Exploring molecular determinants and pharmacokinetic properties of IgG1-scFv bispecific antibodies.

Bispecific antibodies (BsAbs) capable of recognizing two distinct epitopes or antigens offer promising therapeutic options for various diseases by targeting multiple pathways. The favorable pharmacokinetic (PK) properties of monoclonal antibodies (mAbs) are crucial, as they directly influence patient safety and therapeutic efficacy. For numerous mAb therapeutics, optimization of neonatal Fc receptor (FcRn) interactions and elimination of unfavorable molecular properties have led to improved PK properties.

An adapted consensus protein design strategy for identifying globally optimal biotherapeutics.

Biotherapeutic optimization, whether to improve general properties or to engineer specific attributes, is a time-consuming process with uncertain outcomes. Conversely, Consensus Protein Design has been shown to be a viable approach to enhance protein stability while retaining function. In adapting this method for a more limited number of protein sequences, we studied 21 consensus single-point variants from eight publicly available CD3 binding sequences with high similarity but diverse biophysical and pharmacological properties.

Characterization of a Novel Bispecific Antibody With Improved Conformational and Chemical Stability.

Bispecific antibodies containing single-chain variable fragment (scFv) appended to immunoglobulins G offer unique development challenges. Here, we describe the stability of a novel bispecific format, BiS5, where the scFv is tethered to the C3 domain. BiS5 showed an improved conformational and chemical stability compared with that of BiS4 in which the scFv is appended in the hinge region between the F and F.

Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody.

Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling.

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics.

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0).

Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples.

We developed atomic force microscope (AFM)-based protocols that enable isolation and characterization of antibody-based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single-chain antibody fragments (scFvs) that specifically recognize an oligomeric beta-amyloid (Aβ) species correlated with Alzheimer's disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We used several subtractive panning steps to remove all phage binding nondesired antigens and then used a single positive panning step using minimal antigen.

Bispecific tandem single chain antibody simultaneously inhibits β-secretase and promotes α-secretase processing of AβPP.

Misfolding and aggregation of amyloid-β (Aβ) is an important early event in the pathogenesis of Alzheimer's disease. Aβ is produced by sequential proteolysis of the amyloid-β protein precursor (AβPP) by β- and γ-secretases. A third protease, α-secretase, cleaves AβPP in the middle of the Aβ sequence precluding formation of Aβ.

CSF levels of oligomeric alpha-synuclein and beta-amyloid as biomarkers for neurodegenerative disease.

Protein misfolding and aggregation is a critically important feature in many devastating neurodegenerative diseases, therefore characterization of the CSF concentration profiles of selected key forms and morphologies of proteins involved in these diseases, including β-amyloid (Aβ) and α-synuclein (a-syn), can be an effective diagnostic assay for these diseases. CSF levels of tau and Aβ have been shown to have great promise as biomarkers for Alzheimer's disease. However since the onset and progression of many neurodegenerative diseases have been strongly correlated with the presence of soluble oligomeric aggregates of proteins including various Aβ and a-syn aggregate species, specific detection and quantification of levels of each of these different toxic protein species in CSF may provide a simple and accurate means to presymptomatically diagnose and distinguish between these diseases.

Nanobody specific for oligomeric β-amyloid stabilizes nontoxic form.

While accumulation and deposition of beta amyloid (Aβ) is a primary pathological feature of Alzheimer's disease (AD), increasing evidence has implicated small, soluble oligomeric aggregates of Aβ as the neurotoxic species in AD. Reagents that specifically recognize oligomeric morphologies of Aβ have potential diagnostic and therapeutic value. Using a novel biopanning technique that combines phage display technology and atomic force microscopy, we isolated the nanobody E1 against oligomeric Aβ.

Engineered proteolytic nanobodies reduce Abeta burden and ameliorate Abeta-induced cytotoxicity.

Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD), and reduction of Abeta levels in the brain could be a viable therapeutic approach. A potentially noninflammatory route to facilitate clearance and reduce toxicity of Abeta is to degrade the peptide using proteolytic nanobodies. Here we show that a proteolytic nanobody engineered to cleave Abeta at its alpha-secretase site has potential therapeutic value.

Promoting alpha-secretase cleavage of beta-amyloid with engineered proteolytic antibody fragments.

Deposition of beta-amyloid (A beta) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of A beta levels by various therapeutic approaches is actively being pursued. A potentially non-inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze A beta at its alpha-secretase site. We have previously identified a light chain fragment, mk18, with alpha-secretase-like catalytic activity, producing the 1-16 and 17-40 amino acid fragments of A beta 40 as primary products, although hydrolysis is also observed following other lysine and arginine residues.

Detecting morphologically distinct oligomeric forms of alpha-synuclein.

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of alpha-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of alpha-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of alpha-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD.

Anti-oligomeric Abeta single-chain variable domain antibody blocks Abeta-induced toxicity against human neuroblastoma cells.

The Amyloid-beta (Abeta) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Abeta are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Abeta using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM).