High-Throughput Generation of Bipod (Fab × scFv) Bispecific Antibodies Exploits Differential Chain Expression and Affinity Capture.

Generation of bispecific antibodies (BsAbs) having two unique Fab domains requires heterodimerization of the two heavy chains and pairing of each heavy chain with its cognate light chain. An alternative bispecific scaffold (Bipod) comprising an scFv and a Fab on a heterodimeric Fc eliminates the possibility of light chain mispairing. However, unpredictable levels of chain expression and scFv-induced aggregation can complicate purification and reduce the yield of desired Bipod. Here, we describe a high-throughput method for generation of Bipods based on protein A and CH1 domain affinity capture. This method exploits over-expression of the scFv chain to maximize heterodimer yield. Bipods purified by this method have purity suitable for cell-based functional assays and in vivo studies.

Generation of a high-fidelity antibody against nerve growth factor using library scanning mutagenesis and validation with structures of the initial and optimized Fab-antigen complexes.

Nerve growth factor (NGF) is indispensable during normal embryonic development and critical for the amplification of pain signals in adults. Intervention in NGF signaling holds promise for the alleviation of pain resulting from human diseases such as osteoarthritis, cancer and chronic lower back disorders. We developed a fast, high-fidelity method to convert a hybridoma-derived NGF-targeted mouse antibody into a clinical candidate. This method, termed Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope. Functional and structural comparisons between tanezumab and the mouse 911 precursor antibody using neurotrophin-specific cell survival assays and X-ray crystal structures of both Fab-antigen complexes illustrated high fidelity retention of the NGF epitope. These results suggest the potential for wide applicability of the LSM method for optimization of well-characterized antibodies during humanization.

Structural basis of C-terminal ß-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease.

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of ß-amyloid (Aß) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aß40. Ponezumab can label Aß that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aß. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aß present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aß40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aß. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aß40, we determined the X-ray crystal structure of ponezumab in complex with Aß40 and found that the Aß40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AßV40 in the Aß-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aß40 and the brain Aß-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.