Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.

No abstract

centuries-old Haber-Bosch process that suffers from operating under harsh reaction conditions (400-500 °C, 200-250 bar) and needs natural gas as the hydrogen source. [7,8] Therefore, it is highly desired to develop less energy-demanding approaches for sustainable N 2 -to-NH 3 fixation.In biological synthetic processes, certain bacteria are able to convert N 2 into NH 3 with the help of fascinating enzymes under mild conditions. [9][10][11] Electrocatalytic NH 3 synthesis using water as the hydrogen source has been proposed as a sustainable process to make NH 3 at ambient conditions. [12,13] The less abundance and high cost of N 2 reduction reaction (NRR) electrocatalysts based on precious metals (Au, [14,15] Rh, [16] Pd, [17] Ru, [18] and Ag [19] ) push the researchers to explore nonnoble metal alternatives. [20][21][22][23][24][25][26] As carbon materials are mainly composed of carbon and even can be made directly out of biomass, they are obviously "sustainable." The carbonaceous nanomaterials possess wide potential window and structural diversity, which can be active electrocatalysts for the oxygen reduction reaction and CO 2 reduction etc. [27,28] It is well accepted that nonmetal heteroatom doping (e.g., O, B, N, P, and S) can tailor the electronic structure of carbon atoms, which offers an effective method to promote the electrocatalytic performance. [29][30][31] Recent studies have proven that doping of B and N also enhances the NRR performances of carbon catalysts. [32][33][34] In this communication, we report that S-doped carbon nanosphere (S-CNS) acts as a suerb NRR catalyst for ambient N 2 -to-NH 3 conversion with excellent selectivity. In 0.1 m Na 2 SO 4 , the S-CNS achieves a large NH 3 yield of 19.07 µg h −1 mg −1 cat. and a high Faradic efficiency of 7.47% at −0.7 V versus reversible hydrogen electrode (RHE), much higher than those of undoped CNS (3.70 µg h −1 mg −1 cat. , 1.45%). Notably, this catalyst also demonstrates high electrochemical and structural stabilty.S-CNS was prepared via hydrothermal reaction followed by Ar annealing using glucose and benzyl disulfide as carbon and sulfur source, respectively. [35] Figure 1a shows the X-ray powder diffraction (XRD) pattern of S-CNS, and the diffraction peaks at 2θ = 23.8° and 44.0° are attributed to the (002) and (100) reflections of carbon, [36] respectively. Compared with CNS sample (Figure S1, Supporting Information), it can be clearly seen that the diffraction peaks of S-CNS become weaker and broader, confirming S has a great influence on the structure of carbon. [37] Figures 1b and S2 of the Supporting Information Industrial NH 3 synthesis mainly relies on the carbon-emitting Haber-Bosch process operating under severe conditions. Electrocatalytic N 2 to NH 3 fixation at ambient conditions is an attractive approach to reduce energy consumption and avoid direct carbon emission. In this communication, it is reported that the S-doped carbon nanosphere (S-CNS) acts as an efficient and stable nitrogen reduction reaction (NRR) catalyst for ambient N...

(2011) A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity, mAbs, 3:3, 273-288,

After testing 10 parallel experiments, both NH 3 yields (left y-axis) and FEs (right y-axis) show ignorable variation compared with its initial value, demonstrating a good recycle performance. The average of relative standard deviation for FEs is only 5.0% (Figure 3b), implying excellent reproducibility. By varying the N 2 flow rate, NH 3 yields (left y-axis) and FEs (right y-axis) at −0.8 V appear slightly decayed (Figure 3c). Continuous 24 h electrolysis is also steady without any

Therapeutic antibodies directed against the type 1 insulinlike growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting downstream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 M) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy. The type I insulin-like growth factor receptor (IGF-1R)2 is a large transmembrane receptor tyrosine kinase expressed on most somatic cells. IGF-1R is activated by the binding of its constitutive ligands, IGF-1 and IGF-2 (and at a much lower affinity, insulin). Ligand binding to the IGF-1R extracellular domains leads to activation of its cytoplasmic tyrosine kinase domain, receptor autophosphorylation, and phosphorylation of downstream targets such as insulin receptor substrate-1 (IRS-1), the Src homology and collagen domain protein (Shc), and others (1, 2). Phosphorylation of IRS-1 activates the phosphoinositol kinase 3/AKT cellular growth and survival pathways, and Shc phosphorylation leads to the activation of other signal cascades, including the extracellular signal-regulated kinase(Erk)/mitogen-activated protein kinase (MAPK) cellular growth and proliferation pathways (3).Human IGF-1R is synthesized as a 1368-amino acid polypeptide whose primary and tertiary structures have been reviewed (4,5). The N-terminal region (consis...

Currently, industrial-scale NH 3 production almost relies on energy-intensive Haber-Bosch process from atmospheric N 2 with large amount of CO 2 emission, while low-cost and high-efficient catalysts are demanded for the N 2 reduction reaction (NRR). In this study, Mn 3 O 4 nanoparticles@reduced graphene oxide (Mn 3 O 4 @rGO) composite is reported as an efficient NRR electrocatalyst with excellent selectivity for NH 3 formation. In 0.1 M Na 2 SO 4 solution, such catalyst obtains a NH 3 yield of 17.4 µg•h −1 •mg −1 cat. and a Faradaic efficiency of 3.52% at −0.85 V vs. reversible hydrogen electrode. Notably, it also shows high electrochemical stability during electrolysis process. Density functional theory (DFT) calculations also demonstrate that the (112) planes of Mn 3 O 4 possess superior NRR activity. KEYWORDS Mn 3 O 4 @rGO composite, electrocatalyst, NH 3 synthesis, N 2 reduction reaction, ambient conditions Scheme 1 A schematic diagram to illustrate the preparation process of Mn3O4@rGO.