BI-Y, an Neuropilin-1 Antagonist, Enhances Revascularization and Prevents Vascular Endothelial Growth Factor-A Induced Retinal Hyperpermeability in Rodent Models of Retinopathies.

Diabetic retinopathy (DR) is a leading cause of vision loss in working-age adults. Despite an established standard of care for advanced forms of DR, some patients continue to lose vision after treatment. This may be due to the development of diabetic macular ischemia (DMI), which has no approved treatment. Neuropilin-1 (Nrp-1) is a coreceptor with two ligand-binding domains, with semaphorin-3A (Sema3A) binding to the A-domain and vascular endothelial growth factor-A (VEGF-A) binding to the B-domain. Sema3A directs a subset of neuronal growth cones as well as blood vessel growth by repulsion; when bound to Nrp-1, VEGF-A mediates vascular permeability and angiogenesis. Modulating Nrp-1 could therefore address multiple complications arising from DR, such as diabetic macular edema (DME) and DMI. BI-Y is a monoclonal antibody that binds to the Nrp-1 A-domain, antagonizing the effects of the ligand Sema3A and inhibiting VEGF-A-induced vascular permeability. This series of in vitro and in vivo studies examined the binding kinetics of BI-Y to Nrp-1 with and without VEGF-A165, the effect of BI-Y on Sema3A-induced cytoskeletal collapse, the effect of BI-Y on VEGF- A165-induced angiogenesis, neovascularization, cell integrity loss and permeability, and retinal revascularization. The data show that BI-Y binds to Nrp-1 and inhibits Sema3A-induced cytoskeletal collapse in vitro, may enhance revascularization of ischemic areas in an oxygen-induced retinopathy mouse model, and prevents VEGF-A-induced retinal hyperpermeability in rats. However, BI-Y does not interfere with VEGF-A-dependent choroidal neovascularization. These results support further investigation of BI-Y as a potential treatment for DMI and DME. SIGNIFICANCE STATEMENT: Diabetic macular ischemia (DMI) is a complication of diabetic retinopathy (DR) with no approved pharmacological treatment. Diabetic macular edema (DME) commonly co-occurs with DMI in patients with DR. This series of preclinical studies in mouse and rat models shows that neuropilin-1 antagonist BI-Y may enhance the revascularization of ischemic areas and prevents vascular endothelial growth factor-A (VEGF-A)-induced retinal hyperpermeability without affecting VEGF-A-dependent choroidal neovascularization; thus, BI-Y may be of interest as a potential treatment for patients with DR.

Distinct immune stimulatory effects of anti-human VISTA antibodies are determined by Fc-receptor interaction.

VISTA (PD-1H) is an immune regulatory molecule considered part of the next wave of immuno-oncology targets. VISTA is an immunoglobulin (Ig) superfamily cell surface molecule mainly expressed on myeloid cells, and to some extent on NK cells and T cells. In previous preclinical studies, some VISTA-targeting antibodies provided immune inhibitory signals, while other antibodies triggered immune stimulatory signals. Importantly, for therapeutic antibodies, the isotype backbone can have a strong impact on antibody function. To elucidate the mode of action of immune stimulatory anti-VISTA antibodies, we studied three different anti-human VISTA antibody clones, each on three different IgG isotypes currently used for therapeutic antibodies: unaltered IgG1 (IgG1-WT), IgG1-KO (IgG1-LL234,235AA-variant with reduced Fc-effector function), and IgG4-Pro (IgG4- S228P-variant with stabilized hinge region). Antibody functionality was analysed in mixed leukocyte reaction (MLR) of human peripheral blood mononuclear cells (PBMCs), as a model system for ongoing immune reactions, on unstimulated human PBMCs, as a model system for a resting immune system, and also on acute myeloid leukemia (AML) patient samples to evaluate anti-VISTA antibody effects on primary tumor material. The functions of three anti-human VISTA antibodies were determined by their IgG isotype backbones. An MLR of healthy donor PBMCs was effectively augmented by anti-VISTA-IgG4-Pro and anti-VISTA-IgG1-WT antibodies, as indicated by increased levels of cytokines, T cell activation markers and T cell proliferation. However, in a culture of unstimulated PBMCs of single healthy donors, only anti-VISTA-IgG1-WT antibodies increased the activation marker HLA-DR on resting myeloid cells, and chemokine levels. Interestingly, interactions with different Fc-receptors were required for these effects, namely CD64 for augmentation of MLR, and CD16 for activation of resting myeloid cells. Furthermore, anti-VISTA-IgG1-KO antibodies had nearly no impact in any model system. Similarly, in AML patient samples, anti-VISTA-antibody on IgG4-Pro backbone, but not on IgG1-KO backbone, increased interactions, as a novel readout of activity, between immune cells and CD34+ AML cancer cells. In conclusion, the immune stimulatory effects of antagonistic anti-VISTA antibodies are defined by the antibody isotype and interaction with different Fc-gamma-receptors, highlighting the importance of understanding these interactions when designing immune stimulatory antibody therapeutics for immuno-oncology applications.

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.

An optimally designed anti-human CD40 antibody with potent B cell suppression for the treatment of autoimmune diseases.

Antibodies targeting the CD40-CD40L pathway have great potential for treating autoimmune diseases like rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis (LN), and inflammatory bowel diseases (IBD). However, in addition to the known difficulty in generating a purely antagonistic CD40 antibody, the presence of CD40 and CD40L on platelets creates additional unique challenges for the safety, target coverage, and clearance of antibodies targeting this pathway. Previously described therapeutic antibodies targeting this pathway have various shortcomings, and the full therapeutic potential of this axis has yet to be realized. Herein, we describe the generation and characterization of BI 655064, a novel, purely antagonistic anti-CD40 antibody that potently neutralizes CD40-CD40L-dependent B-cell stimulation without evidence of impacting platelet functions. This uniquely optimized antibody targeting a highly challenging pathway was obtained by applying stringent functional and biophysical criteria during the lead selection process. BI 655064 has favorable target-mediated drug disposition (TMDD)-saturation pharmacokinetics, consistent with that of a high-quality therapeutic monoclonal antibody.

Fully human antibodies against the Protease-Activated Receptor-2 (PAR-2) with anti-inflammatory activity.

PAR-2 belongs to a family of G-protein coupled Protease-Activated Receptors (PAR) which are activated by specific proteolytic cleavage in the extracellular N-terminal region. PAR-2 is activated by proteases such as trypsin, tryptase, proteinase 3, factor VIIa, factor Xa and is thought to be a mediator of inflammation and tissue injury, where elevated levels of proteases are found. Utilizing the HuCAL GOLD® phage display library we generated fully human antibodies specifically blocking the protease cleavage site in the N-terminal domain. In vitro affinity optimization resulted in antibodies with up to 1000-fold improved affinities relative to the original parental antibodies with dissociation constants as low as 100 pM. Corresponding increases in potency were observed in a mechanistic protease cleavage assay. The antibodies effectively inhibited PAR-2 mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In addition, the antibodies demonstrated potent inhibition of trypsin induced relaxation of isolated rat aortic rings ex vivo. In a short term mouse model of inflammation, the trans vivo DTH model, anti-PAR-2 antibodies showed inhibition of the inflammatory swelling response. In summary, potent inhibitors of PAR-2 were generated which allow further assessment of the role of this receptor in inflammation and evaluation of their potential as therapeutic agents.