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Discovery of Therapeutic Human Monoclonal Antibiotics through B-cell Immortalization
Dr. Young-Sun Lin
CSO, Medigen Biotechnology Corp. Taipei, Taiwan

The business of drug discovery and development is well known for its uncertainty, simply because of the paramount ethical demand for the safety and efficacy of therapeutics, which more often than not leads to abruptly and obligatorily abandoned once-favored drug candidates, thus accompanied with tremendous financial losses.

Nonetheless, there are a few exceptions to the above-mentioned characteristics of pharmaceutical industry. The category of therapeutic human monoclonal antibody (th-mAb) is a prominent example among those exceptions: the facts of both its origin from and daily utilization by the human body testify to its safety and efficacy for long-term therapeutic applications. In other words, once the bench work (i.e., identification and isolation of a candidate th-mAb) is finished, every other work thereafter (i.e., preclinical animal study and human clinical trials) is largely becoming an expectable due process to routinely comply with the FDA regulations.

Four classes of th-mAb can be derived from potential donors: (1) th-mAb against viral pathogens (such as, HBV, HCMV, HSV1, HSV2, RSV, H5N1…etc), (2) th-mAb against multiple drug-resistant bacteria pathogens (such as, Staphylococcus aureus, Neisseria gonorrhea…etc), (3) th-mAb against cytokines (such as, IL-1, IL2, IL-6, IL-17, IL20, IL-23, TGF-ß, TNF-a…etc) and (4) th-mAb against tumor antigens (such as, Mucin 1, Neu, EGFR…etc). The conceptual simplicity, beauty and feasibility of classes 1 and 2 th-mAbs are self-evident. However, the situation for classes 3 and 4 th-mAb are more complicated and thus need a little more elaboration below.

Patients of autoimmune diseases, and occasionally healthy individuals, produce distinct autoantibodies against their endogenous cytokines (such as, IL-1a, IL-2, IL-6, IL-8, IL-10, GM-CSF and TNF-a [1-13]. Interestingly, the overexpression of some of these cytokines is known to cause many an inflammatory, degenerative or metabolic disease in human. Thus, autoantibodies from donors of autoimmune patients hold great potential for the treatment of distinct human diseases.

As for class 4 th-mAb, it is well known that tumor cells release large quantities of tumor antigens after necrosis and/or apoptosis. Thus, it is reasonable to expect that autoantibodies against them should be made by the patient’s immune system. Indeed, sera derived from cancer patients have been shown to contain many distinct autoantibodies against various cancer antigens [14-37]. Accordingly, autoantibodies from donors of cancer patients should provide precious sources for cancer diagnostics and treatments.

There is one tedious job and one technological bottle-neck to be encountered in the process of cloning a desired th-mAb: the former is screening for suitable donors, while the latter is cloning of corresponding immunoglobulin heavy-chain and light-chain genes from specific memory B cells of the identified donor(s).

The criteria for the former are quite straightforward: donor(s) with serum of high neutralizing activity and no detectable interference of the neutralizing activity by components from other’s serum.

As for the latter, the short life span (just a few days) of memory B cells in vitro has forced researchers to design a variety of approaches to clone th-mAb genes from them as briefly described below:

  1. "Phage display library" exemplified by Cambridge Antibody Technology Group
  2. "Transgenic mouse with human Ig locus" exemplified by Medarex, Abgenix and Regeneron
  3. "Human Ig library by recombinant lentivirus/retrovirus" exemplified by AIMM Therapeutics
  4. "Human hybridoma technology" exemplified by Kenta Biotech and FuCell
  5. "Immortalization of human B cell by EBV" exemplified by Humabs LLC

Each of the above approach has its own technological strengths and weaknesses. As for approach 5, according to the methodology described in “Current Protocols” [38], the efficiency of human B cell immortalization by EBV is as low as 0.01%. Thus, it would be almost impossible to clone a desired Ig gene using the traditional protocol for B cell immortalization by EBV.

Researchers at Medigen have developed an ultra-efficient platform technology for the immortalization of human B cells by EBV (see figures 1 and 2, and table 1 for a brief description). With this proprietary technology at hand, we have routinely immortalized suitable donors’ B cells. Secreted antibodies from the pooled immortalized B cells are subjected to a variety of biochemical, molecular, immunological and cellular assays to determine whether the desired B cell exists in the pooled immortalized B cells. This confirmation step demonstrates another advantage of our protocol — isolation of the specific B cell clone is to be initiated only when the outcome of B cell immortalization is satisfactory.

Accordingly, Medigen has initiated a series of collaboration projects with National Taiwan University Hospital and Taipei Veterans General Hospital, focusing on the following th-mAb:

  1. To isolate neutralizing antibodies from donors recuperating from certain viral and bacterial infectious diseases. The etiological agents include RSV, HBV, HCMV, HSV1, HSV2, H5N1, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Vancomycin- Resistant Enterococci and Neisseria gonorrhea.
  2. To isolate autoantibodies against cytokines from autoimmune patients. The cytokine targets include IL-1, IL2, IL-6, IL-17, IL20, IL-23, TGF-ß, and TNF-a.
  3. To isolate neutralizing antibodies against tumor antigens from cancer patients and/or the general population. The tumor antigens will be predominately focused on Mucin 1, Neu and EGFR.

The global market for monoclonal antibodies was valued at U$27.4 billion in 2008, with a compound annual growth rate (CAGR) of 30.8% between 2000 and 2008. By 2015, due to the expected launch of many new therapeutics such as Denosumab (Amgen) and Teplizumab (Eli Lily), sales of monoclonal antibodies are expected to reach U$67.6 billion, indicating a CAGR of 13.8% between 2008 and 2015.

In short, the th-mAb represents a so-called “blue ocean strategy” in the field of drug discovery and development. In addition to the elimination of uncertainty intrinsic to the pharmaceutical industry, our approach also reduces the financial burden to a minimum: screening, immortalization and confirmation are straightforward and cost little money. Once these works are done, an efficacious therapeutic is surely to be materialized in the not-so-distant future.

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