Monoclonal Antibodies That Target Cancer 11 Magic Bullets, at Last 12 Acknowledgments Magic Bullets and Monoclonals: an Antibody Tale

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Developed by the Federation of American Societies for Experimental Biology (FASEB) to educate the general public about the benefits of fundamental biomedical research. INSIDEthis issue Magic Bullets and Monoclonals: An Antibody Tale

Blood, a natural disinfectant 2 Like Everest to hill climbers 3 B-cell brainteaser: Explaining the antibody response 5 Mystery of diversity 7 Failure that leads to success 8 Monoclonal antibodies that target cancer 11 Magic bullets, at last 12 Acknowledgments Magic Bullets and Monoclonals: An Antibody Tale

Author, Margie Patlak

Scientific Advisor, Dan L. Longo, M.D., National Institute on Aging, National Institutes of Health

Scientific Reviewer, James Allison, Ph.D., Sloan-Kettering Institutes

BREAKTHROUGHS IN BIOSCIENCE COMMITTEE James E. Barrett, Ph.D., Chair, Drexel University College of Medicine

David Brautigan, Ph.D., University of Virginia, School of Medicine

Rao L. Divi, Ph.D., National Cancer Institute, National Institutes of Health

Marnie Halpern, Ph.D., Carnegie Institution of Washington

Tony T. Hugli, Ph.D., Torrey Pines Institute for Molecular Studies

Mary Lou King, Ph.D., University of Miami School of Medicine : Nearly a century of basic discovery in immunol- Fred R. Naider, Ph.D., College of Staten Island, CUNY ogy, cell biology, and cancer biology contributed to today’s remarkable monoclonal antibody therapies, which are pro- Loraine Oman-Ganes, M.D., FRCP(C), CCMG, FACMG, duced through human-mouse cell hybrids. Scientists span- RBC Insurance, Toronto, Canada ning decades pieced together clues about the fundamental Robert N. Taylor, M.D., Emory University School of nature of antibodies, a critical part of our immune response, Medicine in order to develop today’s monoclonal antibody drugs, used to treat cancer, auto-immune disorders, and blood clotting. Credit: Science Photo Library and iStock Photo. BREAKTHROUGHS IN BIOSCIENCE PRODUCTION STAFF

Science Policy Committee Chair: Avrum Gotlieb, M.D., CM, FRCPC, University of Toronto

Managing Editor: Carrie D. Wolinetz, Ph.D., Director of Scientific Affairs and Public Relations, FASEB Office of Public Affairs

Production Staff: Lawrence Green, Communications Specialist Magic Bullets and Monoclonals: MoAn Antibodyn Taleoclonal

In 1890, the German scientist debilitating cases of rheumatoid AEmil Behringnitib stunned his col- arthritis oand inflammatorydies bowel leagues by showing that he could disease. Hundreds more of these protect a guinea pig from the magic bullet drugs are being ravages of diphtheria by merely tested in people with the hope giving it a shot of blood (Figure of providing innovative targeted 1). The blood had been purged of treatments for an even wider its cells, and was gathered from range of disorders. a guinea pig that had recently recovered from the disease. Behring’s fellow scientists at the Institute for Infectious Diseases in Berlin were intrigued by this mysterious invisible substance in the blood that protected the guinea pig from diphtheria, but not from tetanus. One colleague, Paul Ehrlich, postulated that there must be chemicals in the blood that could seek out and destroy specific toxins like “magic bullets.” This led to the suggestion that by harnessing these magic bullets, researchers could develop cures for all kinds of disorders. It took a century for these magic bullets, in the form of monoclonal antibodies, to be developed as effective weapons in combating But the path to the development Figure 1 – Emil von Behring a number of diseases. There are of monoclonal antibody drugs (1854-1917): In 1890, now more than 20 monoclonal was not as straight as you would Behring helped to show that immunity from diphtheria antibody-based drugs on the think, and instead was a global could be achieved by an injec- market, including several block- adventure that depended on the tion of blood serum from an busters, that are used by millions unexpected findings of numerous infected animal. In 1901, he of people worldwide. These curious scientists. These scien- received the first Nobel Prize awarded in physiology or medicines can effectively treat tific explorers pursued such basic medicine for his work. disorders that don’t respond to questions as what “disinfectants” Credit: Science Source / other treatments, such as several lie in the blood?; how do anti- Science Photo Library. common deadly cancers, and bodies work?; how can the body

Breakthroughs in Bioscience 1 create 10 million different antibodies uniquely suited to what- ever foreign invaders it encounters?; and what causes antibodies to improve their ability to latch onto compounds over time? By seeking answers to these questions, researchers from multiple disciplines collectively assembled the basic building blocks that led to the development of the monoclonal antibodies that now underlie many diagnostic tests and important new drugs. Figure 2 – Importance of animal models: Animals such as guinea Blood, a natural pigs, rabbits, horses, disinfectant and mice were critical Behring’s amazing discovery to the basic immu- nological discoveries of the curative powers of blood that led up to today’s led to the first effective treat- monoclonal antibody ments for diphtheria and tetanus, therapies. Credit: and his being awarded, along U.S. Department of Agriculture, with Shibasaburo Kitasato, the Agricultural Research first Nobel Prize in Physiology Service. or Medicine in 1901. But his research had humble origins—the synthetic chemicals he was test- This led Behring to conclude quest to find the perfect disinfec- ing, Behring ran tests in various in his Nobel address “the inter- tant. A military man, Behring’s animals to see if the addition nal disinfection that has been main objective was to find a way of a clear, cell-free component achieved is thanks to the fact that to prevent the scourge of deaths of blood (serum) would affect Nature herself has been taken as a guide…It is one of the most stemming from infections of war the course of a disease, such as injuries. He began his research wonderful things imaginable to diphtheria (Figure 2). But this career exploring in animals see how the supply of poison research had some frustrating whether iodine-like compounds becomes the prerequisite for the moments—although some sera added to wound dressings or appearance of the antidote in the injected could help prevent or was remarkably effective at poisoned living organism.” control infections by blocking the preventing or relieving animals of diphtheria, other sera had no Behring called the natural anti- actions of bacteria. dote “anti-body” and left it up effect. The blood from normal Behring’s research took a revo- to other researchers to answer animals not infected with diph- lutionary turn, however, when the intriguing puzzle of how theria, for example, had no effect, he switched from exploring exactly antibodies work, and of nor did the blood from an animal the power of various chemicals what they are comprised. But one to disinfect to exploring the infected with tetanus. Only an man’s frustration—the specificity power of the blood to disable animal that survived infection of the disinfective properties of microbes. Curious to see if blood with diphtheria or its toxin the blood—proved to be a source might have some of the same could produce serum with of inspiration to others work- anti-bacterial properties as the any anti-diphtheria benefit. ing in this field. It led Ehrlich to

Breakthroughs in Bioscience 2 imagine that the blood must con- antibodies able to latch onto a about antibodies to overcome tain a myriad of antibodies capa- specific microbe could be found these challenges, with the help ble of fitting around and blocking in more than one class of anti- of government funding for their the action of foreign substances bodies grouped by size, shape, or research. Both researchers tack- (antigens) the body encounters. charge. The large size of antibod- led the problem by breaking up He likened the structural affin- ies also made them difficult to antibody molecules into smaller ity an antibody has to antigen to decipher—they were at least 20 components. By comparing what that of a key in a lock. Ehrlich’s times the size of those protein they discovered about those com- contemporary Karl Landsteiner, molecules whose structures had ponents, the investigators were at the Pasteur Institute in Paris, been solved. As Gerald Edelman able to formulate the overall tried to assess the chemical speci- put it, the size of antibody mole- shape and detailed structure of ficity of antibodies. He found that cules was “large enough to strike antibodies. slight alterations in the structure terror in the hearts of the struc- Rodney Porter (Figure 3) of of an antigen changed its three- turally inclined protein chemist… the British National Institute for dimensional shape enough so that Medical Research, who later it no longer bound strongly to the worked at St. Mary’s Hospital same antibody. Medical School in London, found it astonishing, as he put it, that Like Everest to hill the pool of antibodies found in climbers the blood had an infinite ability Many researchers eagerly to combine with any substance, embarked on a quest to discover yet superficially seemed to be the exact structure of antibod- a nearly uniform group of pro- ies and how they formed, so as teins. This confounded him and to figure out how they worked bolstered his determination to so quickly and precisely to fight decipher antibody structure and against invaders. But this quest function. In the late 1950’s, he was a long and difficult jour- took an innovative approach to ney because blood contained the problem. Instead of grap- a confusing jumble of surpris- pling with separating antibodies ingly similar antibodies. Even an according to what they were able animal not actively fighting an to attack, so as to separate and infection will have one percent of accumulate enough of a specific its blood comprised of thousands Figure 3 – Rodney P. Porter (1917- 1985): Porter, a British immunologist, antibody to study, Porter instead of antibodies, which researchers was awarded a Nobel prize in 1972 for assessed the general structural could not distinguish with the his discovery of the chemical structure components of a group of differ- technical tools at hand. of antibodies. Image from the National ent rabbit antibodies. Library of Medicine. Those tools improved with the Porter used an enzyme from advent of spinning devices called It seemed like Everest to local papaya to break apart these ultracentrifuges in the 1930’s, hill climbers. As if that were not antibodies into three major sec- which were modeled after dairy daunting enough, the shape of tions that he separated for fur- cream separators. These devices the mountain—its specificity— ther study. This work, combined separated groups of antibod- seemed almost beyond grasp.” with later work by Edelman of ies by size and shape. Filtering the Rockefeller Institute in New It took Edelman and another techniques developed in the York, revealed that the basic unit dedicated biochemist intrigued 1940’s separated antibodies by of antibodies is shaped like a Y. by the little bit that was known electric charge and/or size. But Despite the mixture of many anti-

Breakthroughs in Bioscience 3 bodies used in his studies, Porter amino acid building blocks of the antibody was virtually identi- found that the stem of the Y was protein chains that make up the cal among individuals, but the remarkably consistent in the antibody so as to determine their V section varied from patient to composition of its protein chains, order in the chains. patient. Later, studies revealed whereas the V portion varied By 1969, Edelman and his col- that it is indeed the V portion of widely. The diverse nature of the leagues had deciphered the entire the antibody that latches on to an V portion led Porter to suggest chemical structure of an antibody antigen. that it is the section of the anti- (Figure 4), the largest molecule But one of the “most satisfying body that locks on to the antigen. to be biochemically unraveled to conclusions” that emerged from Meanwhile, across the Atlantic, that point. His analyses revealed his structural analysis of anti- Edelman’s medical background that two linked protein chains bodies, according to Edelman, made him aware that certain comprised each half of the Y of was that it explained how the cancers of the immune system an antibody divided lengthwise. body could make the millions produce an overabundance of a Edelman’s comparisons of the of antibodies needed to latch on single specific antibody that can chemical structures of the differ- to all the foreign compounds it be easily isolated from the blood or, in incomplete form, from the Antigen urine of patients with these can- Binding Site cers. Such malignancies, known Antigen as myelomas, are tumors of the Variable Binding Site cells that make antibodies. (Only one specific antibody is overpro- Constant duced in a patient because this is the antibody made by the original cell which became cancerous.) Different patients overproduced different antibodies and became useful sources of bulk quantities Light Chain Light Chain of unique antibody proteins. Edelman realized that the large quantities of a specific type of antibody produced by these Heavy Chain Heavy Chain tumors would enable him to do the biochemical studies needed to figure out antibody structure. He used various chemical scalpels recently developed by Swedish Figure 4 – Structure of an antibody: This Y-shaped protein is produced by biochemist Pehr Edman to cut B-lymphocyte white blood cells as part of the immune response. Each antibody con- sists of four polypeptides (protein pieces), two smaller “light” chains and two larger apart the tightly knit antibody “heavy chains.” The amino acid sequence in the tips of the Y (variable region) varies molecule into more components widely among different antibodies and is what confers the antigen specificity to each than Porter’s papaya enzyme antibody. Figure designed by Corporate Press. had done. He then used a new labeling technique, developed by ent antibodies produced by dif- might encounter during a life- his chemist colleagues Stanford ferent patients with myeloma also time. Different combinations of Moore and William Stein at revealed that, as Porter found, the protein building blocks called Rockefeller Institute, to mark the the structure of the base of the amino acids, which make up the

Breakthroughs in Bioscience 4 portions of the V section of an shaped crevasse within the V During this rapid duplication antibody that bind an antigen, region of the antibody jutting process, the cells make subtle could generate the needed anti- from its surface—the binding of genetic mistakes that affect the body diversity. (These minute the antigen to the B cell’s anti- structure of the antibodies they differences in the combination of body triggers the B cell to create amino acids were not significant clones (multiply) and rapidly produce. Some of these changes enough to discernibly affect the secrete an arsenal of its antibod- enable the antibody to bind more size or shape of antibodies, which ies (Figure 5). tightly to the antigen. This tighter is why at first glance, they all appeared so similar.) Both Porter and Edelman were awarded the Nobel Prize in Physiology or Medicine in 1972 for their pioneering work on antibody structure. Antigen

B-cell brainteaser: Explaining the antibody response Still left unexplained was how the body is able to quickly produce an abundance of the specific antibody needed to disable a specific antigen, or as Behring put it, provide an antidote to the poison almost immediately after it enters the body. But this was eloquently explained by a theory formulated Clonal by Danish immunologist Niels Selection Jerne and improved by Australian virologist Sir Frank Burnet. They applied Charles Darwin’s ideas about natural selection of animal Cell species in the environment. Multiplication In their “natural selection” theory, Jerne and Burnet suggested Antibody that each antibody-producing Production white blood cell, called a B cell, can only make one specific antibody. Each of these B cells coats itself with the unique antibody it produces, and then patrols the blood or other body tissues. Figure 5 – Clonal selection: Each B cell has antibody receptors corresponding to When a B cell encounters its a single type of antigen. When the B cells encounters and binds to the antigen it recognizes (clonal selection), it triggers the B cell to multiply, creating many clones matching antigen—the antigen of itself. This new army of clones rapidly releases an arsenal of specific antibodies. that fits best into the uniquely Figure designed by Corporate Press.

Breakthroughs in Bioscience 5 binding leads to greater expan- sion of the clones of cells producing this antibody. A repetition of this scenario over many suc- cessive cell divisions ultimately causes an explosion in the number of cells producing the highest antigen-binding ability (antibody affinity). This could explain why antibodies to a specific toxin or microbe dramatically increase in the blood after the foreign invad- Start Stop er enters the body, and within a short period of time evolve A T G A C G G A T C AG C C G C A A G C G G A A T T G G C G A C A T A A T A C T G C C T A G T C G G C G T T C G C C T T A A C C G C T G T A T T such that they can latch on more strongly to antigens, thereby trig- gering the foreigner’s destruc- TTT Phenylalanine TCT TAT Tyrosine TGT Cysteine tion by other components of the TTC TCC TAC TGC Serine TTA TCA TAA TGA Terminator Threonine immune system. This process is Leucine Terminator TTG TCG TAG TGG Tryptophan Aspartic acid called antibody affinity matura- CTT CCT CAT CGT Glutenine Histidine tion. CTC CCC CAC CGC Leucine Proline Arginine Proline CTA CCA CAA CGA Glutamine Support for the natural selection CTG CCG CAG CGG ATT ACT AAT AGT theory first came in 1958, when Isoleucine Asparagine Serine ATC ACC AAC AGC Threonine Nobel Prize-winning molecular ATA ACA AAA AGA Methionine Lysine Arginine Peptide chains (Initiator) biologist Joshua Lederberg and ATG ACG AAG AGG made up of amino his colleague Gus Nossal showed GT T GCT GAT GGT acids combine Aspartic GTC GCC GAC GGC to make proteins. Valine Alanine Glycine in rabbits that each B cell can GTA GCA GAA GGA Glutamic produce only one specific anti- GTG GCG GAG GGG body. More evidence for the now well-accepted natural selection Three “letter” combinations encoded in DNA correspond theory has accumulated over to different amino acids, the building blocks of protein. the decades. Both Burnet and Jerne won Nobel Prizes for their Figure 6 – Protein is made of sequences of amino acids encoded in DNA: Three letter creative vision of how antibody sequences of nucleotides or bases (the building blocks of DNA) correspond to particu- production is generated and lar amino acids (the building blocks of protein). This is how DNA serves as a genetic improved over time. blueprint for the building of proteins by cellular machinery. See Breakthroughs in Bioscience article “Genetic Research: Mining for Medical Treasures” for more detail on But as often is the case in basic how DNA is translated into protein. Figure designed by Corporate Press. scientific research, when one that the body encounters. And have on hand to defend itself puzzle is solved, another one biologists had offered a plausible from potential intruders? arises. The chemical explorations explanation for how antibodies As the biochemists had shown, undertaken in the first half of are made in response to a bodily the 20th century explained anti- intrusion by an antigen. But an antibody is composed of body structure and even pointed both sets of advances created a several protein chains each com- to the regions of the antibody new puzzle: how could the body prised of amino acids. Genetic responsible for its ability to bind genetically code for the millions studies done in bacteria at that to the many different compounds or more antibodies it needed to time suggested that all proteins

Breakthroughs in Bioscience 6 are made by cellular machinery with modern genetics thought one of hundreds to thousands using a continuous section of this was unlikely, as the known of possible genes coding for the DNA as a pattern or blueprint. number of genes were estimated variable region to form a specific This pattern specifies the order to be far fewer than the num- antibody. But this radical theory, of the amino acids in a cor- ber of antibodies a person was which called for split genes, was responding protein by using an thought able to produce. Another scoffed at by many in the scien- instruction code made from DNA theory was that the genes in B tific community who held to the compounds called bases, which cells changed (mutated) during dogma of one contiguous gene are abbreviated A,T, C or G. The development to form the many equals one protein, apparently not DNA sequence TAC, for exam- antibodies. But if this were the considering that what happens in ple, codes for the amino acid case, why would the portion of bacteria may not also happen in tyrosine, while AAC codes for an antibody gene that coded for mammals. leucine (Figure 6). Just as a string the constant region in the stem of At this point, a Japanese molec- of words spell out a complete the Y stay virtually the same in ular biologist Susumu Tonegawa sentence, it was assumed that a all antibodies, while the part of entered the fray (Figure 7). He contiguous sequence of DNA the same gene that coded for the also was skeptical of the Dreyer- base triplets spelled out a com- variable portions in the ends of Bennett theory, but realized that plete protein. It was also assumed the V mutate wildly? the power of the new genetic that every gene produced only a An out-of-the-box theory was tools that enabled researchers to single protein. needed to explain this conun- sequence genes (spell out their But studies then suggested drum: in the 1960’s, William sequence of A,C,T, and G bases) humans only had about 100,000 Dreyer and Claude Bennett and pinpoint their location on genes in their DNA. (More recent of the California Institute of a DNA strand could shed some estimates are closer to 30,000.) Technology put forth their two- light on this heated debate. Like How could 100,000 genes create gene, one antibody hypothesis. Edelman, Tonegawa’s work was millions of antibodies, in addi- They suggested that one gene for aided by the use of single-spec- tion to providing all the proteins the constant region of the anti- ificity antibodies generated by needed to build a body and body is somehow combined with myeloma tumors. These tumors make it function properly? This paradox was said to be one of the most vexing problems in biology at the time, and it was while trying to solve this new puzzle, that monoclonal antibodies serendipi- tously arrived on the scene.

Mystery of diversity Two competing theories were formulated to explain this paradox in immunology. One theory was that within each sperm and egg, and all the resulting cells of an embryo and then the adult, was an enormous number of Figure 7 – Susumu Tonegawa: Susumu Tonegawa, Director of the Picower Center for genes needed to make millions of Learning and Memory at M.I.T., won the 1987 Nobel Prize for his discovery of the antibodies. Most experts familiar genetic origin of antibody diversity. Credit: Allen Green / Science Photo Library.

Breakthroughs in Bioscience 7 came from Michael Potter of the With this set up, there are tre- Argentinean biochemist Cesar National Cancer Institute, who mendous avenues for antibody Milstein of the publically sup- had an array of experimentally diversity: ported Medical Research Council induced mouse myelomas that he (MRC) in London (Figure 9). • There are several to hundreds used to study the genetic triggers It was while trying to provide of DNA sequence options for of these cancers. proof for this theory, that he and each section of coding DNA that German immunologist Georges In 1976, while working at the is spliced together, which multi- Kohler created compounds that Basel Institute for Immunology plies the possible combinations would revolutionize medicine. (BII) in Switzerland, Tonegawa of amino acids determining anti- was pleasantly surprised to dis- body structure considerably; cover that, in mice, the portion Failure that leads to • Where these sections are of the DNA that encodes the success joined can also vary enough to constant-region stem of Y-shaped Milstein wanted to analyze affect the structure of the result- antibodies is indeed separated the rate and nature of mutations ing antibody; from the DNA that encodes the of antibody-producing cells to variable upper V section, just • The B cell’s genetic machin- see how the mutations affected like Dreyer and Bennett pro- ery makes mistakes in the splic- an antibody’s ability to bind posed. This meant one continu- ing process, especially in the to antigen. For his studies, he ous stretch of DNA could not joining regions. These mistakes used Potter’s mouse myeloma produce an antibody. He also can also affect the structure of tumors to make cell cultures he showed that these two sections of the antibody that forms from the fondly tended like they were his DNA were widely separated on a pattern; and children, one fellow researcher chromosome in mouse embryos noted. But his experiments led to • The main chains that comprise but arranged close together in a frustrating dead end. Contrary an antibody are made separately mouse antibody-producing tumor to Milstein’s expectations, using from different patterns, making cells, suggesting that some genet- these cultures he wasn’t able to even more combinations. ic shuffling had occurred. And in show high mutation rates in the between the constant and variable The end result is that millions variable regions of the antibod- coding sections was a new unex- of combinations are possible, ies these cells produced using pected section, which he named J with each combination these culture. This failure mainly for joining section. generating its own unique stemmed from the fact that his antibody (Figure 8). As Potter put myeloma cell cultures, which had Subsequent studies by it, “Mother Nature pulled out all the longevity needed for labora- Tonegawa at the BII and later the stops” to generate antibody tory studies, produced abnor- at the Massachusetts Institute diversity. At last researchers had mal antibodies that were rather of Technology, as well as those hit upon an explanation for how wimpy when it came to binding of Leroy Hood of California millions of antibodies could form with antigen. Institute of Technology and colleagues, revealed that during the from a small kit of genetic com- Meanwhile, in Switzerland, development of a B cell, several ponents. Tonegawa received the Kohler was having the oppo- different sections of DNA--what Nobel Prize for Physiology or site problem, while also trying one researcher describes as a “kit Medicine in 1987 for his elegant to validate the important role of components”--are stitched work. mutation played in antibody together to form the pattern for A major advocate for the notion structure. Kohler was working the assembly of a specific anti- that genetic changes in antibody with cultured B cells, which had body, with intervening DNA cells enable both antibody diver- great antigen-binding capac- sequences lost in the process. sity and affinity maturation was ity but quickly died out, foiling

Breakthroughs in Bioscience 8 Figure 8 – Avenues for antibody diversity: This figure illus- trates four different ways in which diversity of antibodies occurs, allowing the body to react specifically to innumer- able antigens. Designed by Michael Linkinhoker, Link Studio, LLC.

Breakthroughs in Bioscience 9 ognized, along with Kohler, that the specificity and large production capacity of monoclonal antibodies would make them useful probes. They could be combined with a fluorescent dye or other signaling molecules to detect specific molecules of interest, such as microbial or cancer cell antigens, or hormones. Hundreds of compounds in the body that had previously eluded researchers’ attempts to find and measure them suddenly became detectable and quantifiable. Scientists also discovered many new compounds by making monoclonal antibodies Figure 9 – Cesar Milstein (1927-2002): While studying mechanisms for how antibody to them. diversity is generated, Argentinean scientist Milstein pioneered the fusion technique for producing hybridomas and monoclonal antibodies. For his work, he won a 1984 Nobel In collaboration with a num- Prize. Credit: Dr. Rob Stepney / Science Photo Library. ber of other scientists, Milstein showed the usefulness of mono- his experiments. After hearing those fused cells producing the clonal antibodies in distinguish- Milstein give a talk about his antibody to the injected antigen. ing blood and tissue types needed research, Kohler came to work Much to their surprise and plea- for organ transplants and blood in his lab. The two realized that sure, after years of failed experi- transfusions. The researchers by combining the longevity of ments, the cell fusion worked! also used the antibodies to better myeloma cells with the antigen- With this technique they could diagnose leukemia, more quickly binding capacity of antibodies abundantly produce pure (mono- diagnose pregnancy, which led made by normal B cells, they clonal) antibodies that only to home pregnancy testing kits, could have more fruitful explo- bound to the specific antigen, and to purify and concentrate rations of how B cells generate with which they injected the ani- the infection-fighting compound antibodies that tightly bind to a mals (Figure 10). interferon, which is found in wide variety of antigens. With his monoclonal anti- extremely minute concentrations At the time, a technique for fus- bodies in hand, Milstein put a in the body. ing cells had come onto the sci- research student to work using Milstein also realized that entific scene. The two research- them to detect mutation rates in monoclonal antibodies have the ers used this technique to fuse antibody producing B cells. But capacity to uncover previously antibody-producing B cells from those experiments failed and as unknown cellular components, mouse spleen to mouse myeloma Milstein was fond of saying, “If and by putting them to use in this cells. The mice were previously we didn’t get what we wanted, way they tremendously acceler- injected with a specific antigen, we had to learn to love what we ated the pace of basic research so they would produce large got.” He put his antibody diver- in immunology and other fields amounts of antibodies directed sity experiments aside to explore (much more so than Milstein’s against the antigen. A special the usefulness of monoclonal previous experiments did!) For culture medium developed by antibodies in other areas of basic example, different types of Jerne was then used to select research, and medicine. He rec- white blood cells have different

Breakthroughs in Bioscience 10 when a young oncologist hop- Immunization of mouse to stimulate antibody Tumor cells are ing to cure non-Hodgkin’s lym- production grown in tissue phoma teamed up with an unlikely Antibody-forming culture cells isolated partner—a former classmate who from spleen was trying to make a blockbuster mouthwash to prevent tooth decay.

Monoclonal antibodies that target cancer Antibody-forming cells are fused with cultivated tumor cells to form hybridomas Lee Nadler was working on a clinical fellowship at the Sidney Farber Cancer Institute, (which later became the Dana Farber Cancer Center), when he heard of Milstein and Kohler’s monoclonal antibodies and thought they would be the perfect tool to discover the B cell types that give Hybridomas screened for rise to various immune system antibody production Antibody-producing hybridomas cloned cancers called non-Hodgkin’s Hybridomas screened for antibody production lymphoma. At this point, basic research had revealed that most Figure 10 – Monoclonal antibody production: To produce monoclonal antibodies, first a mouse is immunized (injected) with the antigen of interest to begin production of of these cancers get their start antibodies against that antigen. The antibody producing cells are then isolated from when genetic flaws crop up dur- the mouse’s spleen. Meanwhile, tumor cells known as myelomas are grown in culture ing the duplication of B cells. and then fused with the isolated spleen cells to form a new type of cell called a hybri- These flaws stop the cell’s devel- doma. Hybridoma cells are then grown in culture and then tested individually to see if they are producing antibodies against the antigen of interest. These hybridomas are opment so it never matures and then cloned and cultured, becoming miniature factories for pure, monoclonal antibod- keeps dividing, wreaking havoc ies, which can be isolated. Figure designed by Corporate Press. in the body due to its uncon- trolled growth. antigens on the surface of their cells and linking them to a detect- cell membranes, and these tell- able marker, researchers used the Nadler thought that he could use tale markers change as the cells resulting monoclonal antibod- monoclonal antibodies as basic mature and carry out different ies to probe cells for previously science tools to better know his key functions. But pinpointing undiscovered surface antigens. enemy—to define in what stage these cell membrane proteins had This use of Milstein and Kohler’s of development lymphoma tumor been challenging, in part because monoclonal antibodies set off an cells were in. Armed with this they are present in cells in minis- exciting explosion of research information, he would then be cule amounts. that led to a better understanding able to distinguish the tumors for better diagnosis and treatment. By When mice are injected with of the different white blood cell types in the immune system and knowing what cell markers these whole cells, however, their B tumor cells had, he reasoned he how they functioned. cells produce antibodies to all could also develop a monoclonal cellular components, no matter One practical outcome of this antibody therapy that only tar- how rare, including the distinc- field was the development of the geted the tumor cells, while spar- tive cell membrane antigens. By first monoclonal antibody drug ing most other white blood cells fusing these B cells to myeloma for cancer. This drug got its start needed to fight infections.

Breakthroughs in Bioscience 11 At the time, Nadler’s friend Phil that the researchers were foiled monoclonal antibody didn’t seem Stashenko, who had a Ph.D. in by the same immune system they to trigger an important immune immunology as well as a dental hoped to redirect to the tumor! defense, called the complement degree, was unsuccessfully trying The experimental drug was made system, which normally would to make monoclonal antibodies completely from mouse cells that have led to the destruction of against the bacteria that cause were perceived as foreigners wor- antibody-targeted tumor cells. thy of an antibody attack. This tooth decay with the aim of creat- This finding led Darrell ing a mouthwash that could pre- Anderson and his colleagues vent cavities. Nadler convinced at IDEC Pharmaceuticals Stashenko, then at the Harvard Corporation in San Diego to use Dental School, to drop his project recombinant DNA techniques to and help him make monoclonal develop a monoclonal antibody antibodies that could uncover that combines a mouse (antigen- new cell markers for B cells. binding) V portion of the anti- The two researchers made body with a human constant stem remarkable progress in a short portion. That way the antibody is period of time. Within a few able to activate the human com- years after Milstein and Kohler plement system directed at the first reported they had concocted tumor cells, yet still bind to the monoclonal antibodies, Nadler appropriate antigen. They called and Stashenko and colleagues their part mouse and part human made monoclonal antibodies monoclonal antibody rituximab in their own lab, and then used (Rituxan) (Figure 11). them to discover a B cell antigen that was produced by B cells in Magic bullets, at last most non-Hodgkin’s lympho- Rituximab passed its clini- mas. (Eventually, all four known cal tests on patients with non- human B cell-specific antigens Hodgkin’s lymphoma with flying were discovered in Nadler’s labo- colors. Initial results showed the ratory.) Nadler then became the drug shrank the tumors in half of first in the world to test a mono- Figure 11 – Rituximab: Rituximab, patients with advanced disease clonal antibody on a patient, which is marketed under the names that no longer responded to stan- when in 1979 he gave a mono- Rituxan and MabThera, is a monoclonal dard chemotherapy. Even better antibody used to treat non-Hodgkin’s clonal antibody that targeted a B lymphoma and B cell leukemia. It is cur- results occurred when the drug cell antigen to a 54-year old man rently being studied for its use in treat- was combined with standard che- who had advanced lymphoma ing some autoimmune diseases, to help motherapy and used in younger unresponsive to other treatments. prevent the rejection of transplanted patients with less advanced dis- organs, and also to treat rheumatoid ease. In these patients, rituximab The treatment was a failure. arthritis. Credit: Dr. P Marazzi / Science Photo Library. boosted the rate of complete Although the patient did not remissions by more than 25 per- develop any severe reactions to attack by the patient’s own anti- cent. After 3 years, 79 percent of the treatment, it did not improve bodies blocked the monoclonal patients who received rituximab his condition. Blood and tissue antibody’s actions, and primed were alive and free of any signs samples revealed that there was it for destruction before reach- of the cancer, compared to 59 minimal binding of the monoclo- ing its tumor target. In addition, percent of the chemotherapy-only nal antibody to tumor tissue, and the weak binding of the mouse patients, one study found.

Breakthroughs in Bioscience 12 Rituximab targets B cells in a relieved by standard therapy. The include such widely used medi- stage of development other than drug has also shown promise in cines as trastuzumab (Herceptin) that of those actively secreting the treatment of psoriasis, type 1 for breast cancer, bevacizumab antibodies, and the drug doesn’t diabetes, multiple sclerosis, auto- (Avastin) for colorectal, breast, latch onto stem cells that give immune thrombocytopenia, and brain, and lung tumors, inflix- rise to infection-fighting cells. So is often used to prevent immune imab (Remicade) for rheumatoid the drug does not increase risk rejection after transplant. arthritis and inflammatory bowel of infection as much as standard Rather than relying on mono- disease, and abciximab (Reopro), chemotherapy. The agent does, clonal antibodies to trigger the which is used to prevent blood however, kill normal B cells as immune system to destroy tumor clots. Monoclonal antibodies well as tumor cells, a feature that cells, some researchers took a are also showing promise for has led to its application to treat more aggressive approach. These shepherding various treatments certain autoimmune diseases (see investigators linked monoclonal into the brain, which currently is below). It also has fewer and less antibodies that target the B cell impenetrable to many drugs. severe side effects than tradi- marker found in most lymphoma One expert estimated that it costs tional cancer drugs that are toxic cells to a radioactive chemical, only two million dollars to devel- to any dividing cells, including so that the monoclonal antibody op a monoclonal antibody for those that line the stomach and can mediate tumor destruction clinical testing, versus twenty mil- hair follicles, and bone marrow directly without the help of com- lion for that of a traditional drug. cells that produce the blood cells. plement by delivering its deadly Because monoclonal antibodies This causes nausea and hair loss radioactive baggage to malignant tend to be less toxic, they are also and decreased blood cell counts. tissues. The drug ibritumomab more likely than traditional drugs By contrast, the most common (Zevalin) is made in this man- to clear regulatory hurdles and side effects from rituximab are ner, and researchers have shown enter the market. However, like flu-like malaise, shortness of it shrinks tumors in more than nearly all drugs, monoclonal anti- breath and/or a drop in blood three-quarters of patients who body drugs do cause some side pressure. These can usually be receive it along with rituximab, effects in some people. But these relieved by reducing the infusion with some responses lasting drug reactions are rarely serious rate, and are almost always lim- more than five years. This drug enough that they limit the use of ited to the first course of therapy. came on the market in 2002. monoclonal antibody drugs. For Rituximab first entered the mar- The Pharmaceutical Research example, by disabling an immune ket in 1997 as a lymphoma treat- and Manufacturers of America defense that is overactive in some ment, and its success led to an reported, in 2006, that U.S. disorders, some monoclonal anti- explosion of therapeutic mono- companies had 160 different body drugs have caused some clonal antibodies that researchers monoclonal antibodies in testing serious infections in some people. developed and tested in the clinic or awaiting regulatory approval Heightened vigilance for infection (Table 1). Even rituximab was to enter the market. Drug com- can often prevent this complica- tested further to see what effects panies find they can bring new tion. Some side effects of mono- it might have on autoimmune drugs made with monoclonal clonal antibody drugs are thought disorders, which foster destruc- antibody technology to the mar- to be due to the body’s reaction to tion of normal tissues, often due ket much quicker than those their mouse components. Aimed to overactive B cells. The Food using traditional techniques, at preventing these reactions, and Drug Administration cur- thus explaining why many of the researchers continue to improve rently has approved the use of drugs that entered the market in monoclonal antibodies by making rituximab in the treatment of the last decade have been mono- nearly or completely humanized rheumatoid arthritis patients not clonal antibody drugs. These versions.

Breakthroughs in Bioscience 13 But it is not just monoclonal take it, with some people report- recurrence, when combined with antibody technology that is lead- ing symptom relief within just effective chemotherapy. ing to medical advances. Thanks hours of receiving the medicine. There has also been a produc- to government-funded research, Many of these patients no lon- tive play between monoclonal there have also been stun- ger respond to other rheumatoid antibody diagnostics and thera- ning advances in basic biology arthritis drugs. Infliximab has also peutics. Monoclonal antibody research that have uncovered the proven to be a highly effective probes have fostered a better key compounds these monoclonal treatment for inflammatory bowel molecular understanding of a antibody drugs target. For exam- disease, as well as for psoriasis, number of disorders that, in turn, ple, without understanding that a and is being tested as a treatment has led to the development of compound made by the immune for other autoimmune disorders. monoclonal antibodies to treat system called tumor necrosis fac- Similarly, basic research that the panoply of disorders from tor (TNF) is the kingpin of the uncovered that the growth of allergies and asthma to diabetes, debilitating inflammation seen in certain types of breast cancer are Alzheimer’s disease, and cardio- rheumatoid arthritis, there would fueled by a specific growth factor vascular disorders. And of course be no infliximab, which is com- led to the monoclonal antibody if it wasn’t for the basic research prised of monoclonal antibodies drug trastuzumab, which targets on antibodies that had been pur- that target TNF. Some physicians that growth factor’s receptor on sued by chemists, immunologists, view infliximab as a miracle drug tumor cells. In the quarter of the virologists and other investiga- for their patients’ rheumatoid women with breast cancer who tors around the world, monoclo- arthritis because, unlike other have heightened activity of this nal antibodies wouldn’t be here treatments, it provides significant receptor in their tumors, trastu- today. Cesar Milstein humbly relief of symptoms in more than zumab has proven remarkably sums up what led to these magic three-quarters of patients who effective at preventing disease bullets in his Nobel lecture:

Therapy Disease / Condition Treated Table 1— Some examples of monoclonal antibody Infliximab • Rheumatoid arthritis therapies currently in use Brand name: Remicade • Chrohn’s disease and the conditions treated Rit uximab • Non-Hodgkin’s lymphoma Brand names: Rituxan and MabThera Etanercept • Rheumatoid arthritis Brand name: Enbrel Abciximab • Prevents blood clots in Brand name: Reopro coronary angioplasty Trastuzumab • Specific kind of breast cancer Brand name: Herceptin Bevacizumab • Several types of cancer Brand name: Avastin Basiliximab • Rejection of kidney transplants Brand name: Simulect Paliviziumab • Respiratory syncitial virus (RSV) Brand name: Synagis infections in children Alemtuzuab • B cell leukemia Brand name: Campath

Breakthroughs in Bioscience 14 “The hybridoma [monoclonal detect the mutations that occur antibody] technology was a by- in antibodies as they evolve in product of basic research. Its response to the presence of an success in practical applications antigen? With better techniques, is to a large extent the result Milstein eventually was able to of unexpected and unpredict- use his monoclonal antibodies to able properties of the method. It show this evolution does occur thus represents another clear-cut in the antigen-binding regions of example of the enormous practi- the antibodies, and he reported cal impact of an investment in his results in a paper he submit- research which might not have ted just a week before he died in been considered commercially 2002. So not only did Milstein create monoclonal antibodies, worthwhile, or of immediate but he helped show how “Nature medical relevance. It resulted guides the internal disinfection,” from esoteric speculations, for as Behring put it. His marriage curiosity’s sake, only motivated of basic to applied research led by a desire to understand nature.” to revolutionary new approaches Interestingly, Milstein’s in both biology and medicine— research came full circle by magic bullets in shots that were the end of his life, having gone heard around the world. from basic to applied research, and then back to basic research. Remember his previously failed use of monoclonal antibodies to

Breakthroughs in Bioscience 15 Biographies Margie Patlak writes about biomedical research and health from the Philadelphia The Breakthroughs in Bioscience region. She has written for Discover, Glamour, Physician’s Weekly, Consumer Reports on Health, The Washington Post, Los Angeles Times, Dallas Morning News and numerous series is a collection of illustrat- other publications. She also writes frequently for the National Institutes of Health and the National Academy of Sciences. This is her sixth article in the Breakthroughs in Bioscience ed articles that explain recent series.

developments in basic biomedi- Dan L. Longo, Ph.D. is Scientific Director at the National Institute of Aging at the National Institutes of Health (NIH) and a Senior Investigator in the Lymphocyte Cell cal research and how they are Biology unit, where his research interests encompass the regulation of growth fraction in tumor cells, tumor-induced immunosuppression, and clonal selection of B-cells, among important to society. Electronic other topics. After completing medical school at the University of Missouri, Columbia and internal medicine training at the Peter Bent Brigham Hospital and Harvard Medical School versions of the articles are avail- in Boston, he obtained fellowship and laboratory training at NIH and has been there for 26 years. Before becoming Scientific Director, NIA in 1995, Dr. Longo was the Director, able in html and pdf format at Biological Response Modifiers Program, and Associate Director, Division of Cancer Treatment, National Cancer Institute, Frederick, Maryland. He is the author of over 600 the Breakthroughs in Bioscience articles and book chapters. He is an editor of Harrison’s Principles of Internal Medicine, and Cancer Chemotherapy and Biotherapy. He is an associate editor of Journal of the National website at: Cancer Institute and Clinical Cancer Research and he sits on the editorial boards of six other peer-review journals. He has been cited by Good Housekeeping as one of the “Best Cancer www.faseb.org Doctors in America” and listed in Best Doctors in America.

Breakthroughs in Bioscience 16 Breakthroughs in Bioscience 17 Published 2009

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