Antibody Humanization Paolo Marcatil ipmar/humanization.pdf · Antibody against VEGF developed on...
Transcript of Antibody Humanization Paolo Marcatil ipmar/humanization.pdf · Antibody against VEGF developed on...
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Antibody Humanization
Paolo Marcatili
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HAMA
HUMANIZATION
Murine monoclonal Abs
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Rationale
We can engineer the antibody so that it "looks" human.
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CH2
CL
CH1
CH3
VH
VL
Antibodies are modular
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!
VH VL
ABS
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Chimeric antibody
Human Constant domain Murine variable domains
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Chimeric antibody
Human Constant domain Murine variable domains Still antigenic!
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Chimeric vs humanised
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Chimeric vs humanised
How much can we humanise without loosing affinity?
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Humanization strategies
CDR grafting: Use only the murine CDRS SDR grafting: CDR + structurally important residues (Canonical Structures)
Resurfacing: Change all surface residues (apart to CDR) to human Superhumanization: Check for each segment in the Ab and add mutation
to maximize local similarity to the human
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Humanization strategies
CDR grafting: Use only the murine CDRS SDR grafting: CDR + structurally important residues (Canonical Structures)
Resurfacing: Change all surface residues (apart to CDR) to human Superhumanization: Check for each segment in the Ab and add mutation
to maximize local similarity to the human
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Humanization strategies
CDR grafting: Use only the murine CDRS usually the antibody loses affinity SDR grafting: CDR + structurally important residues (Canonical Structures) often binding is poor
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Antibody Paratope
The binding probability of each residue depends on all the environment. Even distant residues might change the binding mode
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Framework residues affect antigen binding (Mouse)
Antigen
CDR
FR residue
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Framework residues affect antigen binding (Mouse)
FR residue
CDR
Antigen
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Humanization process
“Trial and error cycle” Human
Framework
In vitro evaluation
Back mutations
Regions for grafting
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A novel, integrated method to facilitate and automate the humanization process.
Project Goal
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Known data (Antibody sequences, Antigen contacting
residues)
Machine learning techniques
Prediction method
Input data (Antibody
sequences)
Output data (Antigen contacting
residues)
Project Workflow
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Known data 313 antibody-antigen
complexes Machine learning
Random Forest algorithm
proABC (prediction of
AntiBody Contacts)
Input data (Antibody sequence)
Output data (residue-based
contact probability)
Pipeline
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Performance Evaluation
AUC: 84% MCC: 52%
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Prediction of Antibody Contacts
Olimpieri et al. Prediction of site-specific interactions in antibody-antigen complexes: the proABC method and server. Bioinformatics 29, 2285-2291
www.biocomputing.it/proABC
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Antibody Fingerprint
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Humanization of an Anti-Vascular Endothelial Growth Factor Monoclonal Antibody for the Therapy of Solid Tumors and Other Disorders. Leonard G. Presta, Helen Chen, Shane J. O’Connor et al. Cancer Res 1997; 57:4593-4599 Antibody against VEGF developed on mouse Drafting of the CDRs Test of affinity Back mutation (8 rounds)
proABC for humanization Humanization of an Anti-Vascular Endothelial Growth Factor Monoclonal
Antibody for the Therapy of Solid Tumors and Other Disorders.
Leonard G. Presta, Helen Chen, Shane J. O’Connor et al.
Cancer Res 1997; 57:4593-4599
Why this paper?
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1 6 12 18 24 30 34 40 46 52 55 61 67 73 79 83 89 95 100C 100U 108
020
4060
80Mouse
Pro
babi
lity
(Per
cent
age)
Position
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1 6 12 18 24 30 34 40 46 52 55 61 67 73 79 83 89 95 100C 100U 108
020
4060
80Grafted vs Mouse
Pro
babi
lity
(Per
cent
age)
Position
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1 6 12 18 24 30 34 40 46 52 55 61 67 73 79 83 89 95 100C 100U 108
020
4060
803 back mutations
Pro
babi
lity
(Per
cent
age)
Position
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1 6 12 18 24 30 34 40 46 52 55 61 67 73 79 83 89 95 100C 100U 108
020
4060
808 back mutations
Pro
babi
lity
(Per
cent
age)
Position
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Humanization process
“Trial and error cycle” Human
Framework
In vitro evaluation
Back mutations
Regions for grafting
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The Tabhu web server
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Template Selection
www.biocomputing.it/tabhu
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The Tabhu web server
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Antibody humanization is needed for any Ab drug It is still a "trial and error" procedure non CDR residues can greatly affect the binding Structural analysis is extremely important
Conclusion