A Novel Process for Developing Fully Human Monoclonal Antibodies

The authors describe a process for generating high affinity, fully human antibodies in culture. Specially selected splenocytes are incubated with cytokines and low levels of antigen, yielding affinity-matured, class-switched B cells. The fully human, specific antibodies produced by these cells can then be cloned and expressed for further characterization.
Nov 01, 2012
Volume 2012 Supplement, Issue 6

Monoclonal antibodies (mAbs) are a rapidly growing class of human therapeutics representing approximately 25% of drugs under development (1). By 2014, 34 therapeutic mAbs are predicted to be on the market for treating cancer, autoimmune diseases, and infectious diseases (1). The commercial market for therapeutic mAbs grew to $32 billion in 2008, and is expected to reach $58 billion by 2014 (1).

Since their discovery in 1975, mAbs have been described as "magic bullets" with the potential to seek out and bind targets with high affinity and specificity (2). The potential of using mAbs to treat human disease was immediately apparent. Early treatment efforts using rodent systems to develop mAbs for use in humans proved ineffective because of the strong immune responses they elicited in humans, limiting their half-life in the body and potentially causing anaphylaxis (3–5). Strategies to overcome this obstacle have included humanization of rodent-derived mAbs and development of fully human mAbs (hu-mAbs).

Currently, there are 27 mAb therapeutics on the market, and mAbs account for more than 25% of the drugs in the FDA pipeline and approximately 50% of all new drug launches (6, 7). Despite significant promise as therapeutic agents, many challenges potentially block mAbs' successful clinical implementation, including low affinities of mAbs for their targets, allergic reactions to mAbs, and high clearance rates. To address these challenges, there is a need to develop new therapeutic mAbs that are fully human. Hu-mAbs advance through clinical trials quickly because of their high specificity and typically predictable toxicity (1). Murine antibodies previously used as therapeutics (e.g., Johnson and Johnson's Orthoclone OKT3) had to be withdrawn from the market because of immunogenicity issues, short half-life, and side effects. Humanized mAbs, such as Genetech/Roche's Herceptin, and hu-mAbs, such as Abbott's Humira, are clearly the more desirable antibody therapeutics.

Methods for fully human antibody development

Several technologies exist for developing hu-mAbs, including:

  • Complementarity-determining region (CDR) engraftment: This method entails preparation of a cDNA library, amplification of immunoglobulin (Ig) CDRs from a mouse hybridoma cell line, and engineering these sequences into the human variable light and heavy chain framework (8-10).
  • Use of transgenic mice with human Ig genes: This method uses traditional hybridoma fusion techniques or molecular techniques to produce hu-mAbs (2). The transgenic mice have lost endogenous murine Ig production, and instead express DNA encoding human Ig genes (11–17). An immunized transgenic mouse is induced to develop an antigen-specific human immune response. Its B cells are then isolated and fused with a myeloma cell line to produce a mouse hybridoma that secrets a hu-mAb.
  • Display technologies such as phage, yeast, and ribosome: These methods rely upon selection of desired binding characteristics of a particular antibody or scaffold protein expressed on the surface of a bacteriophage or other format (e.g., yeast cell or mammalian cell) and recovery of the encoding genes (10).

Current technologies for hu-mAb development are suboptimal in that the therapeutics derived from these approaches are either not fully human, resulting in increased human antimouse immune response, or require several rounds of maturation to achieve high affinity. The high cost of developing fully hu-mAbs using these available technologies, prevents many therapeutics researchers from employing them in preclinical testing against novel targets.

Figure 1: Method for production of fully human monoclonal antibodies.
A reactivation strategy has been developed by Neoclone Biotechnology for generating fully human mAbs (see Figure 1). The technology employs in vitro culturing of immunized, specially-selected splenocytes with cytokines and low levels of antigen (Ag). This reactivation process yields affinity-matured, class-switched B cells. NeoClone has already developed and commercialized a murine mAb technology that employs in vitro reactivation for the custom mouse mAb market and is currently testing a novel hu-mAb development technology using therapeutically relevant cancer Ags.

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