Holly Prentice

Targeting the Lymphotoxin-β Receptor with Agonist Antibodies as a Potential Cancer Therapy

The lymphotoxin-beta receptor (LT beta R) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LT beta R monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LT beta R caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LT beta R activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LT beta R with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

Improving Performance of Mammalian Cells in Fed-Batch Processes through "Bioreactor Evolution"

The amount of recombinant product obtained from mammalian cells grown in a bioreactor is in part limited by achievable cell densities and the ability of cells to remain viable over extended periods of time. In an attempt to generate cell lines capable of better bioreactor performance, we subjected the DG44 Chinese Hamster Ovary (CHO) host cell line and a recombinant production cell line to an iterative process whereby cells capable of surviving the harsh conditions in the bioreactor were selected. This selective process was termed “bioreactor evolution”. Following the selective process, the “evolved” host cells attained a 2‐fold increase in peak cell density and a 72% increase in integral cell area. Transient transfection experiments demonstrate that the evolved cells have the same transfection efficiency and the same secretory potential as the initial cells. The “evolved” host was also found to contain a large subpopulation of cells that did not require insulin for growth. From this, a new population of growth‐factor‐independent cells was obtained. These improvements in host properties should prove beneficial in the expression of recombinant proteins in fed‐batch processes. The selective process was also applied to a recombinant production cell line. The evolved cells from this selection exhibited a 38% increase in peak cell density, a 30% increase in integral cell area, and a 36% increase in product titer. These increases were obtained without any appreciable impact on product quality, demonstrating the usefulness of this simple approach to improve the performance of recombinant cell lines.

The effect of antiapoptosis genes on clarification performance

Optimal bioreactor harvest time is typically determined based on maximizing product titer without compromising product quality. We suggest that ease of downstream purification should also be considered during harvest. In this view, we studied the effect of antiapoptosis genes on downstream performance. Our hypothesis was that more robust cells would exhibit less cell lysis and thus generate lower levels of cell debris and host‐cell contaminants. We focused on the clarification unit operation, measuring postclarification turbidity and host‐cell protein (HCP) concentration as a function of bioreactor harvest time/cell viability. In order to mimic primary clarification using disk‐stack centrifugation, a scale‐down model consisting of a rotating disk (to simulate shear in the inlet feed zone of the centrifuge) and a swinging‐bucket lab centrifuge was used. Our data suggest that in the absence of shear during primary clarification (typical of depth filters), a 20–50% reduction in HCP levels and 50–65% lower postcentrifugation turbidity was observed for cells with antiapoptosis genes compared to control cells. However, on exposing the cells to shear levels typical in a disk‐stack centrifuge, the reduction in HCP was 10–15% while no difference in postcentrifugation turbidity was observed. The maximum benefit of antiapoptosis genes is, therefore, realized using clarification options that involve low shear, <1 × 106 W/m3 and minimal damage to the cells.

Why choose mammalian cells for protein production

Publisher Summary There are many different types of hosts to use for production of natural or recombinant proteins: mammalian cells; bacteria, including Gram-negative, Gram-positive, and L-form; filamentous fungi and yeast, including Saccharomyces cerevisiae and Pichia pastoris ; insects, including Drosophila melanogaster , Aedes albopictus , Spodoptera frugiperda , and Bombyx mori ; Dictyostelium ; Xenopus oocytes, and other types of cells, as well as plant tissue culture, transgenic animals and transgenic plants. Progress is continuing in the development of cell-free systems consisting of purified components. The choice of a suitable host cell or expression system for protein production depends on many considerations, such as cell growth characteristics, the ability to effect extracellular expression, post-translational modifications, folding and biological activity of the protein of interest, as well as regulatory and economic issues in the large-scale production of therapeutic proteins. The economics of the selection of a particular expression system requires a cost breakdown in terms of process, design, and other considerations. Key advantages of mammalian cells over other hosts are the ability to carry out proper protein folding, and complex N -linked and authentic O -linked glycosylation of mammalian proteins. Also, mammalian cells posses an extensive post-translational modification machinery, including the ability to produce mature proteins through proteolytic processing.