Structure-Based Prediction of Asparagine and Aspartate Degradation Sites in Antibody Variable Regions

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs

Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases

Anti-angiogenic therapies using biological molecules that neutralize vascular endothelial growth factor-A (VEGF-A) have revolutionized treatment of retinal vascular diseases including age-related macular degeneration (AMD). This study reports preclinical assessment of a strategy to enhance anti-VEGF-A monotherapy efficacy by targeting both VEGF-A and angiopoietin-2 (ANG-2), a factor strongly upregulated in vitreous fluids of patients with retinal vascular disease and exerting some of its activities in concert with VEGF-A. Simultaneous VEGF-A and ANG-2 inhibition was found to reduce vessel lesion number, permeability, retinal edema, and neuron loss more effectively than either agent alone in a spontaneous choroidal neovascularization (CNV) model. We describe the generation of a bispecific domain-exchanged (crossed) monoclonal antibody (CrossMAb; RG7716) capable of binding, neutralizing, and depleting VEGF-A and ANG-2. RG7716 showed greater efficacy than anti-VEGF-A alone in a non-human primate laser-induced CNV model after intravitreal delivery. Modification of RG7716’s FcRn and FccR binding sites disabled the antibodies’ Fc-mediated effector functions. This resulted in increased systemic, but not ocular, clearance. These properties make RG7716 a potential nextgeneration therapy for neovascular indications of the eye

A Novel Angiopoietin-2 Selective Fully Human Antibody with Potent Anti-Tumoral and Anti-Angiogenic Efficacy and Superior Side Effect Profile Compared to Pan-Angiopoietin-1/-2 Inhibitors

<div><p>There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.</p> </div

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions

Anti-tumor activity of LC06 and LC08 in s.c. Colo205 and orthotopic KPL-4 xenograft tumors.

<p>(<b>A</b>) Colo205 tumor (mean 100 mm³) growth curves in female SCID beige mice receiving LC06 and LC08 (10 mg/kg) once weekly i.p. (n = 10, *p<0.0001, Student's t-test). (<b>B</b>) Total Colo205 tumor weight in tumor bearing mice treated with LC06 and LC08 (n = 10, *p<0.0001 compared to control, Student's t-test). (<b>C</b>) KPL-4 orthotopic tumor (mean 90 mm³) growth curves in mice receiving LC06 and LC08 (10 mg/kg) once weekly i.p. (n = 10, *p<0.05 compared to control, Student's t-test). Error bars represent ± SEM. Arrows indicate start of treatment. The results were confirmed in 3 independent experiments.</p

Effect of LC06 and LC08 treatment on MVD (A and B), vascular coverage (C), vessel area (D), perfusion (E) and vessel branching (F) in Colo205 tumors.

<p>(<b>A</b>) Representative pictures of CD34-stained Colo205 tumors (mean 800 mm³). (<b>B</b>) Quantitative analysis of tumor microvessel density (MVD). MVD was quantified in CD34-stained whole tumor slides. LC06 and LC08 treatment led to a significant reduction of intratumoral MVD (n = 5, *p<0.05 compared to control, Student's t-test). (<b>C</b>) Quantification of vessel coverage calculated as the percentage of desmin-positive vessels in relation to CD34-positive endothelial cells staining in six random regions of 1000×1000 µm per tumor slide. LC06 and LC08 treated tumors showed increased vessel coverage by desmin positive pericytes (n = 5 mice per group, *p<0.05 compared to control, Student's t-test). (<b>D</b>) The average vessel area of intratumoral microvessels were significantly reduced in tumors treated with LC06 and LC08 (n = 5, *p<0.05 compared to control, Student's t-test). (<b>E</b>) Perfusion was assessed based on analysis of TRITC-lectin perfusion and CD34-positive staining in six random regions of 1000×1000 µm per tumor slide. Almost all remaining intratumoral microvessels were perfused in LC06 (93%) and LC08 (97%) treated tumors compared to control (56%) (n = 5 mice per group, *p<0.01). (<b>F</b>) Number of branched intratumoral microvessels was counted in six random regions of 1000×1000 µm per tumor slide and calculated per mm² and was significantly reduced in tumors treated with LC06 and LC08. LC06 treatment resulted in stronger inhibition of vessel branching compared to pan-Ang-2/-1 treatment via LC08 (n = 5, *p<0.01 compared to control; ^#p<0.01 compared to LC08, Student's t-test). Results are expressed as mean ± SEM. Scale bars represent 500 µm.</p

Tumor necrosis in LC06 and LC08 treated Colo205 tumors.

<p>(<b>A</b>) Quantitative image analysis shows 27% of necrotic area in vehicle treated, 51% in LC06 treated and 43% in LC08 treated tumors (n = 5, *p<0.05 compared to control, Student's t-test). Results are expressed as mean ± SEM. Two independent experiments were performed to confirm the results. (<b>B</b>) Representative mosaic images (10x) of vehicle, LC06 or LC08 treatments. Scale bar represents 1.3 mm.</p