Jill Carton

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095–2, displays specificity for IdeS-generated F(ab’)2 fragments, but not for full-length IgG or for closely-related F(ab’)2 fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095–2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab’)2 fragment. Similarly, 2095–2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab’)2 fragment. mAb 2095–2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab’)2 fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095–2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095–2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab’)2 fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095–2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab’)2 fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095–2 to F(ab’)2, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.

Generation of a Protective T-Cell Response Following Coronavirus Infection of the Central Nervous System Is Not Dependent on IL-12/23 Signaling

The functional role of IL-12 and IL-23 in host defense and disease following viral infection of the CNS was determined. Instillation of mouse hepatitis virus (MHV, a positive-strand RNA virus) into the CNS of mice results in acute encephalitis followed by a chronic immune-mediated demyelinating disease. Antibody-mediated blocking of either IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) signaling did not mute T-cell trafficking into the CNS or antiviral effector responses and mice were able to control viral replication within the brain. Therapeutic administration of either anti-IL-23p19 or anti-IL-12/23p40 to mice with viral-induced demyelination did not attenuate T-cell or macrophage infiltration into the CNS nor improve clinical disease or diminish white matter damage. In contrast, treatment of mice with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition of the autoimmune model of demyelination, experimental autoimmune encephalomyelitis (EAE). These data indicate that (1) IL-12 and IL-23 signaling are dispensable in generating a protective T-cell response following CNS infection with MHV, and (2) IL-12 and IL-23 do not contribute to demyelination in a model independent of autoimmune T-cell-mediated pathology. Therefore, therapeutic targeting of IL-12 and/or IL-23 for the treatment of autoimmune diseases may offer unique advantages by reducing disease severity without muting protective responses following viral infection.

Protein therapeutics (introduction to biopharmaceuticals)

Abstract Throughout human history, the morbidity and mortality associated with human disease has driven medical science into an ever-expanding quest for treatment and cure. Over the past century, a therapeutic approach complementing chemical drugs has been developing which uses proteins and peptides in the treatment of disease. Many innovative protein therapeutic platforms are currently being employed and continue to be developed to attain cures in areas of unmet medical need; these include direct copies of natural protein structure and function as well as proteins with completely novel functionality. Today, protein therapeutics represents the fastest growing sector in the pharmaceutical industry and comprises 16% of prescription drug sales in 2011.

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 A190: Colon tumor cells expressing CD24 have oncogenic properties and are inhibited by monoclonal antibody immunotherapy.

CD24 is a heavily glycosylated glycosylphosphatidylinositol- anchored protein that is overexpressed in many different tumor types, including colon cancer (>80 %), and has been shown to correlate with shortened patient survival. CD24 plays a role in regulating cancer cell proliferation and tumor microenvironment interactions. The mechanism by which CD24 regulates cell survival and proliferation is not very well understood. What is better known is that CD24 modulates cancer cell adhesion to the vasculature wall and cancer cell-platelet thrombi formation by its binding to P-selectin expressed on activated platelets and endothelial cells. CD24 was also reported to be a functional marker for liver, colon and pancreatic cancer stem cells. All of these functions may contribute to tumor growth and metastasis. The aim of this study was to investigate the function of CD24 as a target for colon cancer therapy. In order to confirm that CD24 expressing cells acquire oncogenic properties, we transfected the CD24 gene into the CD24− colon cancer line SW480. We confirmed that CD24 overexpression induces SW480 tumor growth in vivo. In order to understand the mechanism by which CD24 promotes tumorigenesis, we found that CD24 overexpression activates several oncogenic pathways. Previous publications have shown that CD24 increases expression of p-Raf, p-ERK, and p-JNK in SW480CD24+ cells. Here, using a reporter assay, we show that CD24 expression activates not only ERK and JNK but also the Wnt pathway. It was also shown in the past that treatment of HT-29 colon cancer cells with the antiproliferative anti-CD24 mAb SWA11 caused a decrease in hypoxia and VEGF pathways. We show here that CD24 induced VEGF as well as FGF-2, IL-10, and MMP2 expression and activation of the hypoxia pathway. Finally, we have tested the tumor inhibitory effect of two different anti-CD24 antibodies, SWA11 (mouse IgG2a) and ALB9 (mouse IgG1). Results suggest that both antibodies bind specifically to the CD24 protein core as shown by competition assays with CD24 Fc and binding assays to the deglycosylated CD24. We show that SWA11 significantly inhibited both HT-29 and SW480CD24+ tumor growth in vivo while ALB9 inhibited SW480CD24+ tumor growth only. SWA11 didn9t show any neutralizing activity in vitro in inhibiting cell proliferation or CD24 expression but showed effector function activity that may account for its mechanism of action in vivo. ALB9 mechanism of action was not explored in this study, but as it is a mouse IgG1 antibody, ADCC activity is not likely to be part of its mechanism of action. Together, these results provide support for the hypothesis that CD24 has oncogenic properties and that CD24-expressing tumor can be inhibited with antibody immunotherapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A190. Citation Format: Luciana F. Macedo, Elizabeth Kaiser, Haiyan Jiang, Hillary Millar, Diana Wiley, Adam Cotty, Fred Kaplan, Barry Morse, Jill M. Carton, Michael F. Naso, Randall Brezski, Allison Oberholtze, E. Christine Pietsch, Li Yingzhe, Debbie Marshall, Linda A. Snyder. Colon tumor cells expressing CD24 have oncogenic properties and are inhibited by monoclonal antibody immunotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A190.