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8/7/2019 CNT0607 Solubilities
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Structure-based engineering of
a monoclonal antibody for
improved solubilitySheng-Jiun Wu, Jinquan Luo, Karyn T.ONeil, James Kang, Eilyn
R.Lacy, Gabriela Canziani, Audrey Baker, Maggie Huang, Qing MikeTang, T.Shantha Raju, Steven A.Jacobs, Alexey Teplyakov, Gary
L.Gilliland and Yiqing Feng
Protein Engineering, Design and Selection (2010) 23 (8): 643-651.doi: 10.1093/protein/gzq037
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IL-13 and anti-IL-13 mAb
� Interleukin-13 (IL-13) is an important component intriggering the allergic responses by inducing T helper 2(TH2) cells and promoting the production of IgE.
� IL-13 plays a critical role in the pathogenesis of asthma. In
humans, IL-13 levels are upregulated both systemically andin the lungs during asthmatic attacks.
� Neutralizing anti-IL-13 monoclonal antibody (mAb) cantherefore provide therapeutic benefits to asthmaticpatients.
� Yang et al. in 2004 proved that neutralizing anti-IL-13antibody can significantly suppress eosinophil infiltration,proinflammatory cytokine and serum IgE production, andairway remodeling.
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The problem with CNTO607
� CNTO607 is a potent neutralizing anti-IL-13 mAb
� It is open for glycosylation when expressed in
mammalian cells
± presence of a consensus N-linked glycosylation site
(53NSS55) in heavy-chain CDR2 (H-CDR2)
� N53 was replaced with D to improve homogeneity.
� Results in mAb with retained high affinity for IL-13 but with low solubility and aggregation near
neutral pH.
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The problem with CNTO607
� Poor antibody solubility can lead to poor
cellular distribution, immunogenicity and
unwanted side effects.
� Protein aggregates can trigger immune
response.
� It is essential to improve CNTO607 solubility to
ensure effective therapy.
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Project Aims
� To improve anti-IL-13 mAb CNTO607 solubility
whilst maintaining the high affinity binding to
IL-13 using 3 structure-based protein
engineering approaches:
± Altering the isoelectric point (pI)
± Decreasing the overall surface hydrophobicity
± Re-introducing an N-linked carbohydrate moiety
within a complementarity-determining region
(CDR)
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Structure of antibody
Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal
hemoglobinuria. P Rother et al. Nature Biotechnology 25, 1256 - 1264 (2007)doi:10.1038/nbt1344
Mutation site for
pI alteration
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Solubility = maximum protein concentration
that can be achieved
before precipitation is observed
Highest concentration of CNTO607 in PBS
buffer at pH 7.2 is ~13 mg/ml
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The effect of pI
� Solubility of a protein is often lowest near its pI,therefore changing the pI would give a moresoluble antibody.
� Mab II variant was constructed with the lightchain variable fragment (Fv) sequences alteredat
±
framework region 1: E3 V3 and± framework region 3: 77GTQAE81 77RVEAG81,
� Mab II was predicted to have a pI of 7.9
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Mab II variant
Average pI of ~7.8
Solubility increased up to
~29mg/ml
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Hydrophobicity of CNTO607
Interact with high affinity to IL-13 (18 pM) but
also favours non-specific protein aggregation.
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The effect of surface hydrophobicity
� Residues in L-CDR 3 is less important for binding to IL-13
� Replaced to hydrophilic residues to give 3 more variants with similarbinding affinities to CNTO607: Mab III, Mab IV and Mab V
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The effects of L-CDR3 hydrophobicity
� Hydrophobicity of the variants were determined usinghydrophobic interaction chromatography (HIC).
� Mab III, IV and V were eluted earlier than CNTO607.
� However, their solubility were not impressivelyimproved.
± Mab III: ~25 mg/ml
± Mab IV: ~12 mg/ml
±
Mab V: ~ 29 mg/ml� L-CDR3 hydrophobicity was not important for
aggregation of CNTO607.
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Aggregation Hot Spot
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Experiment
� H-CDR3
� Mutated the triad by mutating all of them to
alanine (99FHW100a
99AAA100a)� Result:
± Mab VI
± Highly soluble in PBS (>160 mg/ml)
± But, no longer binds to IL-13
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Aggregation hot-spot
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Conclusion
� The aromatic triad in the H-CDR3 sequence is
the hot spot for both AGGREGATION and
INTERACTION with IL-13
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Experiment
� Find a way to disrupt the hot spot without
disrupting antigen binding
�
D53 of H-CDR2� Mutated to N
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Addition of N-glycan
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Result
� Mab VII
± Solubility: >110 mg/ml
± Bound to IL-13 with significant affinity
� Higher molecular weight band on SDS-PAGE
� Significant increase in pI
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Conclusion
� N-linked carbohydrate existed and prevented
aggregation of the antibody while allowing it
to interact with the antigen with high affinity
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Thermal Stability
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Experiment
� to determine whether low thermal stability
contributed to the low solubility of some of
the variants by measuring the melting
temperature
� Differential Scanning Calorimetry
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Conclusion
� No correlation between solubility and thermal
stability
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References
� G. Yang et al. Anti-IL-13 monoclonal antibody
inhibits airway hyper-responsiveness,
inflammation and airway remodeling (2004).
Cy tokine 28(6), p224-232
� A. S. Rosenberg (2006) Effects of Protein
Aggregates: An Immunologic Perspective.
AAPS Journal 8(3), e501-507
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