Dataset Information

CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery.

ABSTRACT: While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

SUBMITTER: Fennell BJ

PROVIDER: S-EPMC3896602 | biostudies-literature | 2013 Nov-Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery.

Fennell Brian J BJ McDonnell Barry B Tam Amy Sze Pui AS Chang Lijun L Steven John J Broadbent Ian D ID Gao Huilan H Kieras Elizabeth E Alley Jennifer J Luxenberg Deborah D Edmonds Jason J Fitz Lori J LJ Miao Wenyan W Whitters Matthew J MJ Medley Quintus G QG Guo Yongjing J YJ Darmanin-Sheehan Alfredo A Autin Bénédicte B Shúilleabháin Deirdre Ní DN Cummins Emma E King Amy A Krebs Mark R H MR Grace Christopher C Hickling Timothy P TP Boisvert Angela A Zhong Xiaotian X McKenna Matthew M Francis Christopher C Olland Stephane S Bloom Laird L Paulsen Janet J Somers Will W Jensen Allan A Lin Laura L Finlay William J J WJ Cunningham Orla O

mAbs 20130821 6

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulati ...[more]

PMID: 23995618

Similar Datasets

Project description:BACKGROUND:Neuroblastoma is the most common pediatric solid tumor. About fifty percent of high risk patients die despite treatment making the exploration of new and more effective strategies for improving stratification mandatory. Hypoxia is a condition of low oxygen tension occurring in poorly vascularized areas of the tumor associated with poor prognosis. We had previously defined a robust gene expression signature measuring the hypoxic component of neuroblastoma tumors (NB-hypo) which is a molecular risk factor. We wanted to develop a prognostic classifier of neuroblastoma patients' outcome blending existing knowledge on clinical and molecular risk factors with the prognostic NB-hypo signature. Furthermore, we were interested in classifiers outputting explicit rules that could be easily translated into the clinical setting. RESULTS:Shadow Clustering (SC) technique, which leads to final models called Logic Learning Machine (LLM), exhibits a good accuracy and promises to fulfill the aims of the work. We utilized this algorithm to classify NB-patients on the bases of the following risk factors: Age at diagnosis, INSS stage, MYCN amplification and NB-hypo. The algorithm generated explicit classification rules in good agreement with existing clinical knowledge. Through an iterative procedure we identified and removed from the dataset those examples which caused instability in the rules. This workflow generated a stable classifier very accurate in predicting good and poor outcome patients. The good performance of the classifier was validated in an independent dataset. NB-hypo was an important component of the rules with a strength similar to that of tumor staging. CONCLUSIONS:The novelty of our work is to identify stability, explicit rules and blending of molecular and clinical risk factors as the key features to generate classification rules for NB patients to be conveyed to the clinic and to be used to design new therapies. We derived, through LLM, a set of four stable rules identifying a new class of poor outcome patients that could benefit from new therapies potentially targeting tumor hypoxia or its consequences.

Dataset Information

CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery.

Publications

CDR-restricted engineering of native human scFvs creates highly stable and soluble bifunctional antibodies for subcutaneous delivery.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets