A novel platform to produce human monoclonal antibodies

Abstract

A new technology has been developed that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps, and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.

Acknowledgements

The authors thank W. Guy Bradley for graciously assisting with the flow cytometry and ELISpot experiments contained in this manuscript. We would also like to thank the members of our Scientific Advisory Board for assisting in the preparation of this manuscript.

Conflict of Interest

All three authors are full-time employees of Immunologix, Inc.

Figures and Tables

Figure 1 Enrichment of naïve B cells from human tonsil tissue. Mononuclear cells (MC) prepared from a Ficoll-Hypaque gradient were enriched for B cells using negative magnetic separation. Samples of the mononuclear cells (A–C) and the enriched B-cells (D–F) were phenotyped by gating for CD45 positive cells and staining for CD19 and CD20 (A and D) and also by staining for surface IgD, IgM and/or IgG expression (B, C, E and F). Results from a representative sample are shown.

Figure 2 (A) Representative EliSpots displaying IgG secretion. (i) Freshly isolated naïve B cells from human tonsil [mean = 0.33 cells (0.0% positive)]. (ii) Total mononuclear cell population [mean = 17 cells (0.23% positive)]. (iii) B-cell population after undergoing Immunologix platform technology [mean = 438 cells (14.6% positive)]. All samples plated at 3 × 10³ cells/well and done in triplicates. (B) Profile of IgG subclasses expressed by B-cell libraries. Duplicate experiment with two libraries.

Figure 3 Primary screening of human B-cell supernatants. After at least 12 days, supernatant from each well of the 96-well plates containing transformed human B cells was added to microtiter plates on which antigen was immobilized and captured human IgG detected using an HRP conjugated anti-IgG-Fc antibody. The data from the wells were sorted in descending order and expressed as a line graph. Supernatants from at least four B-cell libraries (48 plates total) are represented for each antigen. The inset indicates the percentage of supernatants that returned an optical density (OD) of greater than 1.0.

Table 1 Cellular yields from tonsil tissue

Table 2 B-cell transformation efficiency using spinfection with 10× concentrated EBV supernatant