February 1, 2005 (Vol. 25, No. 3)

The Search for Cancer-Related Markers Continues

Recombinant antibody therapeutics are moving to a next generation of products, according to a keynote address by Ronald Levy, M.D., given at IBC’s recent meeting held in San Diego, “Antibody Engineering and Therapeutics.”

Dr. Levy is a professor of medicine at Stanford, and was responsible for one of the early successes of antibody therapeutics. In 1982 he and his colleagues reported successful treatment of a chemotherapy-refractory patient with low-grade follicular lymphoma using high doses of anti-idiotypic monoclonal antibodies.

This strategy takes advantage of the fact that certain tumors of the immune system display antibody molecules on their surface, and these antibody molecules possess unique CDR regions against which antibodies can be produced. In this case a murine anti-idiotypic antibody was injected into the patient, resulting in a complete remission.

The mechanism of action was unknown at the time, and apparently involved activation of immune networks that provided the patient with long-term protection, long after the antibody was eliminated from the patient’s circulation.

This early success was a bright light in a dark period of anticancer antibody development, at a time when few positive results were observed in antibody trials, and the business and scientific communities were expressing a pessimistic view of the technology.

The introduction of recombinant antibody technologies and the development of human and humanized antibodies in the late 80s and 90s have moved many products through clinical trials and into the marketplace. Now the early successes are being followed by a new generation of improved products, as Dr. Levy discussed in the case of Genentech’s Rituxan.

This antibody is approved for the treatment of B-cell lymphomas, and is directed against the CD20 protein, present on the surface of >95% of B cell lymphomas. In a clinical trial, a 48% overall response rate was achieved.

Today, Rituxan is used alone or in combination with a number of different chemotherapeutic treatments. When combined with fludarabine or fludarabine and cyclophosphamide, a high frequency of complete responses and prolonged progression-free survival are observed without significant toxicity.

However, many basic questions remain, including the critical issue of why only a certain subset of patients respond to the drug. One possible mechanism of action of Rituxan is through the mediation of natural killer cells and macrophages. This entails the binding of the Rituxan antibody to the FcgR II A and FcgR III A receptors.

Dr. Levy and his colleagues have determined that a genetic polymorphism exists for the receptor, and that different mutations carried in the general population dramatically increase chances for a favorable outcome. This means that by identifying important binding motifs it is possible to modify the antibody so it shows binding to a larger array of receptor phenotypes.

In fact, an anti-CD20 antibody with a modified Fcg region initiates antibody-dependent cell cytotoxicity much better than does Rituxan. Clinical trials with these antibodies are either in the planning stages or have been initiated.

CBAS Technology

Meanwhile, the search for new cancer-related markers continues. Affitech (Oslo, Norway) uses a procedure known as CBAS (Cell Based Antibody Screening) Technology, according to R&D director Bjorn Cochlovius, Ph.D. This approach uses phagemid display of antibodies from libraries and harkens back to the days of the 1980s when laboratories screened vast numbers of hybridoma fusions in a search for cancer-specific monoclonals.

Despite huge expenditures of resources, such Herculean efforts yielded very few useable antibodies. The screening systems were slow and cumbersome, and it was difficult to evaluate the very large number of candidates without robotics. Most of the antibodies, on careful screening against many human cell types, failed to demonstrate the level of specificity necessary for anticancer therapeutic performance.

The CBAS approach identifies antibodies from libraries specific for native cell surface antigens. Initially the antigen on the surface of the cancer cell is unknown. By isolating the antigen-antibody binding pairs, the target protein can be identified through a variety of methods.

The technology allows removal of irrelevant binders by negative selection on noncancer cells. The resulting clones can then be screened against a large panel of cell types. Using fluorescence activated cell sorting, the Affitech research team identified antibodies that react with mammary carcinoma cell lines.

These antibodies are not directed against the best known mammary cancer proteins, including Her-2/neu, EGF-R, CEA, nor EpCAM.

It will be necessary to carefully evaluate these interesting antibodies against a wide range of normal human tissues, since in the past, anticancer antibodies have frequently proven to be unworkable because of cross reactivity against noncancerous cells, but if Affitech’s initial observations survive preclinical screening, they could prove to be an important addition to the currently approved anticancer antibodies.

Improving Affinity

In recent years, antibody engineering technology has evolved increasingly elegant means to improve affinity of recombinant antibody molecules, including phage and ribosomal display, and rational design of antibody framework and antigen combining regions.

However, there may be a physical limit to the degree that improvements in affinity can be engineered into a single molecular species. Several groups are addressing this problem through different strategies for the development of recombinant antibody cocktails.

James Marks, Ph.D., of the University of California, San Francisco, described his work with recombinant antibodies against botulism toxin. He and his colleagues have developed single chain antibodies against the toxin with Kd values in the range of 10-8 to 10-12.

While single antibodies, even of very high affinity, do not protect animals challenged with low doses of toxin, combinations of as few as three antibodies binding nonoverlapping epitopes protect animals challenged with large toxin doses.

Synergy of antibody combinations resulted from accelerated clearance of toxin from the circulation, an increase in functional affinity, and steric interference with toxin binding to cellular receptors.

With single antibodies, clearance of the toxin is slowed, but this simply prolongs the inevitable death of the host, even with high affinity individual antibodies. When multiple antibody cocktails are used, protection is much better, as the synergy of the molecular interactions yields the required potency.


H. R. Hoogenboom, Ph.D., CSO of Merus Biopharmaceuticals (Driebergen, The Netherlands), discussed his company’s plans for development of defined recombinant antibody mixtures, using cell lines engineered to simultaneously produce several different antibodies.

Traditionally, companies have shied away from the use of antibody mixtures because of cost, and the complexity of manufacturing and regulatory issues. Merus seeks to develop and commercialize proprietary technology for human antibody-based therapeutics using Oligoclonics, defined mixtures of human antibodies.

The Oligoclonic technology is designed to achieve the performance of polyclonal antibodies combined with consistency and cost saving of monoclonal antibodies. This format places multiple genes in the same host cell, rather than growing a number of cell lines concurrently, where they might compete with one another.

A clonal cell line capable of generating different antibodies is produced by simultaneously transfecting cells with multiple antibody heavy and light encoding genes (in one or more expression vectors) and selecting for stable integration.

By carefully screening a number of cell lines, clones are identified that express multiple antibodies, and at a total expression level typical for a monoclonal antibody. Biological assays are used to find the most optimal composition of each of the antibody components in the mixture.

The Merus team demonstrated that when two antibodies sharing the same light chain are encoded in a single host cell (PER.C6), the two homodimers will be produced as well as the heterodimer, composed of the two dissimilar heavy chains and coding for a bifunctional antibody.

In cell lines producing three different antibodies the different species can be distinguished in isoelectric focusing gels. Moreover, peptides encoded by the different antibody genes can be separated out using mass spectrometry.

Using these tools, it was demonstrated that some of the cell lines stably produced the different antibody for 30 generations of cell culture growth. Therefore the assumptions upon which the technology is based appear to be valid.

There are a variety of potential applications of the Merus technology. Oligoclonics cell lines could effectively target multiple epitopes on the same protein target, or they could target different antigens, such as CD20 and CD22 on the same leukemic cell line.

Other potential applications include multiple cytokines or a collective group of antigens on an infectious disease entity. Moreover, this technology has the potential to generate higher affinity and avidity antibodies that could function more vigorously in a variety of tasks, including synergy in apoptosis and antigen-dependent cell cytotoxicity, and suppression of multiply redundant cellular pathways.

This approach has the potential to serve as a means to boost antibody efficacy yet retain a natural, nonmodified antibody format and build on existing antibody production facilities.

While the technology has great appeal, there are substantial challenges that the Merus team is addressing. These include establishment of stability of the antibody-producing cell lines, proof of performance as compared to standard monoclonal and polyclonal antibodies, and the formidable issue of overcoming regulatory hurdles.

Symplex Technology

Another approach to polyclonal recombinant antibody technology is the basis of Symphogen (Lyngby, Denmark), according to John Haurum, M.D., CSO. He discussed the use of Symplex technology, which recreates the natural immune response in vitro.

Thus, antibody-producing B lymphocytes are isolated by cell sorting from humans that have been exposed to desired target antigens, and cognate pairs of variable light and variable heavy regions are isolated using PCR primers that preserve the original pairing of the light and heavy chains.

The product consists of the variable heavy chain gene joined to a variable light chain and constant light chain gene through a linker section. These recombinant antibodies are then cloned into a Fab expression vector and the clones screened without phage display.

In a typical protocol, tetanus toxoid antibodies were isolated from recently vaccinated individuals, and ca. 8% of the clones were tetanus toxoid-specific. Sequencing of the light and heavy chains revealed 75 completely unique, high affinity antibodies from two donors.

“The sequence analysis showed that the original heavy and light chain pairing from the donor B lymphocytes was preserved and the antibodies were of high affinity,” Dr. Haurum stated.

These clones can be used to produce a polyclonal cocktail of fully human recombinant antibodies. After cloning of the antibody drug leads, Symphogen utilizes a proprietary manufacturing technology for production of therapeutic recombinant polyclonal antibodies, based on site-specific integration of the antibody genes into a host cell line carrying a preformed targeting site.

The clones are grown together as a mixture, and because the antibody genes are integrated at the same point on the genome, the cells have identical growth rates. In this fashion the mixtures retain their polyclonal complexion through a number of generations of growth, and batch-to-batch stability and reliability is maintained.

The company’s Sympress polyclonal manufacturing technology is robust in terms of in-process compositional stability, as well as batch-to-batch consistency, and our most advanced drug development project is approaching clinical trials.

“The commercial potential of symphobodies is expected to be unique,” according to Dr. Haurum, and this assessment is reflected in the acquisition of $50M in venture capital. The objective of Symphogen is to build a long-term dominant position within the field of polyclonal antibody therapy.

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