes are taken up (endocytosed) on
on side of an epithelial cell and directed to
theother side, where they are exocytosed
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Multigene organization of Ig genes light
chains V, J (variable) and C (constant) heavy
chain V, D, J, (variable) C (constant) Mechanis
m of Ab gene rearrangement Recombination signal
sequences (RSS)flank V, D, J gene
segments V-RSS------RSS-D-RSS---------RSS-J
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IgGkappa gene rearrangement
SOMATIC HYPERMUATION
SPLICING
SPLICING
SOMATIC HYPERMUATION
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Antibodies can participate in host defense in
three main ways
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Got this far
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ADCC antibody-dependent cell-mediated
cytotoxicity
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MAb therapy targets Inflammation Autoimmune
disease Graft rejection Heart disease
(thrombosis) Cancer Viral infection
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Therapeutic strategies Mabs straight Mabs fused
to other protein binders (e.g., soluble
receptors) Mabs fused to cytotoxic agents
(toxins, radionuclides) Toxins ricin (stops
protein synthesis) calicheamicin (DNA
breaks) Radionuclides 90Y yttrium 111I
indium
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• Problems of mouse MAbs
• Fc portion limited in its ability to interact
  with Fc receptors of human cells.
• Lower serum half-life
• Development of human anti-mouse antibodies (HAMA)
• Retreatment results in allergy or anaphylactic
  shock
• Retreatment is less effective

Breedveld, Lancet 2000 3559205

• Solutions via recombinant DNA genetic engineering
  
• Chimeric mouse-human antibodies Hu V-region
  fused to mouse C regions
• Humanized mouse antibodies, Parts of V-region
  from human interspersed with mouse CDR V-regions
• Human antibodies (fully), via transgenic mice
  carrying human immunoglobulin genes(Medarex,
  Abgenix, Kirin)

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MAbs approved for human therapy
IL-2 immunosupressant
Respiratory infection Synciitial Virus
HER-2/neu (EGF2) breast
cancer
CD33 leukemia (AML)
IgE asthma
EGF-R colon cancer
VEGF colon cancer
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• Monoclonal antibody generation
• - Cells needed myeloma cells, mouse spleen cells
  
• - antigen administration Kohler and Milstein
• - hybridoma formation via cell fusion
• selection mutants required defer
• - antibody generation cDNA cloning
• - engineered MAbs expression vectors
• - refinement chimeric, humanized, human (defer)

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Monoclonal antibodies via cell hybridization
Selects for rare hybrid cells
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Reduced myeloma hybrid
Unreduced myeloma hybrid
Isoelectric focusing immunoglobulins made
in hybridoma cells
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Mab Fusion Proteins Other protein-binding
proteins natural receptors in soluble
form Analogous to MAbs and make use of the Fc
portion of the antibody molecule Example
Enbrel (etanercept) Anti-rheumatoid arthritis
drug Soluble TNF receptor fused to the Fc IgG1
domain (TNF tumor necrosis factor) Ties up TNF,
blocking its inflammatory function Fc domain
dimerizes the receptor, which increases its
affinity for TNF. Fc domain increases the
half-life of the protein in the bloodstream Amgen
Wyeth
Still experimental anti HIV drug PRO 542
Soluble CD4 (HIV receptor) fused to IgG2.
Tetrameric (4 V-regions replaced) Reduced Fc
function (since IgG2 lt IgG1), Better
half-life Progenics
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Single chain antibodies (scFv)
Ag binding site
15 AA linker