Rebecca Croasdale

A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties.

Background: Bispecific antibodies are currently emerging as a promising new class of cancer therapeutics. Results: The novel one-arm single chain Fab IgG bispecific antibody (XGFR) targeting IGF-1R and EGFR demonstrated potent signaling inhibition and enhanced ADCC induction. Conclusion: XGFR has shown in vitro and in vivo anti-tumor activity in pancreatic, lung, and colorectal mouse xenograft tumor models. Significance: Rational design can help to overcome low expression yields and impaired effector functions of bispecific antibodies. In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the “knob-into-hole” technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics.

Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s−1. We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the 15N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.

A structural and functional dissection of the cardiac stress response factor MS1.

MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F‐actin binding ability, located to the C‐terminal half of the protein, which promotes stabilization of F‐actin in the cell thus releasing myocardin‐related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor. Initial attempts to purify the protein only resulted in heavily degraded samples that showed distinct bands on SDS gels, suggesting the presence of stable domains. Using a combination of combinatorial domain hunting and sequence analysis, a set of potential domains was identified. The C‐terminal half of the protein actually contains two independent F‐actin binding domains. The most C‐terminal fragment (294–375), named actin binding domain 2 (ABD2), is independently folded while a proximal fragment called ABD1 (193–296) binds to F‐actin with higher affinity than ABD2 (KD 2.21 ± 0.47 μM vs. 10.61 ± 0.7 μM), but is not structured by itself in solution. NMR interaction experiments show that it binds and folds in a cooperative manner to F‐actin, justifying the label of domain. The architecture of the MS1 C‐terminus suggests that ABD1 alone could completely fulfill the F‐actin binding function opening up the intriguing possibility that ABD2, despite its high level of conservation, could have developed other functions. Proteins 2012.

A structural and functional dissection of the cardiac stress response factor MS1

MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F‐actin binding ability, located to the C‐terminal half of the protein, which promotes stabilization of F‐actin in the cell thus releasing myocardin‐related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor. Initial attempts to purify the protein only resulted in heavily degraded samples that showed distinct bands on SDS gels, suggesting the presence of stable domains. Using a combination of combinatorial domain hunting and sequence analysis, a set of potential domains was identified. The C‐terminal half of the protein actually contains two independent F‐actin binding domains. The most C‐terminal fragment (294–375), named actin binding domain 2 (ABD2), is independently folded while a proximal fragment called ABD1 (193–296) binds to F‐actin with higher affinity than ABD2 (KD 2.21 ± 0.47 μM vs. 10.61 ± 0.7 μM), but is not structured by itself in solution. NMR interaction experiments show that it binds and folds in a cooperative manner to F‐actin, justifying the label of domain. The architecture of the MS1 C‐terminus suggests that ABD1 alone could completely fulfill the F‐actin binding function opening up the intriguing possibility that ABD2, despite its high level of conservation, could have developed other functions. Proteins 2012.