A method is described for obtaining the axes of the diagonal paramagnetic susceptibility tensor for the low-spin cyanide complexes of distal point mutants of ferric sperm whale myoglobin (metMbCN). It relies on using the crystal coordinates of the wild-type (WT) protein for that portion of the molecule unperturbed by the point mutation, together with the experimental dipolar shifts, to search for the Euler rotation that correctly converts the crystal coordinates to the magnetic axes. The complete set of NMR dipolar shifts is shown to lead to the determination of the magnetic anisotropies as well as the orientation of the magnetic axes for WT metMbCN. Various sets of input NMR dipolar shifts for protons not only emphasizing the proximal side of the heme but also considering distal backbone protons and the structurally conserved Phe CD1 are shown to lead to well-defined magnetic axes for WT metMbCN with closely clustered Euler angles. The tilt of the major magnetic axis from the heme normal, the projection of this tilt on the heme plane, and the position of the rhombic axies projected on the heme plane range only over 1.5°, ~10°, and ~10°, respectively, for nine different data sets comprising as many as 37 to as few as five input dipolar shifts. The NMR spectra of the metMbCN complexes of a strongly perturbed (His E7 -*• Gly) and a minimally perturbed (Arg CD3 -* Gly) point mutant are analyzed to yield the assignments necessary to define the magnetic axes. Using a variety of input data sets of dipolar shifts limited to the residues expected to be unperturbed by distal point mutation, the magnetic axes were determined by a three parameter least-square search for both the His E7 -*• Gly and Arg CD3 -Gly mutants. For the E7 Gly mutant, the major magnetic axis tilt is minimally altered, but the projection of the tilt is rotated by ~45°; the CD3 Gly mutant yields a magnetic axes orientation within the range defined by different data sets of WT metMbCN. However, simulation of the predicted dipolar shift based on systematic changes is used to show that the axes of the CD3 Gly mutant differ from those of the WT by a very small (2°) rotation of the projection of the tilt of the major axis, rather than from a change in tilt. Inasmuch as the orientation of the magnetic axes can be related to distal steric tilt of the isostructural Fe-CO unit in WT MbCO, the present demonstration that magnetic axes can be determined for point mutants has significant implications for the elucidation of steric constraints on bound ligands in a variety of low-spin hemoproteins.

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Soluble forms of transforming growth factor-a (TGFa) are derived by proteolytic processing of an integral membrane glycoprotein precursor (proTGFa). Previous studies indicated that phorbol ester-induced cleavage of proTGFa in CHO cells is dependent on the presence of a valine residue located at the carboxyl terminus of the precursor's cytoplasmic domain. We reassessed this requirement with epitope-tagged constructs introduced into transformed rat liver epithelial cells that normally express and process TGFa. We found that proTGFa mutants lacking the terminal valine residues showed greatly reduced maturation to the fully glycosylated form. Additionally, they were present at substantially reduced levels on the cell surface and, instead, accumulated in the endoplasmic reticulum. Consistent with these results, enzyme-linked immunosorbant assay (ELISA) and Western blot analyses revealed little or no soluble TGFa in medium conditioned by cells expressing the mutant constructs. Finally, a truncated proTGFa mutant lacking most of the cytoplasmic domain but retaining a carboxylterminal valine was processed and cleaved in a near-normal manner. These results, some of which were reproduced in CHO cells, indicate that the predominant effect of the carboxyl-terminal valines is to ensure normal maturation and routing of the precursor.

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A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling.