(2015) Improving target cell specificity using a novel monovalent bispecific IgG design, mAbs, 7:2, 377-389, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the C H 1-C L interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.

Two new seco-prezizaane-type sesquiterpenes, 1,2-dehydroneomajucin (1) and jiadifenin (2), were isolated from the methanol extract of the pericarps of Illicium jiadifengpi, indigenous to the southern part of China. Their structures were elucidated on the basis of NMR data. Compound 2, which is an equilibrated mixture of the epimers 2a and 2b on the C-10 acetal carbon, is the first example of a majucin-type seco-prezizaane with an oxo-function at the C-10 position. The proposed structure for 2 was unambiguously confirmed by chemical conversion of the known sesquiterpene (2S)-hydroxy-3,4-dehydroneomajucin (5) to 2. Compounds 2 and 5 were found to significantly promote neurite outgrowth in primary cultures of fetal rat cortical neurons at concentrations from 0.1 to 10 microM.

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Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected.

'Acqua (2015) Insights into the molecular basis of a bispecific antibody's target selectivity , mAbs, 7:3, 461-469,

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The alphaviruses are composed of two icosahedral protein shells, one nested within the other. A membrane bilayer derived from the host cell is sandwiched between the protein shells. The protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. We have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with other virus proteins in an attempt to understand the role this domain plays in virus assembly and function. Through incremental deletions, we have reduced the length of a virus membrane protein transmembrane domain from its normal 26 amino acids to 8 amino acids. We examined the effect of these deletions on the assembly and function of virus particles. We found that progressive truncations in the transmembrane domain profoundly affected production of infectious virus in a cyclic fashion. We also found that membrane composition effects protein-protein and proteinmembrane interactions during virus assembly.Sindbis virus is an alphavirus and a member of the arboviruses, a group of viruses which are propagated in nature via a complicated life cycle involving insect vectors and mammalian hosts. Sindbis virus is simple in its composition but complex in its structure (42). The virion contains three structural proteins, E1, E2, and Capsid (C). These proteins are organized into two geometrically identical Tϭ4 icosahedral shells (32). The outer protein shell is composed of the glycoproteins E1 and E2, organized into trimers of heterodimers (2, 9, 34), and surrounds the inner shell, composed of protein C, which is assembled around the viral RNA. A host-derived membrane bilayer is situated between the concentric shells and is penetrated by the transmembrane (TM) domain anchors of each glycoprotein (19,36,41). The E2 glycoprotein contains a 33-amino-acid endodomain which specifically interacts with a hydrophobic cleft in the capsid protein (17,18,30,31). The interaction between E2 and C gives stability to the structure of the virus and plays a critical role in the formation of the outer virus protein shell around the preformed inner protein shell as the process of envelopment takes place (13). The membrane glycoprotein E1 is incorporated into the icosahedral outer protein shell in a highly constrained, energy-rich conformation (9, 28). This stored energy likely drives the process of cell penetration during infection. Physical and chemical treatments of the virus result in the conversion of the protein to a low-energy, nonnative configuration, which results in the loss of virus infectivity (3,12,28).The three virus structural proteins are synthesized from a subgenomic polycistronic message in the sequence NH-C-PE2

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