Download Monoclonal Antibodies

Monoclonal Antibodies
Past Present and Future
Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Clinical Applications
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Transplantation – muronomab (OKT3) 1986, basiliximab 1998
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Cardiovascular disease – abciximab 1994
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Cancer – rituximab 1997, trastuzumab 1998
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Viral infection – palivizumab 1998
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Inflammatory diseases – infliximab 1998, etanercept 1999
Side effects:
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Transfusion reactions (any adverse event which occurs because of a blood
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Infections, immunosuppression
Cardiac, respiratory arrest (discontinuation of breathing)
Pharmacological toxicity
transfusion)
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Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Production
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Monoclonal antibodies results from a clone of a B lymphocyte producing a
single antibody which will bind to a specific epitope of an antigen.
Monoclonal antibodies are produced:
– Fusion of a myeloma (B cell which has become cancerous) with a
spleen cell that is immunized with a specific antigen.
The resulting hybridomas are tested for the production of a monoclonal
antibodies.
Diluted to one cell cultures
Hypoxanthine Guanine Phosphoribosyl Transferase (HGPT) negative
myeloma cells
Grown in Hypoxanthine Aminopterin Thymidine (HAT) medium
Only successful fusion cells grow (rare)
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Therapeutic Agents
Recombinant Proteins
Monoclonal
Antibodies
Production:
Major Problems with non-human ABs:
Immunological Responses
 ”Humanization” of ABs
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Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Chimeric Ab
Humanized Ab
DNA that encodes the binding
portion of monoclonal mouse
antibodies and merges it with
human antibody-producing DNA.
65 – 90% human and consist
of the mouse variable regions
and substituting the mouse
Fc region of the antibody
with that from human
95% human, and are made by
grafting the hypervariable
region (or CDR) of the
chimeric antibody –which
determines Ab specificity
Use mammalian cell cultures to
express this DNA and produce
these half-mouse and half-human
antibodies. (Bacteria cannot be
used for this purpose, since they
cannot produce this kind of
glycoprotein.) Depending on how big
a part of the mouse antibody is
used, one talks about chimeric
antibodies or humanized
antibodies.
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Therapeutic Agents
Recombinant Proteins
Antibodies for human therapy derived without using mice:
-> uses various "display" methods
(primarily phage display) as well as
methods that exploit the elevated Bcell levels that occur during a human
immune response.
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Create new variations of antibodies
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New combinations of heavy and light
chains
mRNA from immunized individual
PCR H and L chains
Clone into vector in new H/L
combinations
-> create library -> screening by display
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Therapeutic Agents
Recombinant Proteins
Antibodies for human therapy derived without using mice:
Screening by Phage display
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Therapeutic Agents
Recombinant Proteins
Antibodies for human therapy derived without using mice:
Single-chain Fixed variable
only one heavy-chain variable
domain and one light-chain variable
domain covalently linked by peptide
Single-Chain Combinatorial Antibody Library
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Therapeutic Agents
Recombinant Proteins
Monoclonal Antibodies
Application of single-chain fixed variable-> Immunotoxins
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Protein toxin connected to Fv region
Single-chain or S-S linked Fv region
Toxin localized to antigen-expressing cells
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