Identification and engineering of potent bispecific antibodies that protect against herpes simplex virus recurrent disease.

Herpes simplex virus (HSV) causes lifelong infections, including oral and genital herpes. There is no vaccine, and current antivirals are only partially effective at reducing symptoms and transmission. Therapeutic antibodies offer a potentially long-acting treatment option, although efforts to pursue this have been limited.

Trop-2 expression in non-small cell lung cancer.

Trophoblast cell-surface antigen 2 (Trop-2) is highly expressed in non-small cell lung cancer (NSCLC) and has become an attractive target for antibody-drug conjugates (ADCs). ADC tumor target expression is essential in investigating the predictive value of Trop-2 and Trop-2 ADC efficacy. Although Trop-2 mRNA expression in NSCLC has been described, protein-level expression is poorly understood.

Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy.

Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 () are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo.

Rapid Development of Glaucoma Via ITV Nonselective ANGPT 1/2 Antibody: A Potential Role for ANGPT/TIE2 Signaling in Primate Aqueous Humor Outflow.

Purpose: Investigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys.

Methods: ITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice.

Results: Following the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning.

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.

"Two-in-One" approach for bioassay selection for dual specificity antibodies.

Dual specific antibodies and bispecific antibodies that recognize two different antigen targets are currently being regarded as very effective therapeutics for complex human diseases. While effective, designing and developing a bioassay strategy for dual specific antibodies that is reflective of the mechanism of action (MoA) and also measures the dual activities of antibodies pose unique and exciting challenges. An important question asked while developing a bioassay for dual specific antibodies is, "How many bioassays will be needed, one bioassay or two separate bioassays?" Here we present an approach of using one bioassay for a dual specific antibody that targets two receptors in signaling pathways.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function.

Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics.

Purpose: To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody.

Methods: Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites.

A two-in-one antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions.

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only.

Inhibition of VEGF-C modulates distal lymphatic remodeling and secondary metastasis.

Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastatic cells. This notion is supported by preclinical findings that inhibition of pro-lymphangiogenic signaling during tumor development reduces cell spread to sentinel lymph nodes (SLNs). However, it is unclear how lymphatics downstream of SLNs contribute to metastatic spread into distal organs, or if modulating distal lymph transport impacts disease progression.

Translation levels control multi-spanning membrane protein expression.

Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli).

A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies.

Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions.

Assessing therapeutic responses in Kras mutant cancers using genetically engineered mouse models.

The low rate of approval of novel anti-cancer agents underscores the need for better preclinical models of therapeutic response as neither xenografts nor early-generation genetically engineered mouse models (GEMMs) reliably predict human clinical outcomes. Whereas recent, sporadic GEMMs emulate many aspects of their human disease counterpart more closely, their ability to predict clinical therapeutic responses has never been tested systematically. We evaluated the utility of two state-of-the-art, mutant Kras-driven GEMMs--one of non-small-cell lung carcinoma and another of pancreatic adenocarcinoma--by assessing responses to existing standard-of-care chemotherapeutics, and subsequently in combination with EGFR and VEGF inhibitors.

Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site.

The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF).

Improving antibody binding affinity and specificity for therapeutic development.

Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification.

Interaction between bevacizumab and murine VEGF-A: a reassessment.

Purpose: Bevacizumab is a humanized anti-human VEGF-A monoclonal antibody (mAb) approved by the United States Food and Drug Administration for cancer therapy and used off label to treat neovascular age-related macular degeneration. Earlier studies characterized bevacizumab as species specific and lacking the ability to neutralize murine (m) VEGF-A. However, a recent study reported that bevacizumab is a potent inhibitor of hemangiogenesis and lymphangiogenesis in murine models.

Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade.

Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes.

Mice expressing a humanized form of VEGF-A may provide insights into the safety and efficacy of anti-VEGF antibodies.

VEGF-A is important in tumor angiogenesis, and a humanized anti-VEGF-A monoclonal antibody (bevacizumab) has been approved by the FDA as a treatment for metastatic colorectal and nonsquamous, non-small-cell lung cancer in combination with chemotherapy. However, contributions of both tumor- and stromal-cell derived VEGF-A to vascularization of human tumors grown in immunodeficient mice hindered direct comparison between the pharmacological effects of anti-VEGF antibodies with different abilities to block host VEGF. Therefore, by gene replacement technology, we engineered mice to express a humanized form of VEGF-A (hum-X VEGF) that is recognized by many anti-VEGF antibodies and has biochemical and biological properties comparable with WT mouse and human VEGF-A.

Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells.

BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor.

Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the Avastin Fab.

In the quest to discover new research tools and to develop better agents in the fight against cancer, two antibodies, G6 and B20-4, were isolated from synthetic antibody phage libraries. Unlike the AVASTINtrade mark antibody, a recently approved agent for the treatment of patients with colorectal cancer, B20-4 and G6 bind and block both human and murine vascular endothelial growth factor (VEGF). Here we have analyzed and compared the binding epitopes on VEGF for these three antibodies using alanine-scanning mutagenesis and structural analyses.

Cross-species vascular endothelial growth factor (VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF.

To fully assess the role of VEGF-A in tumor angiogenesis, antibodies that can block all sources of vascular endothelial growth factor (VEGF) are desired. Selectively targeting tumor-derived VEGF overlooks the contribution of host stromal VEGF. Other strategies, such as targeting VEGF receptors directly or using receptor decoys, result in inhibiting not only VEGF-A but also VEGF homologues (e.

High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold.

Phage-displayed synthetic antibody libraries were built on a single human framework by introducing synthetic diversity at solvent-exposed positions within the heavy chain complementarity-determining regions (CDRs). The design strategy of mimicking natural diversity using tailored codons had been validated previously with scFv libraries, which produced antibodies that bound to antigen, murine vascular endothelial growth factor (mVEGF), with affinities in the 100nM range. To improve library performance, we constructed monovalent and bivalent antigen-binding fragment (Fab) libraries, and explored different CDR-H3 diversities by varying the amino acid composition and CDR length.

Bivalent antibody phage display mimics natural immunoglobulin.

We report the development of a system for displaying bivalent antibody fragments on M13 bacteriophage in a manner that effectively mimics the binding behavior of natural antibodies. In the "bivalent display" format, two copies of antigen binding sites are displayed on the coat of a single phage particle. Bivalent display was first achieved by the insertion of a dimerization domain, consisting of an IgG1 hinge region and a homodimerizing GCN4 leucine zipper, between a Fab and the C-terminal domain of the M13 gene-3 minor coat protein.