Read Therapeutic monoclonal antibodies text version

Therapeutic monoclonal antibodies

F. Edward Boas 2005-03-01

Antibody structure

IgG

PDB code: 1IGT

Hybridomas that secrete monoclonal antibodies

MBOC, 3rd edition, pg. 188

FDA-approved therapeutic antibodies

Hudson and Souriau. (2003) Nature medicine. 9(1): 129-34.

· 30 antibodies now in late stage clinical trials · $5.1 billion in annual sales

Problems with therapeutic antibodies

Problems Immunogenicity Affinity / stability Difficult binding targets Antibody size prevents tissue permeation

Solutions Humanization (loop grafting, transgenic mice) Selections on phage display libraries Use other scaffolds (camelid antibodies) Use Fab fragments

Humanization

Protein design labs

Binding loops from a murine antibody are transferred to a human antibody.

Biovation

Predicted T-cell epitopes are mutated and screened for activity and immunogenicity.

www.pdl.com

Mouse antibody Human antibody Modified residues

Fully human antibodies produced by transgenic mice

Xenomouse (Abgenix)

Jakobovits (1995) Curr. Opin. Biotech. 6: 561-6.

Techniques for affinity maturation

Hudson and Souriau. (2003) Nature medicine. 9(1): 129-34.

Difficult binding targets for mammalian antibodies

Deep pockets Prions

Camelid antibodies can reach deep into the active site of lysozyme, something mammalian Amyloid antibodies can't do. Camelid antibodies can inhibit formation of amyloid from lysozyme with the amyloidogenic mutation D67H.

HIV gp120

This protein's flexibility somehow misleads the immune system into producing antibodies that bind but do not neutralize the virus.

Specificity

Desmyter et al. (1996) Nature structural biology. 3(9): 803-811. Lysozyme -helix Lysozyme -sheet Camelid antibody constant region Camelid antibody CDR3 loop

Michaud et al. (2003) "Analyzing antibody specificity with whole proteome microarrays" Nature biotech. 21(12): 1509.

Antibody fragments

Fragments are smaller, easier to express, and penetrate tissues better. Avidity can be recovered by using multivalent fragments.

Hudson and Souriau. (2003) Nature medicine. 9(1): 129-34.

Multifunctional antibodies

Antibody anti-CD20 (various) (various) anti-ganglioside GD2 anti-HER2 (various)

Conjugate / modification Covalently attached tiuxetan (yttrium-90 chelator) Covalently attached PEG Mutant Fc domain with higher affinity for Fc receptors IL-2 fusion Anchored to a liposome containing doxorubicin Enzyme fusion

Application Radiotherapy for non-Hodgkin lymphoma (Zevalin) Increased serum half life Increased serum half life Recruit T-cells to tumor In animal model of breat cancer, worked better than anti-HER2 or doxorubicin alone. Prodrug activation (esp. for cancer)

Multifunctional non-antibody proteins (Avidia)

Avidia evolves binding sites into human, non-glycosylated (easier to express), non-antibody (not covered by patents) scaffold proteins, then links several monomers into a multifunctional protein. Protein monomers EGF-like domain Kringle fibronectin BPTI Trefoil domain C-type lectin von Willebrand factor SH2 SH3

US patent application 20030082630 (Cominatorial libraries of monomer domains)

www.avidia.com

Interesting selections

Internalization

Present phage display library to a tumor cell line, and select internalized phage. Selected phage targeted transferrin receptor and ErbB2. They did not internalize into non-cancer cells.

MA Poul et al. (2000) "Selection of tumor-specific internalizing human antibodies from phage display libraries" Journal of molecular biology. 301: 1149-61.

Aggregation

Heat denature phage display library of antibody heavy chains, then select for binders.

L Jespers et al. (2004) "Aggregation-resistant domain antibodies selected on phage by heat denaturation" Nature biotech. 22(9): 1161.

Computational protein design: Beyond stability, affinity, and specificity

Binding sites in non-antibody scaffolds

Fix some of the problems with antibody scaffolds: glycosylation, size, stability/aggregation.

Aggregation

Model assuming that aggregate consists of buried -sheets correctly predicts aggregation from primary sequence with 87% accuracy

AM Fernandez-Escamilla et al (2004). Nature biotech. 22(10): 1302.

Immunogenicity

Model based on antigenic propensities of surface amino acids predicts antigenic sites with 75% accuracy.

AS Kolaskar and PC Tongaonkar (1990). FEBS. 276: 172-4.

Information

Therapeutic monoclonal antibodies

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