Matrixed CDR grafting: A neoclassical framework for antibody humanization and developability.

Discovery and optimization of a biotherapeutic monoclonal antibody requires a careful balance of target engagement and physicochemical developability properties. To take full advantage of the sequence diversity provided by different antibody discovery platforms, a rapid and reliable process for humanization of antibodies from nonhuman sources is required. Canonically, maximizing homology of the human variable region (V-region) to the original germline was believed to result in preservation of binding, often without much consideration for inherent molecular properties. We expand on this approach by grafting the complementary determining regions (CDRs) of a mouse anti-LAG3 antibody into an extensive matrix of human variable heavy chain (VH) and variable light chain (VL) framework regions with substantially broader sequence homology to assess the impact on complementary determining region-framework compatibility through progressive evaluation of expression, affinity, biophysical developability, and function. Specific VH and VL framework sequences were associated with major expression and purification phenotypes. Greater VL sequence conservation was correlated with retained or improved affinity. Analysis of grafts that bound the target demonstrated that initial developability criteria were significantly impacted by VH, but not VL. In contrast, cell binding and functional characteristics were significantly impacted by VL, but not VH. Principal component analysis of all factors identified multiple grafts that exhibited more favorable antibody properties, notably with nonoptimal sequence conservation. Overall, this study demonstrates that modern throughput systems enable a more thorough, customizable, and systematic analysis of graft-framework combinations, resulting in humanized antibodies with improved global properties that may progress through development more quickly and with a greater probability of success.

Sema3A Antibody BI-X Prevents Cell Permeability and Cytoskeletal Collapse in HRMECs and Increases Tip Cell Density in Mouse Oxygen-Induced Retinopathy.

Purpose: Semaphorin 3A (Sema3A) is an axonal guidance molecule that inhibits angiogenesis by vasorepulsion and blocks revascularization in the ischemic retina. BI-X is an intravitreal anti-Sema3A agent under clinical investigation in patients with proliferative diabetic retinopathy (PDR) and diabetic macular ischemia (DMI). Methods: Surface plasmon resonance was used to determine binding affinity of BI-X to human and murine Sema3A. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to assess effects of BI-X on cell permeability and cytoskeletal collapse induced by Sema3A. In vivo, intravitreal BI-X or an anti-trinitrophenol control antibody was administered in both eyes in mice with oxygen-induced retinopathy (OIR). Retinal flat mounts were prepared, and avascular area and tip cell density were determined using confocal laser-scanning microscopy. Results: Dissociation constants for BI-X binding to human and murine Sema3A were 29 pM and 27 pM, respectively. In vitro, BI-X prevented HRMEC permeability and cytoskeletal collapse induced by Sema3A. In vivo, BI-X increased tip cell density by 33% (P < 0.001) and reduced avascular area by 12% (not significant). A significant negative correlation was evident between avascular area and tip cell density (r2 = 0.4205, P < 0.0001). Conclusions: BI-X binds to human Sema3A with picomolar affinity and prevents cell permeability and cytoskeletal collapse in HRMECs. BI-X also enhances revascularization in mice with OIR. Translational Relevance: BI-X is a potent inhibitor of human Sema3A that improves revascularization in a murine model of OIR; BI-X is currently being investigated in patients with laser-treated PDR and DMI.

Optimizing NBE PK/PD assays using the Gyrolab Affinity Software; conveniently within the bioanalyst's existing workflow.

AIM: The fully automated microfluidics-based Gyrolab is a popular instrument for the bioanalysis of protein therapeutics; requiring minimal sample and reagent volumes. Gyros offers affinity software for determining binding affinity in solution using a high-throughput method and miniaturized reactions. RESULTS: Using this affinity software, multiple CTGF-targeting reagents were characterized on the Gyrolab after <100% target coverage was seen in a cynomolgus pharmacokinetic/PD study dosed with anti-CTGF antibodies. The results uncovered magnitude differences in binding affinities between the dosed antibody, target and assay reagents. CONCLUSION: The binding affinity values were used to investigate reduced target coverage and results highlight potential of the affinity software for incorporation into the bioanalyst's existing Gyrolab workflow for characterizing reagents and optimizing pharmacokinetic/PD bioanalytical assays.

Unexpected Potency Differences between B-Cell-Activating Factor (BAFF) Antagonist Antibodies against Various Forms of BAFF: Trimer, 60-Mer, and Membrane-Bound.

Therapeutic agents antagonizing B-cell-activating factor/B-lymphocyte stimulator (BAFF/BLyS) are currently in clinical development for autoimmune diseases; belimumab is the first Food and Drug Administration-approved drug in more than 50 years for the treatment of lupus. As a member of the tumor necrosis factor superfamily, BAFF promotes B-cell survival and homeostasis and is overexpressed in patients with systemic lupus erythematosus and other autoimmune diseases. BAFF exists in three recognized forms: membrane-bound and two secreted, soluble forms of either trimeric or 60-mer oligomeric states. To date, most in vitro pharmacology studies of BAFF neglect one or more of these forms. Here, we report a comprehensive in vitro cell-based analysis of BAFF in assay systems that measure all forms of BAFF-mediated activation. We demonstrate the effects of these BAFF forms in both a primary human B-cell proliferation assay and in nuclear factor κB reporter assay systems in Chinese hamster ovary cells expressing BAFF receptors and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI). In contrast to the mouse system, we find that BAFF trimer activates the human TACI receptor. Further, we profiled the activities of two clinically advanced BAFF antagonist antibodies, belimumab and tabalumab. Unexpectedly, we revealed differences in inhibitory potencies against the various BAFF forms, in particular that belimumab does not potently inhibit BAFF 60-mer. Through this increased understanding of the activity of BAFF antagonists against different forms of BAFF, we hope to influence the discovery of BAFF antagonist antibodies with distinct therapeutic mechanisms for improvement in the treatment of lupus or other related autoimmune pathologies.

Lead identification to generate 3-cyanoquinoline inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. Inhibitors of this receptor are believed to provide a new target in cancer therapy. We previously reported an isoquinolinedione series of IGF-1R inhibitors. Now we have identified a series of 3-cyanoquinoline compounds that are low nanomolar inhibitors of IGF-1R. The strategies, synthesis, and SAR behind the cyanoquinoline scaffold will be discussed.

Lead identification to generate isoquinolinedione inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment.

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.

Combinatorial synthesis of substituted 3-(2-indolyl)piperidines and 2-phenyl indoles as inhibitors of ZipA-FtsZ interaction.

The ZipA-FtsZ protein-protein interaction is a potential target for antibacterial therapy. The design and parallel synthesis of a combinatorial library of small molecules, which target the FtsZ binding area on ZipA are described. Compounds were demonstrated to bind to the FtsZ binding domain of ZipA by HSQC NMR and to inhibit cell division in a cell elongation assay.

Design and synthesis of indolo[2,3-a]quinolizin-7-one inhibitors of the ZipA-FtsZ interaction.

The binding of FtsZ to ZipA is a potential target for antibacterial therapy. Based on a small molecule inhibitor of the ZipA-FtsZ interaction, a parallel synthesis of small molecules was initiated which targeted a key region of ZipA involved in FtsZ binding. The X-ray crystal structure of one of these molecules complexed with ZipA was solved. The structure revealed an unexpected binding mode, facilitated by desolvation of a loosely bound surface water.

Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction.

A fluorescence polarization competition assay has been developed to screen for inhibitors of the Escherichia coli FtsZ/ZipA protein-protein interaction. A previously published X-ray costructure demonstrated that a 17-amino-acid peptide, corresponding to FtsZ C-terminal residues 367-383 (FtsZ(367-383)), interacts with the C-terminal FtsZ binding domain of ZipA (ZipA(185-328)). Phage display was employed to identify a unique but related peptide which when further modified and labeled was shown to have a higher affinity to ZipA(185-328) than the FtsZ(367-383) peptide and binds to the same site. This peptide had a six fold increase in fluorescence polarization upon binding to ZipA(185-328) compared to a two fold increase for the FtsZ(367-383) fluorophore. As a result, assay parameters using the phage display peptide were further optimized and adapted for the high-throughput screen. A high-throughput screen of 250,000 compounds identified 29 hits with inhibition equal to or greater than 30% at 50 microg/ml. An X-ray costructure of a promising small molecule in this library complexed with ZipA(185-328) (KI=12 microM) revealed that the compound binds to the same hydrophobic pocket as the FtsZ(367-383) peptide.

Structure-based design of carboxybiphenylindole inhibitors of the ZipA-FtsZ interaction.

Structural features of two weak inhibitors of the ZipA-FtsZ protein-protein interaction which were found to bind to overlapping but different areas of the key binding site were combined in one new series of carboxybiphenyl-indoles with improved inhibitory activity.