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



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.



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.



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.



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)



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



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





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



Mab II variant

Average pI of ~7.8

Solubility increased up to

~29mg/ml



Hydrophobicity of CNTO607

Interact with high affinity to IL-13 (18 pM) but

also favours non-specific protein aggregation.



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



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.







Aggregation Hot Spot



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



Aggregation hot-spot



Conclusion

� The aromatic triad in the H-CDR3 sequence is

the hot spot for both AGGREGATION and

INTERACTION with IL-13



Experiment

� Find a way to disrupt the hot spot without

disrupting antigen binding

�

D53 of H-CDR2� Mutated to N



Addition of N-glycan



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





Conclusion

� N-linked carbohydrate existed and prevented

aggregation of the antibody while allowing it

to interact with the antigen with high affinity



Thermal Stability



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





Result



Conclusion

� No correlation between solubility and thermal

stability



Summary



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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CNT0607 Solubilities