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. However, many BsAbs exhibit unfavorable PK, which has precluded their development as drugs. In this report, we present studies on the molecular determinants underlying the distinct PK profiles of three IgG1-scFv BsAbs. Our study indicated that high levels of nonspecific interactions, elevated isoelectric point (pI), and increased number of positively charged patches contributed to the fast clearance of IgG1-scFv. FcRn chromatography results revealed specific scFv-FcRn interactions that are unique to the IgG1-scFv, which was further supported by molecular dynamics (MD) simulation. These interactions likely stabilize the BsAb FcRn interaction at physiological pH, which in turn could disrupt FcRn-mediated BsAb recycling. In addition to the empirical observations, we also evaluated the impact of in silico properties, including pI differential between the Fab and scFv and the ratio of dipole moment to hydrophobic moment (RM) and their correlation with the observed clearance. These findings highlight that the PK properties of BsAbs may be governed by novel determinants, owing to their increased structural complexity compared to immunoglobulin G (IgG) 1 antibodies.

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. All single-point consensus variants retained CD3 binding and performed similarly in cell-based functional assays. Using Ridge regression analysis, we identified the variants and sequence positions with overall beneficial effects on developability attributes of the CD3 binders. A second round of sequence generation that combined these substitutions into a single molecule yielded a unique CD3 binder with globally optimized developability attributes. In this first application to therapeutic antibodies, adapted Consensus Protein Design was found to be highly beneficial within lead optimization, conserving resources and minimizing iterations. Future implementations of this general strategy may help accelerate drug discovery and improve success rates in bringing novel biotherapeutics to market.

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 CH3 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 Fab and Fc. By switching the location of the scFv from hinge region to the CH3, there was an improved stabilization of CH2 and scFv domains. Interestingly, no noticeable impact was observed on the conformational stability of CH3 and Fab domains. BiS4 and BiS5 showed different aggregation and fragmentation rates under accelerated temperature stress conditions. BiS4 showed higher fragmentation rates compared with BiS5 likely owing to fragmentation in the linker region on either side of the scFv while BiS5 is more resistant toward fragmentation owing to tethering of scFv to the CH3 domain at its N and C terminus. In conclusion, the location of scFv affects both aggregation and fragmentation kinetics. These insights into the molecular structure and correlations with their physical and chemical stability will help formulation development of these novel bispecific antibodies.

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 BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.

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 (Aß) 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. We also used AFM to characterize the specificity of the isolated clones, again using minimal material, selecting the C6 scFv based on expression levels. We show that C6 selectively binds cell and brain-derived oligomeric Aß. The protocols described are readily adapted to isolating antibody-based reagents against other antigenic targets with limited availability.

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). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.

Nanobody specific for oligomeric ß-amyloid stabilizes nontoxic form.

While accumulation and deposition of beta amyloid (Aß) is a primary pathological feature of Alzheimer's disease (AD), increasing evidence has implicated small, soluble oligomeric aggregates of Aß as the neurotoxic species in AD. Reagents that specifically recognize oligomeric morphologies of Aß 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 Aß. Here we show that E1 specifically recognizes a small oligomeric Aß aggregate species distinct from the species recognized by the A4 nanobody previously reported by our group. While E1, like A4, blocks assembly of Aß into larger oligomeric and fibrillar forms and prevents any Aß induced toxicity toward neuronal cells, it does so by binding a small Aß oligomeric species, directing its assembly toward a stable nontoxic conformation. The E1 nanobody selectively recognizes naturally occurring Aß aggregates produced in human AD brain tissue indicating that a variety of morphologically distinct Aß aggregate forms occur naturally and that a stable low-n nontoxic Aß form exists that does not readily aggregate into larger forms. Because E1 catalyses the formation of a stable nontoxic low-n Aß species it has potential value as a therapeutic reagent for AD which can be used in combination with other therapeutic approaches.

Bispecific tandem single chain antibody simultaneously inhibits ß-secretase and promotes α-secretase processing of AßPP.

Misfolding and aggregation of amyloid-ß (Aß) is an important early event in the pathogenesis of Alzheimer's disease. Aß is produced by sequential proteolysis of the amyloid-ß protein precursor (AßPP) by ß- and γ-secretases. A third protease, α-secretase, cleaves AßPP in the middle of the Aß sequence precluding formation of Aß. The levels of Aß generated from AßPP can therefore be controlled by tailoring activity of these proteases toward AßPP. We previously showed that ß-secretase proteolysis of AßPP could be selectively inhibited using the single chain antibody fragment (scFv) iBSEC1, which blocks the cleavage site on AßPP, and α-secretase proteolysis of AßPP could be selectively enhanced using a proteolytic scFv (Asec1A) engineered to have α-secretase-like activity. Here we show that DIA10D, a novel tandem bispecific scFv combining iBSEC1 with the ASec1A can control amyloidogenic processing of AßPP by simultaneously inhibiting ß-secretase and increasing α-secretase processing of AßPP. When expressed in H4 (neuroglioma) cells overexpressing AßPP, DIA10D potently reduces levels of extracellular Aß by around 50% while also increasing levels of neuroprotective sAßPPα. DIA10D activity has been designed to selectively target AßPP, so this modulation of AßPP processing should not affect endogenous activity of α-and ß-secretases towards other substrates.

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 ß-amyloid (Aß) and α-synuclein (a-syn), can be an effective diagnostic assay for these diseases. CSF levels of tau and Aß 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 Aß 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. Here we show that the presence of different protein morphologies in human CSF samples can be readily detected using highly selective morphology specific reagents in conjunction with a sensitive electronic biosensor. We further show that these morphology specific reagents can readily distinguish between post-mortem CSF samples from AD, PD and cognitively normal sources. These studies suggest that detection of specific oligomeric aggregate species holds great promise as sensitive biomarkers for neurodegenerative disease.

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. The Asec-1A proteolytic nanobody, derived from a parent catalytic light chain antibody, prevents aggregation of monomeric Abeta, inhibits further aggregation of preformed Abeta aggregates, and reduces Abeta-induced cytotoxicity toward a human neuroblastoma cell line. The nanobody also reduces toxicity induced by overexpression of the human amyloid precursor protein (APP) in a Chinese hamster ovary (CHO) cell line by cleaving APP at the alpha-secretase site which precludes formation of Abeta. Targeted proteolysis of APP and Abeta with catalytic nanobodies represents a novel therapeutic approach for treating AD where potentially harmful side effects can be minimized.

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. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking alpha-secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for A beta. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the alpha-secretase site of A beta. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3- and 6-fold increase in catalytic activity (k(cat)/K(M)) toward the synthetic A beta substrate compared to the original scFv primarily due to an expected decrease in K(M) rather than an increase in k(cat). This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for A beta. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble A beta levels in vivo.

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. Reagents that can interact with specific aggregate forms of alpha-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of alpha-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of alpha-synuclein in vitro, blocks extracellular alpha-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.

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). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Abeta, we identified an scFv that bound oligomeric Abeta specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Abeta aggregation and toxicity, and reduces the toxicity of preformed oligomeric Abeta towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Abeta scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Abeta in AD brains.