Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas

Abstract

Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igα and Igβ of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igα expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).

Introduction

Monoclonal antibodies (mAbs) are essential biotechnology reagents widely used in every phase of the biomedical field from discovery research and diagnosis to therapeutics. The power of mAb based therapeutics lies primarily in their specificity and strong affinity for a target antigen as well as their ability to mediate immune effector functions such as Antibody Dependent Cell Mediated Cytotoxicity and the Complement Cascade. The expansion of downstream technologies, most notably the humanization of mouse mAbs, and resultant low immunogenicity, have greatly expanded the therapeutic applications for mAbs (Milstein, 1999, Glennie and van de Winkel, 2003, Jakobovits et al., 2007). Consequently, there are 21 therapeutic mAb-based reagents currently approved for clinical use, five with annual sales in excess of $3B, and many others in various stages of clinical trials for a variety of diseases (Maggon, 2007) (www.fda.gov/cder/biologics/default.html, www.pipelinereview.com/index.php?option=com_performs&formid=7). In addition, mAbs account for a major market share in clinical and outpatient products and even over-the-counter diagnostics. However, current hybridoma-based mAb development technologies are cumbersome and confounding to the demands of high-throughput facilities. Further, alternative methodologies that avoid hybridomas (e.g. XenoMax and Phage Display), while offering certain advantages, carry burdens of expense and proprietary issues and have their own limitations (Marks et al., 1991, Babcook et al., 1996). Hence, we set out to develop new scientific and technical tools for rapid hybridoma detection and isolation.

Kohler and Milstein's (1975) seminal publication describes the generation and selection of hybridoma cells, heterokaryons resulting from the fusion of mouse B-lymphocytes and immortal myeloma cells, for the production of mAbs. The relevant hybridoma cells producing the mAb of choice are separated into individual clones using Limiting Dilution Subcloning (LDS). Recovering the hybridomas using cell cloning by LDS is perhaps the most problematic, time consuming, and labor-intensive step in generating mAbs (Antczak, 1982, O'Reilly et al., 1998). The fused hybridoma cells are deposited into a few thousand microtiter plate wells containing media supplemented with HAT (hypoxanthine, aminopterin, thymidine). HAT selects for hybridoma cells by killing unfused myeloma cells. The desired hybridomas are identified by screening for the reactivity of mAb secreted into the media using well-known methods such as an Enzyme-Linked Immunosorbent Assay (ELISA). Each HAT resistant cell population testing positive for secreted target mAb must be processed by reiterative cycles of LDS until the progeny of a positive cell is mathematically identified as clonal (Staszewski, 1984). Proposed solutions to this limitation including soft agar culture techniques (Draber et al., 1980), robotics to conduct the repeated cycles of LDS (Wewetzer and Seilheimer, 1995) and micro-encapsulation technologies that trap and assay the secreted Ab in the media around cells (Prokop et al., 2004, Hanania et al., 2005) have attempted to address the various weaknesses of LDS, but are expensive or offer little improvement in the efficiency.

The LDS process could be eliminated if all the desired hybridoma cells expressed the membrane Ig form of the secreted mAb. Cells could then be purified using Fluorescence Activated Cell Sorting (FACS). A few early attempts to use FACS for hybridoma cell cloning (Parks et al., 1979, Meilhoc et al., 1989), while promising, lacked efficiency because most hybridomas poorly express surface Ig (Matsuuchi et al., 1992, Seegmiller et al., 2007). Thus, the immediate objective of our research was to generate hybridomas that would consistently express membrane Ig on the cell surface and thereby facilitate efficient clonal selection by FACS.

We saw two potential obstacles to developing DiSH technology. The first of these was expression of the B-cell receptor subunit proteins Igα (CD79a, NP_031681) and Igβ (CD79b, NP_032365) necessary for assembly and trafficking of a functional BCR complex to the cell surface. Expression of membrane immunoglobulin on the surface of myeloma cells was obtained by transfecting lymphoid cells with cDNAs encoding the membrane isovariant of the antibody heavy chain (HC_m) and the Igα and Igβ receptor proteins (Hombach et al., 1990). A diagram of the proposed natural arrangement of these proteins on the cell surface as they are positioned in the B-cell antigen receptor (BCR) complex is shown in Fig. 1A This experimental observation of engineered BCR complex presentation on the cell surface was extended to non-lymphoid cells using a pituitary cell line that is active in secretory functions, but normally would not synthesize the BCR nor express it on its cell surface (Matsuuchi et al., 1992). Once again, transgenic expression of the associated Igα and Igβ proteins, and light chain (LC) and HC_m was sufficient to restore cell surface expression of the complete BCR. Thus, the first obstacle to developing hybridomas that efficiently express membrane immunoglobulin on their surface might be the limiting expression of Igα and Igβ proteins, and perhaps other accessory factors necessary for assembly and trafficking of a functional BCR complex.

The second potential obstacle to developing hybridomas that efficiently express membrane Ig might be a deficiency in the expression of the membrane isovariant of the Ig heavy chain (HC_m). It is unclear if hybridoma cells express the longer HC_m isovariant that is normally part of the BCR complex, along with the usual high levels of HC_s, the secreted form. HC_m contains the C-terminal peptide partly responsible for efficient insertion of the Ig molecule into the membrane. As B-cells develop into plasma cells an alteration in the post-transcriptional processing of the HC nuclear transcript (Fig. 1B) is reported to result in a switch from HC_m to HC_s expression. This switch plus an increase in the rate of transcription results in robust antibody (Ab) secretion (Milcarek and Hall, 1985, Genovese et al., 1991, Lassman et al., 1992). HC_s protein lacking the membrane-spanning domains is thought to be unable to bind the associated receptor proteins, Igα and Igβ, and therefore is secreted. Thus, we explored HC_m, Igα, and Igβ protein expression as possible barriers to obtaining high level BCR expression in hybridomas.

We found that Igα expression was the major limitation to the efficient expression of membrane-associated Ig for typical mAb secreting hybridoma cells. Furthermore, hybridomas derived from a myeloma parent engineered to over-express Igα displayed significant amounts of the BCR complex on the cell surface. Using this engineered myeloma, hundreds of hybridomas making Abs to four diverse antigens were rapidly and efficiently identified and recovered by FACS. DiSH technology greatly simplifies and accelerates mAb development and should help in developing targeted therapeutics and meet the ever-increasing demand for these valuable reagents.