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From : Scientific American



Magic Bullets Fly Again



Molecular guided missiles called monoclonal antibodies were poised to shoot down cancer and a host of other diseases--until they crashed and burned. Now a new generation is soaring to market

By Carol Ezzell

The unbridled optimism that surrounded monoclonal antibodies in the 1980s was



infectious. You had to be the world's toughest cynic not to be dazzled. Got cancer?

No problem. Like heat-seeking missiles, monoclonal antibodies tipped with poisons

or radioactive isotopes would home in on malignant cells and deliver their deadly

payloads, wiping out cancer while leaving normal cells intact. How about an

infectious disease? All would be well. Monoclonals would surround marauding

viruses and bacteria like goombahs from Tony Soprano's crew, muscling them into

secluded byways where killer cells of the immune system would make them an offer

they couldn't refuse.

If only things had been so simple. Monoclonal antibodies are highly pure

populations of immune system proteins that attack specific molecular targets.

Unfortunately, people who received infusions of the early therapeutic monoclonal

antibodies tended to develop their own antibodies against the foreign ones, which

caused them to become even sicker for reasons that are not entirely clear. And

the liver showed a predilection for these early monoclonals, sopping them up

before they could target their quarries. Clinical trials failed. Stocks plunged.

Millions of dollars were lost. And a generation of scientists and biotechnology

Business people developed the skepticism shared only by the once burned, twice

shy.

Luckily, some of those individuals soldiered on despite the bad news and found

ways to overcome the failings of the early versions of the drugs. Now many are

hoping that 2001 will be the Year of the Monoclonals, when their perseverance

will pay off in the form of lots of effective monoclonal antibody-based drugs

approved or under evaluation by the U.S. Food and Drug Administration. "Antibodies

will be surging ahead," says Franklin M. Berger, a biotech analyst with JP Morgan

Securities. He predicts that soon there will be so many monoclonal antibodies

awaiting approval by the FDA that they will cause a bottleneck in the review

process.

Ten monoclonals have reached the market, and three await FDA approval, including

the first two that would be equipped to deliver a dose of radiation [see table].

Another 100 or more antibodies are being tested in humans, having already shown

promise in tests involving animals. But this summer the FDA sent a message that

could slow the monoclonal juggernaut. In July the agency told Genentech, located

in South San Francisco, Calif., that it would have to present additional data

from human (clinical) trials to prove the long-term safety of its monoclonal

antibody for asthma, Xolair, which mops up the antibodies that play a role in

asthma and allergies. Some observers have interpreted the move as an indication

that the FDA might be particularly rigorous in scrutinizing the side effects of

monoclonal antibodies, especially those that patients would take for years for

chronic conditions. The announcement sent a brief chill through investors, who

drove down the stocks of monoclonal developers for a week or so.

Nevertheless, the advantages of monoclonals are hard to ignore. Donald L. Drakeman,

president and CEO of monoclonal maker Medarex in Princeton, N.J., says that

antibodies are simply easier to develop than traditional drugs composed of small,

inorganic molecules. Because they are large molecules, they might not be suitable

for every disease, but he emphasizes that it takes only one or two years to come

up with a monoclonal antibody suitable for testing, versus the five years required

for small molecules. That speed translates into savings: it costs only $2 million

to ready a monoclonal antibody for clinical testing, Drakeman estimates, compared

with $20 million for a traditional drug. And despite the FDA's hesitancy to approve

Genentech's asthma therapy, he states that monoclonals have so far had a higher

success rate than small-molecule drugs in clearing regulatory hurdles.

"Antibodies are almost never toxic," he explains.

Ironically, monoclonals might be victims of their own success: market analysts

are predicting that companies won't have sufficient production facilities to make

them all. But the biotechnology industry has anticipated this problem. Some of

the more inventive proposals include the manufacture of monoclonals in the milk

of livestock or in plants.





Monoclonal Methods

The past failure of monoclonals stemmed in part from the way they were originally

made. The classic manufacturing technique was devised in 1975 by immunologists

Georges J. F. Kler and Car Milstein of the Medical Research Council's

Laboratory of Molecular Biology in Cambridge, England, who were awarded the 1984

Nobel Prize in Physiology or Medicine for their innovation. The basic process

involves injecting an antigen--a substance the immune system recognizes as

foreign or dangerous--into a mouse, thereby inducing the mouse's

antibody-producing cells, called B lymphocytes, to produce antibodies to that

antigen. To harvest such antibodies, scientists would ideally pluck only the B

cells that make them. But finding the cells and getting them to make large

quantities of the antibodies takes some doing.

Part of the complex procedure involves fusing B cells from the mice to immortalized

(endlessly replicating) cells in culture to create cells called hybridomas

[see illustration]. The drawback of these particular hybridomas is that they

produce murine antibodies, which the human immune system can perceive as

interlopers. Patients who have received infusions of murine monoclonals have

experienced a so-called HAMA response, named for the human anti-mouse antibodies

they generate. The HAMA response includes joint swelling, rashes and kidney

failure and can be life-threatening. It also destroys the antibodies.

To avoid both the HAMA response and the premature inactivation of mouse antibodies

by the immune system, scientists have developed a variety of techniques to make

murine antibodies more human. Antibodies are Y-shaped molecules that bind to

antigens through the arms, or FAb regions, of that Y. The stem of the Y, the Fc

region, interacts with cells of the immune system. The Fc region is particularly

important in eradicating bacteria: once antibodies coat a bacterium by binding

to it through their FAb regions, the Fc regions attract microbe-engulfing cells

to destroy it.

One approach involves replacing all but the antigen-binding regions of murine

monoclonals with human components. Four of the monoclonals now for sale in the

U.S. are such chimeric--part mouse, part human--antibodies. Among them is ReoPro,

made by Centocor in Malvern, Pa., which prevents blood clots by binding to a

specific receptor on platelets; it had sales last year of $418 million. (The body

usually doesn't make antibodies targeted to healthy tissues, or autoimmune

disease would result. But such antibodies, delivered as drugs, can help treat

certain disorders.)

Another strategy, called humanization, is behind five more products on the market,

including Herceptin, the breast cancer-targeting monoclonal antibody developed

by Genentech. Humanization entails using genetic engineering to selectively

replace as much as possible of the murine antibodies--including much of their

antigen-binding regions--with human protein [see illustration].



Campath--thought by its maker, Millennium Pharmaceuticals in Cambridge, Mass.,

to be the first humanized antibody ever made--received FDA approval in May for

people with B cell chronic lymphocytic leukemia for whom other therapies haven't

worked. Campath binds to a receptor found on various types of normal and cancerous

immune cells, but patients make more of the normal cells after treatment ends.

The other monoclonal on the market is a purely murine antibody.

After more than 25 years of trying, researchers have also finally fused human

B cells to immortalized cells to create hybridomas that generate fully human

antibodies. In February, Abraham Karpas of the University of Cambridge and his

colleagues reported accomplishing the feat, although it is too soon to tell

whether the monoclonals made using human cells will be safer, more effective or

cheaper to manufacture than those generated using other technologies.

Medarex and Fremont, Calif.-based Abgenix have devised ways to induce mice to

produce fully human antibodies. The companies genetically alter the mice to

contain human antibody genes; when they inject the mice with antigens, the animals

produce antibodies that are human in every way. "The technology to humanize or

make fully human monoclonal antibodies was one of those changes that made

[monoclonals] more commercially viable," suggests Walter Newman, senior vice

president of biotherapeutics and nonclinical development for Millennium

Pharmaceuticals, which is also developing monoclonal antibody therapeutics.

Abgenix is conducting clinical tests of a fully human antibody against

interleukin-8 (IL-8), a naturally occurring chemical known as a cytokine that

normally activates cells of the immune system. When the body produces too much

IL-8, inflammatory autoimmune diseases such as rheumatoid arthritis or psoriasis

can result. Medarex has a variety of clinical trials of fully human monoclonals

ongoing for cancer and autoimmune conditions. It is also developing so-called

designer antibodies that have been engineered either to deliver a toxin directly

to a diseased cell or to recruit immune cells specifically to attack tumors.

Other investigators are attempting to mass-produce monoclonals without the aid

of mice. Cambridge Antibody Technology in England and MorphoSys AG in Munich are

using a technique called phage display that does just that--and also helps to

find the most specific monoclonals against a particular antigen

Phage display takes advantage of a long, stringy virus called a filamentous phage

that infects bacteria. Researchers can isolate DNA from human B lymphocytes (each

cell of which makes antibodies against only one antigen), insert this DNA into

bacteria such as Escherichia coli and then allow filamentous phages to infect

the bacteria. As the phages produce new copies of themselves, they automatically

make the proteins encoded by the antibody genes of the various B lymphocytes and

add them to the surfaces of newly forming phage particles. Scientists can then

use the antigen they intend to target, such as a receptor on cancer cells, to

fish out the phages containing the gene for the most specific antibody to that

antigen. To produce a lot of that antibody, they can either have one phage infect

more bacteria or insert the antibody gene into cultured cells.



Zeroing In on the Targets

Together the newer forms of monoclonals--chimeric, humanized and human--are

looking good against an array of diseases. Two such drugs, if they pass muster

with the FDA this year as expected, will finally fulfill the dream of deploying

so-called conjugated monoclonals--ones that carry radioactive chemicals or

toxins directly to tumors as a new form of cancer therapy. Zevalin (developed

by San Diego-based IDEC Pharmaceuticals and Schering AG) and Bexxar (devised by

Corixa in Seattle and GlaxoSmithKline) both target an antigen called CD20 on the

surfaces of B lymphocytes, cells that grow uncontrollably in the cancer known

as non-Hodgkin's lymphoma. And both pack a hot punch: Zevalin totes an isotope

of yttrium (90Y), and Bexxar carries a radioactive form of iodine (131I).

Many other monoclonals now in clinical trials also target molecules on immune

cells that play a role in a variety of diseases. For example, Genentech is in

the late stages of testing Xanelim, its monoclonal against CD11a. This protein

exists on the surfaces of T lymphocytes and helps them to infiltrate the skin

and cause the inflammation of psoriasis, which afflicts an estimated seven million

people in the U.S. In a study of nearly 600 psoriasis sufferers that was reported

at the American Academy of Dermatology conference in July, researchers found that

57 percent of patients on the highest dose of the drug experienced at least a

50 percent decrease in the severity of their disease. Several companies are also

pursuing monoclonals against CD18, a protein on T lymphocytes that underlies

inflammation as well as the tissue damage resulting from a heart attack.

A molecule called the epidermal growth factor (EGF) receptor is an additional

tempting target for monoclonal developers. One third of patients with solid tumors

make excess EGF receptors; indeed, the much heralded small-molecule drug Gleevec,

sold by Novartis, interferes with the ability of cancer cells to receive growth

signals from those receptors. Anti-EGF receptor monoclonals might best be

administered in combination with traditional chemotherapies. At the American

Society of Clinical Oncology conference in May, researchers reported that

cetuximab, an anti-EGF receptor antibody produced by ImClone Systems in New York

City, helped chemotherapy to start working again in 23 percent of patients with

advanced colorectal cancer who had stopped responding to chemotherapy alone.

Other companies are focusing on making monoclonal antibodies to molecules on the

surfaces of the cells that line the blood vessels. Certain types of these

molecules, such as avb3, play a role in angiogenesis, the growth of new blood

vessels, which is a crucial step in the development of tumors. A hugely successful

monoclonal antibody drug now on the market, Remicade, targets tumor necrosis

factor (TNF), a molecule the body uses to juice the cellular arm of the immune

system but that also plays a role in inflammatory diseases. According to company

reports, Remicade, which is on pharmacy shelves for Crohn's disease (an

inflammatory bowel disease) and rheumatoid arthritis, made $370 million last year

for its developer, Centocor. Therapies that wipe out TNF have a potential

$2-billion annual market, according to Carol Werther, managing director of equity

research at the investment bank Adams, Harkness and Hill. (Enbrel, an anti-TNF

drug developed by Immunex in Seattle that was approved in 2000 for patients with

moderate to severe rheumatoid arthritis, is not technically a monoclonal

antibody, because only part of an antibody--the backbone--is used; that backbone

is linked to a piece of another kind of molecule, the normal cellular receptor

for TNF.)

Emerging Issues

With all these good opportunities facing them, biotechnology and pharmaceutical

companies might be expected to be ramping up their production lines in

anticipation of a big market surge. But worldwide just 10 large-scale antibody

plants are now operating.

Part of the problem is financial: banks don't want to lend the hundreds of millions

of dollars it takes to build a state-of-the-art monoclonal production facility

unless the likelihood that the plant will generate profits is all but guaranteed.

Many of them look back on the 1980s, when drug manufacturers built facilities

that operated for years at only partial capacity.

The gold standard for producing monoclonals from hybridomas relies on enormous

tanks called bioreactors. V. Bryan Lawlis, chairman of Diosynth ATP in Cary, N.C.,

estimates that one giant, 60,000-liter bioreactor plant would be able to

(hypothetically) accommodate only four products. Assuming that 100 monoclonals

will be on the market by 2010, as analysts predict, Lawlis calculates that the

industry will need to build at least 25 new facilities or "we can't satisfy all

the needs." Those production plants would require $5 billion or more and between

three and five years to be built and certified by the FDA--a prospect no one thinks

is going to happen.

To fill the void, some companies are turning to transgenic animals and plants,

those organisms engineered to carry genes for selected antibodies. Transgenic

mammals that secrete monoclonals in their milk can generate one gram of antibody

for roughly $100--one third the cost of traditional production methods, industry

officials say. Centocor and Johnson & Johnson are looking into producing Remicade

using transgenic goats, and Infigen in DeForest, Wis., intends to make monoclonals

in cow's milk, although no such products have yet received FDA approval. Moreover,

it isn't clear how many companies will be willing to turn to these transgenic

options over the standard bioreactors.

Newman concedes that transgenic animals are attractive alternatives, but he adds

that companies must still undergo the sometimes tedious step of isolating the

monoclonals from the milk proteins. "You have purification problems, but you don't

have the expense of 10,000-liter bioreactors," he says. Genetically engineering

and breeding the animals can also take years.

Mich B. Hein, president of Epicyte in San Diego, sees green plants as the answer

to the monoclonal production shortfall. "It's pretty clear that the production

facilities will not meet the demand for even the most promising molecules," he

says. Plants have the advantages of being economical and easily scalable to any

level of demand: they can yield metric tons of monoclonal products. But

purification problems remain, and it is still unclear how the FDA will regulate

pharmaceuticals produced by transgenic plants.

Epicyte has teamed up with Dow to produce corn plants able to generate monoclonal

antibodies that will be formulated as creams or ointments for mucosal surfaces

such as the lips and genitalia or as orally administered drugs for

gastrointestinal or respiratory infections. Hein predicts that by the end of next

year Epicyte will seek FDA clearance to begin clinical trials of corn-produced

monoclonals to prevent the transmission of herpes simplex between adults and

during childbirth. The company is also developing monoclonals that bind to sperm

as possible contraceptives, as well as antibodies that could protect against human

papillomavirus, which causes genital warts and cervical cancer.

Whether they come from cattle, goats, corn or bioreactors, monoclonal antibodies

are set to become a major part of 21st-century medicine.



Further Information:

Monoclonal Antibodies: A 25-Year Roller Coaster Ride. N. S. Halim in The

Scientist, Vol. 14, No. 4, page 16; February 21, 2000.

A Human Myeloma Cell Line Suitable for the Generation of Human Monoclonal

Antibodies. A. Karpas, A. Dremucheva and B. H. Czepulkowski in Proceedings of

the National Academy of Sciences USA, Vol. 98, No. 4, pages 1799-1804; February

13, 2001.

Biotech Industry Faces New Bottleneck. K. Garber in Nature Biotechnology, Vol.

19, No. 3, pages 184-185; March 2001.

Abstracts of scientific presentations at the 2001 annual meeting of the American

Society of Clinical Oncology are available at virtualmeeting.asco.org/vm2001/



The Author

Carol Ezzell is a member of the board of editors.