Jennifer F. Nemeth

A fully integrated multi-column system for abundant protein depletion from serum/plasma

This work details the transformation of a conventional HPLC system to a low back pressure liquid chromatography set-up for automated serum/plasma depletion and fractionation. A Dionex U3000 HPLC was converted to low back pressure operation (125 psi max) by replacing all narrow-bore lines to larger inner-diameter tubing. The system was configured to use two immunoaffinity columns, first for depletion of the top 14 most abundant proteins (Seppro IgY14), then for the next 200-300 proteins (Seppro SuperMix). The autosampler was dual-purposed for both injection and fraction collection. Both the flow-through and SuperMix bound proteins were collected in an automated fashion. Three samples could be depleted consecutively before the system required user intervention, and up to nine samples could be depleted within a 24 h period. This study documents the validation of the instrument performance with a 90-patient sample set, demonstrating overall CVs for 86 of the 90 samples to be within the 95% confidence intervals. Additionally, there was excellent reproducibility within the same patient (biological replicates) across days.

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.

Expression, refolding and purification of a human interleukin-17A variant.

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.

Synthesis by native chemical ligation and crystal structure of human CCL2

The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP‐1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester‐peptide segment was synthesized using the sulfonamide safety‐catch linker and 9‐fluorenylmethoxycarbonyl (Fmoc) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full‐length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP‐1 cells, as expected. CCL2 was crystallized and the structure was determined by X‐ray diffraction at 1.9‐Å resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic twofold axis. The dimer interface involves residues Val9‐Thr10‐Cys11, which form an intersubunit antiparallel β‐sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety‐catch linker and NCL.

Probing the solution structure of tumor necrosis factor-α homotrimer and heterotrimer after complex perturbation using electrospray ionization mass spectrometry†

There are a number of proteins whose active forms are non‐covalent multichain complexes. Therapeutic intervention involving such complexes has been proposed through the use of muteins to form heterostructures. These resulting structures would either not be recognized by receptors or would be inactive competitive inhibitors to wild‐type (wt) proteins. We have used tumor necrosis factor‐α (TNF‐α) to establish that it is possible to use mass spectrometry to monitor the non‐covalent solution structure of therapeutically relevant proteins and correlate the results with binding data. Mass spectrometry is shown to be able to directly monitor the state of the solution complexes to within 5 Da errors mass accuracy of theoretical mass at 50 kDa, as well as to resolve homocomplex from heterocomplex. Furthermore, it was determined that perturbation of the TNF‐α complex, at or below pH 4.0, results in monomers that cannot reform into the multimeric complex, and the resulting protein solution can no longer bind to an anti‐TNF‐α antibody. Dissociation and re‐association of the trimer was possible with the use of dimethyl sulfoxide at pH 5.5 and allowed for the resulting detection of both homotrimer and heterotrimer in solution with no impact on antibody binding. This work demonstrates that mass spectrometric techniques offer a means to monitor native solution interactions of non‐covalent complexes and to differentiate multiple complexes from each other in solution. This method has applicability in the biopharmaceutical arena for monitoring engineering non‐covalent drug complexes for the purpose of altering biological activity. Copyright

The role of interchain disulfide bond in a recombinant human interleukin-17A variant

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1=isoform 2<isoform 4<isoform 3. In addition, compared to covalent dimers (isoform 3 and 4), the non-covalent dimers (isoforms 1 and 2) are slightly less active in a receptor-binding assay but at least 5-fold less active in a cell-based assay.

Abstract 1492: Development of a CD123xCD3 bispecific antibody to treat acute myeloid leukemia (AML)

AML is a heterogeneous disease characterized by uncontrolled clonal expansion of leukemic stem cells (LSCs). Current therapies for AML are not curative, in part due to their inability to eradicate LSCs from the bone marrow. T cell redirection has been shown to be effective in heme malignancies and represents a promising approach to treat AML by targeting markers differentially expressed on the cell surface of cancer cells. One marker, CD123 (α-chain of the interleukin-3 receptor) is often present on AML LSCs and blasts. We developed a human bispecific antibody (CD123xCD3; Ab-178) capable of binding to the extracellular domain of CD123 and the e chain of CD3 on T cells to induce T cell–mediated tumor cell killing. This bispecific IgG4 antibody can recruit T cells to CD123 + AML cells (MOLM-13, KG-1 and OCI-AML5) and induce T cell activation as evidenced by CD69 and CD25 up-regulation on T cells. Ab-178 potently killed these CD123 + AML cell lines in vitro (EC 50 = 0.51-0.91 nM) but not a CD123 − cell line (JIM3). Ab-178 was also able to induce tumor inhibition (MOLM-13 cells) and regression (KG-1 cells) in murine xenograft CD123 + AML models in the presence of human PBMCs. Furthermore, this antibody was able to kill AML blasts in primary AML blood samples ex vivo in the absence of exogenous T cells (autologous setting; EC 50 = 0.83 nM). Related bispecific antibodies directed against a viral epitope (nullxCD3 or CD123xnull) did not activate T cells or cause tumor cell killing in the various assays tested. Ab-178 had no impact on T cell activation when incubated with T cells alone. These results indicate that Ab-178 can potently and specifically activate T cells in the presence of CD123 + AML cells and induce their killing. Furthermore, because of the antibody format, this molecule is expected to have a longer half-life compared to smaller bispecific biologic scaffolds. Ab-178 is currently being evaluated pre-clinically for its potential to treat patients with AML. Citation Format: Francois Gaudet, Jennifer F. Nemeth, Ronan McDaid, Yingzhe Li, Benjamin Harman, Hillary Millar, Alexey Teplyakov, John Wheeler, Jinquan Luo, Susan Tam, Sheng-Jiun Wu, Emily Chen, Alexander Babich, Yusri Elsayed, Ricardo Attar. Development of a CD123xCD3 bispecific antibody to treat acute myeloid leukemia (AML). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1492.

Biologics and molecular recognition: Mike Brigham‐Burke memorial issue

This special issue of the Journal of Molecular Recognition is dedicated to Dr Michael Brigham-Burke, Principal Investigator, Centocor R&D, Inc. (Centocor), a division of Johnson & Johnson Pharmaceutical Research & Development, L.L.C. It showcases antibody drug research and discovery, an area that was the focus of his interests in the years before his passing in November 2008. We miss Mike’s leadership, his knowledge of people, and his commitment and tenacity in the field of the biophysical analysis of proteins. Mike championed the characterization of the biochemical and biophysical properties of the therapeutic antibody by promoting structural and thermal stability studies. He is remembered for his enthusiastic, positive mental outlook despite the challenges presented by his deteriorating health. Nonetheless, inside and outside the R&D organization, Mike is remembered for his riveting, almost profane, sense of humor. For those who did not know him personally, he was an avid reader of world history, capable of sustaining a conversation with anyone on any political, religious, or social issue. He also shared his love for horses with his wife Lauren, who teaches horseback riding in Pennsylvania. Mike had a long-running career in pharmaceutical research, which spanned more than 20 years. He started his career in industry in 1984 when he joined GlaxoSmithKline. As an investigator there, he applied analytical and synthetic chemistry, peptide synthesis, protein chemistry, and protein interaction measurement methods to the drug discovery process. In addition to providing critical data, Mike was highly innovative. He was constantly developing new approaches for addressing difficult scientific questions. These efforts have resulted in several scientific publications and patents. In 2002, Mike joined Centocor as a Principal Research Scientist to head up the Molecular Interactions Laboratory. Early on, he integrated an analytical ultracentrifuge into the existing core molecular interaction facility to enhance the analytical offerings of the group. His research group routinely participated in discovery R&D projects through lead candidate selection. The strong leadership of his team and his scientific prowess assured that his group delivered consistent measurements and data analyses of the highest quality that were critical for maintaining the high standards needed for the success of the research programs. Mike also offered internal consultation to all program teams regarding protein expression hurdles, immunogenicity concerns, and aggregation issues. Before his passing, the group was poised to establish guidelines on target affinity improvement as well as stability of antibody leads in serum and formulation components. Dr Michael Brigham-Burke Principal Research Scientist 1961–2008