Bovine apolipoprotein B-100 is a dominant immunogen in therapeutic cell populations cultured in fetal calf serum in mice and humans

Abstract

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine embryonic stem cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100, which binds to abundant low-density lipoprotein receptors on the cell surface and is internalized. Here we show that in the majority of patients administered 3 different types of cell-based therapies using cells grown in fetal calf serum-containing media, an antibody response to bovine apolipoprotein B-100 develops after the second infusion and is the dominant specificity. The known and potential clinical effects of such antibodies are discussed.

Figure 1

Mice immunized with embryonic stem cells produce antibodies. (A) FACS analysis of antiserum from FVB/N mice immunized with BL6.9 ES cells. Sera from mice before and after the second, third, and fourth immunizations were analyzed (solid line); secondary antibody only (dotted line). (B) Indicated tumor cell lines, C57BL/6 and BALB/c splenocytes, and human peripheral blood lymphocytes were stained with serum from ES cell immunized FVB/N mice (solid line), with serum from nonimmunized FVB/N mice (dotted line), and (C) with monoclonal antibody 3E8.1 (solid line) and isotype-matched antibody (dotted line).

Figure 2

Identification of the ligand detected by 3E8.1 mAb and antiserum from BL6.9 immunized mice. Western blot analysis of ES cell antiserum identified a unique band (BW lysate, left). The same molecular-weight band was precipitated from both surface-biotinylated BW cell (BW lysate, middle) and BL6.9 cell lysates (right panel) with the 3E8.1 mAb. The band is approximately 520 kDa (BW lysate, right).

Figure 3

The target of antibody responses is bovine apoB. (A) 3E8.1 binding to BW cells (thick solid lines) is blocked with antimouse LDL receptor antibody and heparin (dotted lines) but the binding of H-2Kk antibody is not blocked. (B) Sera from mice, adult cows, and human donors were immunoprecipitated by 3E8.1 or isotype-matched control. Human serum was immunoprecipitated by antihuman apoB antibody (bottom, right). “C” refers to immunoprecipitation using isotype matched control antibody and “3” to immunoprecipitation with 3E8.1 mAb. (C) BW cells cultured with 10% FCS (dark solid line) or 10% lipoprotein deficient (LD) medium (dotted line) were stained with indicated antibodies. Isotype-matched antibody (thin solid line) and normal rabbit Ig (dashed line) are negative controls. (D) BW cells incubated for 1 hour in PBS containing various concentrations of FCS were stained with indicated reagents.

Figure 4

Bovine apoB-100 is present on the cell surface and is found intracellularly. (A) Trypsin-treated BW and (B) BL6.9 ES cells were stained by 3E8.1-FITC with or without saponin permeabilization. The staining medium contained PBS only. (C) Permeabilized BW cells were stained with 3E8.1-FITC or isotype-matched control antibodies and then stained with Alexa Fluor 488 conjugated goat antimouse IgG.

Figure 5

Bovine apoB-100 is the predominant target of the antibody response in patients treated with FCS cultured cells. (A) FCS was immunoprecipitated by isotype-matched control IgG1, 3E8.1 mAb, serum samples from 2 healthy human volunteers, or 2 serum samples from ADA-SCID patients in a gene therapy trial. Samples were analyzed by SDS-PAGE. (B) BW cells were incubated with the FITC-conjugated IgG fraction of serum from patient 2 with (dotted light line) or without (thick solid line) 3E8.1 mAb. (C) FCS treated with ( + ) or without (−) neuraminidase was immunoprecipitated by 3E8.1 or by serum from patient 2 and analyzed by SDS-PAGE (lower panel) or by Western blotting with anti-Neu5Gc antibody (upper panel). (D) FCS immunoprecipitated by serum from a healthy human volunteer, by serum from patient 2, or by serum samples from 4 patients in the breast cancer vaccine trial, were analyzed by SDS-PAGE. The definitions of + and − are in “Materials and methods, Patient sera/plasma from clinical trials and healthy donor cells.”