Although some claimed it could not be done, industry scientists reported recently in Cancer Immunology, Immunotherapy that they have identified a novel cell-surface antigen on small-cell lung cancer cells, marking one of the firstif not the firstunique molecular target to be found on these tumors, which account for about 20% of lung cancers.
And the news gets even better. The team found that, when bound by an antibody, this still nameless antigen is then internalized by the tumor cell, suggesting that it might be an ideal port of entry for a deadly immunotoxin to enter and kill the cancer cells. Whats more, they also found that this antigen sheds into the blood of people with established and possibly developing small-cell lung cancer, meaning it could have important implications for future diagnostic and prevention strategies.
As intriguing as this finding sounds, dont expect to find the antigen listed on the CancerTrials web site any time soon as a potential drug target. Molecular Discoveries, LLC, the small, start-up company that spearheaded the research, shut down its laboratory in Tarrytown, N.Y., 2 years ago, another victim of the boom-gone-bust biotechnology industry.
Although the company still exists on paper, waiting for a corporate partner to restart its stalled research engine, what makes this story unique is why Molecular Discoveries has had no takers. Its antigen is a protein-carbohydrate complex, as, in fact, most antigens are in the body. But most companies today are eager to dive into proteomicsthe study of protein structurenot the carbohydrates that decorate them and influence their biological function.
As industry and academia invest billions in the promise of proteomics and ultimately molecular based medicine, the plight of Molecular Discoveries raises a crucial question: What constitutes a valid target for clinical development? Must the target first have been trapped on a 2-D gel, a common proteomic medium, and be based on protein structure alone? Or, are non-proteomic approaches that offer different and perhaps more complete biological perspectives fair game?
It is an issue that has some scientists concerned. "Proteomics by itself cannot solve any biological problem," said Senitiroh Hakomori, M.D., Ph.D., a scientist at the Pacific Northwest Research Institute in Seattle. "The majority of proteinsabout 65% of total proteins and over 80% of cell-surface proteins are highly glycosylated [covered in sugars]. Without knowledge of glycosylation, protein function and their interaction with antigens or antibodies cannot be understood."
Cryptic Antigens
When the immune system mounts a response, it generally goes for the molecular equivalent of the sure thing: It targets the most dominant antigens on the cell surface. Although this strategy keeps most of us alive and well, it presents a challenge for scientists interested in identifying low abundance, or so-called "cryptic," antigens, where many believe the subtle and potentially most exploitable differences between normal and tumor cells lie.
To discover these cryptic antigens, scientists traditionally have had to cast a wide and inefficient immunologic net. They inject a laboratory animal with tumor cells to elicit an immune response, then haul in for analysis all of the antibodies that its immune system produces. The problem is, because tumor cells and normal cells share the same dominant antigens and the immune system tends to target the sure thing, the vast majority of antibodies bind to the dominant antigens. That leaves the scientists with the tedious task of essentially throwing the vast majority of antibodies back into the water and, of the hundreds that might be left, screening them one by one to test whether they selectively bind to tumor cells and leave normal cells alone. Often, all of the hard work produces little or nothing new.
In the late 1990s, Cohava Gelber, Ph.D., who had been studying autoimmunity at Stanford University in Palo Alto, Calif., made what she considered an intriguing discovery. She and her colleagues had pieced together a "virtual lymph node"a perforated, half-centimeter piece of plastic tubing with a bit of sponge stuffed insidethat allowed them to trap immune cells and measure their activity over time against individual antigens.
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Addition By Subtraction
The group called the concept Differential Immunization for Antigen or Antibody Discovery, or DIAAD. Although the acronym might be new, the general concept, called subtractive immunization, is not. Introduced in the 1980s by developmental biologists, efficiently subtracting out the dominant antigens from an immune response, according to many, is somewhat of an artform, which has restricted its use to a few dedicated laboratories.
Gelber said that she and her colleagues quickly recognized the potential utility of DIAAD for discovering unique tumor antigens. "I talked to a few companies, and one of them actually came with a list of different tumor types that had been the most challenging in the field," she said.
At the top of the list was small-cell lung cancer, which involves neuroendocrine cells in the lungs. "Over the last 20 years, scientists have been given a lot of grants and put a lot of energy into attempting to find a unique small-cell lung cancer antigen that would not cross react with normal neuroendocrine-derived tissues," Gelber said. "We actually put a grant into the NIH, claiming that we could do it, and we were denied based on the fact that it had been tried before, and they didnt believe it would work."
Around this time, Gelber met with Leonard Gordon, a New York venture capitalist and founder of two medical device companies, who liked what he heard. In mid-1999, with Gordons financial backing, Molecular Discoveries LLC was born. "Cohava gave me the lowest budget and shortest time frame that I have ever heard in this business," said Gordon, noting the time frame was 6 months. "And she brought it in."
Assembling a team of six scientists, Molecular Discoveries first generated in vitro data on a monoclonal antibody, VAC69, that selectively bound to multiple myeloma and ovarian cancer cells, leaving normal cells untouched. According to Gelber, the group also found, after transplanting multiple myeloma or ovarian cancer cells into mice, that all of the animals responded to the antibody. Gelber also said the novel antigen, a single-chain glycoprotein, was found in the blood of a preliminary sampling of ovarian cancer patients, suggesting it might be useful in diagnosing or staging the condition.
Meanwhile, the group also succeeded in raising five monoclonal antibodies that identified a unique antigen, a single-chain glycoprotein, on the surface of small-cell lung cancer cells. "It was very difficult because the overlap between small-cell lung cancer and the neuroendocrine tissues was more than 99%," said Gelber.
Gelber said, once the protocol clicked, it really clicked. She and her colleagues found five different antibodies specific to small-cell lung cancer in one 96-well plate, a success rate unheard of in monoclonal laboratories. "We had so many clones in the end that we just didnt have the resources to follow all of them," said Gelber, noting that the antibodies also bound to pancreatic cancer cells. "We basically just froze them down."
Out of Money
Because of the initial successes, however, the company now needed a sustained and more substantial investment to move its discoveries into clinical development and ultimately human trials. As Gordon explained, the timing couldnt have been worse. "The market turned to hell," he said. "Im really excited about it, but nobody seems to be interested in a product that is 3 to 5 years out with possibly $20 million in expenses. And, of course there is always a question mark with any antibody, although outside laboratories have now validated these antibodies."
In April 2001, Molecular Discoveries folded down its laboratory, though keeping the company afloat on paper should a corporate partner appear.
Recently, Gelber and colleagues summarized the data on the small-cell lung cancer work and published it in Cancer Immunology, Immunotherapy. Their data on the multiple myeloma antibodies was published in the Journal of Immunotherapy in 2001. Although several companies have considered partnering with Molecular Discoveries, Gelber said some have stepped away, in part, because of the chemical nature of the antigens. "Companies have a lot of information about antibodies directed against proteins," she said. "But there are very, very few companies that specialize in carbohydrates. So, when you have a target that a company is not familiar with, the level of risk increases, even though its a valid target."
As Gelber noted, these antigens could not have been found with popular gene profiling strategies. "Our technique can look at glycolipids, glycoproteins, sugar residues, and so on," said Gelber. "It is so specific that it can pick up on subtle changes involving one molecule. By gene profiling, if the same gene is there, and there is no dramatic change in the amount of RNA, for instance, you wouldnt know that there is differential expression."
Nor would the antigens have been detected with proteomic approaches. "We know that most of the antigens of progressive cancer cells are not pure proteins," she said. "They are highly glycolsylated, and you wouldnt see them. The protein sequence of the antigen might be the same in a normal person, but the amount and types of sugars on the antigen will be different." In fact, Gelbers group reported that their monoclonal antibody only worked in the small-cell lung cancer cells when the carbohydrate chain was present. Remove it, and the antibody was ineffective. Although the group could not rule out a protein-carbohydrate interaction as key for antibody recognition, they also could not dismiss the carbohydrate as the pivotal epitope.
James Quigley, Ph.D., a cancer researcher at The Scripps Research Institute in La Jolla, Calif., who uses subtractive immunization in his laboratory to study metastasis, said he agrees that genomic and proteomic approaches do not tell the whole molecular story. "By screening monoclonal antibodies, by nature of their specificity, you do end up with things that are more relevant," he said. "Monoclonals as therapeutics and diagnostics certainly shouldnt be looked askance at, but, right now, proteomics and genomics are whats in vogue."
"There is no doubt about the fact that there are fads in science, as in all aspects of life, and proteomics is one of them at present," said Christian Raetz, M.D., Ph.D., a biochemist at Duke University Medical Center in Durham, N.C., who previously has worked at Merck & Co., Inc. ". . . With regard to drug discovery, we already had many genetically validated targets in the 1980s, but that did not ensure finding a drug for each of those targets. The research community will eventually figure all this out once the fad has passed, and there will be winners, but not necessarily those people who were pursuing the fads."
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