section 1 7-8

Chapter 7.  THE IMMUNE SYSTEM

• Tutorial: Our skin and intestinal track create barriers that protect our bodies from a world full of bacteria viruses, and the other microscopic creatures. 
• Immune cells float through our blood and lymph and identify, imprison, and destroy invaders. 
• In the process of protecting a body they can unleash an inflammatory attack that is painful and debilitating. 
• At times defenders mistake good guys for bad guys and attack joints (rheumatoid arthritis), the intestine (Crohn’s), the kidney (lupus) or the nervous system (multiple sclerosis.). 

There are over ten billion B lymphocytes in the blood and lymphatic systems of each human body.  Like a hive of bees, they are an ecosystem.³ Each can identify one and only one unique sequence of alien DNA or RNA.  When a B cell encounters its fated invader it rapidly clones itself, makes a huge number of carbon copies.  Some of the offspring become memory cells.   Most, now called “plasma cells”, fabricate free floating antibodies that attach themselves to the foreign protein, and mark it for destruction. 

Some immune cells are sentinelsthat recognize and ingest foreign protein and “process” it.   Called dendritic or antigen presenting cells they don’t destroy, they display the distilled protein on their outer membrane in an area called the “MHC complex.” 

The T lymphocyte has receptors that recognize the offering and grab it.  Some T-lymphocytes exterminate viruses; others destroy malignant cells. 

Macrophages “surround and kill microorganisms and remove dead cells.”  Much as a caterpillar turns into a butterfly, macrophages begin life as monocytes.²⁸

Immune cells communicate and influence one another by secreting small molecules called cytokines.² Some of these play a role in the inflammation that protects us from invaders.  Others are a major contributor to the pain and damage caused by one of several autoimmune diseases.

We can usually temporarily control immunologic assaults with cortisone derivatives.  To block the inflammatory cytokines physicians are increasingly using monoclonal antibodies. 

TNF—tumor necrosis factor—is a misleading, inappropriate name—for a family of cytokines that is the major cause of the pain, swelling and inflammation suffered by people who have any of a number of auto-immune diseases. The name was chosen by researchers who were trying to understand how some malignancies were cured when they were intentionally infected with virulent bacteria.³¹

Intentionally infect a cancer?   Some doctors had tried it here and there for a few centuries, but it wasn’t studied and promoted before William Coley, a physician at a major New York hospital became a believer. 

An upper “crusty,” Coley could trace his American lineage to the Mayflower era.  He graduated from Harvard Medical School in 1988 and during his apprentice years learned that half the surgical repairs of abdominal hernias in kids didn’t work very long. (Hernias are weak areas the belly wall that intestines can protrude through.). He introduced the European approach, using sutures and sewing and resewing.  He was successful and “admired.”³²

He started suspecting that infections can lead to a cure for cancer when he saw a malignancy disappear after a man with cancer developed erysipelas, a streptococcal infection of the face.  The cancer was infected and it faded away. Years later Coley located the man and the cancer was still gone.  An influential surgeon Coley decided to infect the throat tumor of an Italian immigrant who couldn’t speak or eat.  Making small incisions in the growth, Coley rubbed streptococcus into the wound.  At one point “the patient became extremely ill and looked like he might die.”  But he survived and the tumor “liquefied.”      Coley published a case report and promoted the approach. As head of the bone tumor service at New York hospital he injected streptococcus into 1000 malignant sarcomas. After they were infected  about 10 percent of them regressed and disappeared.²⁹  In subsequent years the drug company Parke-Davis marketed a mixture of two virulent bacteria that could be used to treat cancers.  In 1962 the government clamped down on medications that weren’t proven safe and effective. Coley couldn’t prove his approach worked and Parke-Davis stopped marketing the bacteria.

Decades later a team of researchers in Belgium led by Walter Fiers discovered a cytokine that eradicated human tumors that were planted into laboratory mice.  They named the cytokine family TNF—tumor necrosis factor. 

Researchers have developed antibodies that block TNF cytokines.  The medications they developed are among the most costly and profitable pharmaceuticals of the day.  Humira generated $19.9 billion in 2018.  Enbrel/etanercept had $7.1 billion in revenue.  Remicaid/infliximab-$5.9 billion.

The story of the cytokine TNF and the creation of antibodies that block their action starts in 1980.  A researcher named Hilary Koprowski, “a colorful, prominent Polish-born virologist” patented a process he had used in his research—a method for making monoclonal antibodies.⁴

The technique was developed 6 years earlier in Cambridge England by George Kohler and Cesar Milstein.  They won a Nobel Prize for the process, but they didn’t bother to file a patent. 

Their project started when they injected purified protein into a mouse.  One of the lymphocytes floating in the creature’s blood realized the injected amino acid was foreign and it had to be destroyed.  The lymphocyte started cloning, making huge numbers of copies of itself.  The numerous identical lymphocytes all made the same antibody to the foreign protein.  Days went by.  Then one of the researchers drew blood from the animal’s spleen, a large blood filled organ..  As expected, a sizeable proportion of the mouse’s lymphocytes were now clones of the original cell, and each of the lymphocytes made the same antibody. So far nothing that happened was exceptional.  

At this point they fused some of the lymphocytes to mouse myeloma cells, malignant plasma cells that keep reproducing and don’t die.  The hybrid they created made and kept making large quantities of one and only one antibody.  .  

As Kohler, a shy, gentle Swiss German immunologist later explained, the fusion approach was new and unique.  “If by blind chance the right lymphocyte, the one producing the antibody against the injected antigen had fused with the myeloma cell and was forming daughter cells that were locked into producing the same pure antibody. …it was a long shot.”  Around Christmas 1974 Kohler added the antigen to the fused cells and went home.  If the experiment worked the antibodies produced by the fused cells would combine with the antigens and they would precipitate.  Halos would form around the cells.  He returned hours later and fearing failure brought his wife along to console him.  They looked in the window, saw the halos and were elated.  “I kissed my wife.  I was all happy.²⁶”

Kohler’s partner, Milstein was a Jewish researcher from Argentina.  His 14 year old father had exited Russia the year before the country became embroiled in the First World War.  His Argentina born mother was the head mistress of a school and encouraged her son to study hard and to go to the University of Buenos Aires.  At one point she helped type his PhD thesis.  Married and a post doc researcher, Milstein spent three years in the 50s working at a lab in Cambridge England. He returned to Argentina in 1961 as head of a university department, but a military coup had taken control of the country.  It conducted a campaign against political dissenters, and Millstein had been a prominent anti – Peron student when he was an undergraduate.  It was also targeting Jews.  Milstein felt unsafe and returned to the lab at Cambridge.

In the 1960s and 70s a number of scientists developed mouse myeloma cells (malignant plasma cells) that could be grown in tissue culture and were “immortal”..They or their progeny survived indefinitely.  Milstein learned how to turn two small myeloma cells into one larger cell.  In 1974 he was joined by Georges Kohler, a Swiss postgraduate researcher who was also interested in fusing myeloma cells.  Together they developed the first “hybridoma”—part lymphocyte—part myeloma cell—the first “factory” that produced monoclonal antibodies.  

With a patent in hand, Koprowski owned the process for making monoclonal antibodies.  Along with an entrepreneur named Michael Wall, he formed a company named Centacor, and they tried to figure out how turn mouse monoclonal antibodies into gold. 

In the summer of 1982 Michael re-met Jan Vilcek, a man who studied cytokines and who worked at a New York hospital.  A Czech researcher, Jan was a 6 year old Jewish kid in 1939 when his country was occupied by Nazi Germany.  During the next few years the Nazis rounded up and killed Jews.  Vilcek wrote that he and his parents survived in a hostile environment because they had “a complicated attitude toward their Jewishness.”  At some point they converted to Catholicism.  Later they moved. Jan’s father joined the underground.  One way or another they managed to avoid the death camps.  After the Second World War Russia took control of Czechoslovakia, and the country became part of the Eastern Bloc.  The Soviet Union and the U.S. feared one another, built nuclear missiles, and created armies that could defend their nation.  Travel between the Soviet Bloc and the West was restricted and immigration forbidden.

Vilcek married and became a virology researcher.  When he was in his 20s, fed up with the Czech Communist government, he wanted to “relocate.”    In 1964 the couple received permission to cross the iron curtain for a three day vacation in Vienna. They traveled by auto.  It was October, still warm, and they brought their heavy winter coats.  When they reached the border and their car was being searched Jan worried that the coats would be a giveaway– that the inspectors would realize that Jan and his wife were trying to escape.  He waited while the border guards “hesitated for the longest minutes of his life before letting them pass.⁴”   Once across the line that divided the countries they, of course, didn’t go back.  After the couple reached Germany, life was rough, but within a year Vilcek was hired by NYU, New York University.

After spending a number of years studying interferon, one of the body’s cytokines, Vilceck attended a workshop on a poorly understood immune regulator called Tumor Necrosis Factor. 

In 1984 Genentech scientists determined and published the complete amino acid composition of TNF.  They purified the human TNF protein and they gave some of it to NYU.  Vilcek and his colleagues accepted the gift and “felt like kids in a candy store. –what should we try first?” 

The cytokine turned out to play a role in a body’s ability to fight viral infections.  It had so many actions that one of Vilcek’s graduate students quipped “TNF should stand for too numerous functions.” 

Cytokines are groups of special proteins.  They are discharged by immune cells and they act as chemical messengers.   After they are secreted by a cell, cytokines bind to receptors on the surface of other cells and they regulate the immune response.  They can work alone, work together, or they can work against one another.

In the 80s Centacor (still struggling) on a whim, a hope, produced a next generation monoclonal antibody that would block or inactivate TNF.  It was “chimeric”, a protein that was part human and part mouse.  The development took experts at Centacor 6 months and its patent was owned by NYU (an independent private research University) and Centacor.  The antibody didn’t (as Centacor had hoped) help people with sepsis.  But blocking TNF hindered one of the cascades of pro-inflammatory cytokines. It stopped or hindered inflammation.

When London doctors (Feldman and Maini) injected the medication into the swollen inflamed joint of a person with Rheumatoid Arthritis it usually helped.  The effect lasted three months.  A repeat injection was also successful.  In 1993 a physician from Holland used the antibody to treat a desperately ill 12 year old girl with a severe case of Crohn’s disease, a chronic inflammation of the small and sometimes large bowel.  The disease can cause diarrhea, pain, bowel blockage and fistulas, connections between the intestine and the skin or an organ.  The infusion was very effective for 3 months and it helped 8 of 10 additional people with severe Crohn’s. 

10 years after the original mouse antibody to TNF was generated in Jan Vilcek’s NYU lab, doctors had a tool that helped them treat a number of auto immune disorders.⁴

Part of the research was funded by the NIH (the taxpayer).  Part by Centacor. There was a lot of luck and serendipity along the way.⁵  Both Centacor and NYU were rewarded.  The FDA approved the drug for use in inflammatory bowel disease (for Crohn’s they say it has a positive effect 60 to 70 percent of the time),–and it can be used for ulcerative colitis, rheumatoid arthritis, ankylosying spondylitis and various manifestations of psoriasis.

In 1999 Johnson and Johnson bought Centacor for $4.9 billion.  Revenues from the drug (per J and J) rose annually between 2009 and 2016—from $4.3 billion in 2009 to $7 billion in 2016.⁶

Humira—adalimumab,  another antibody that blocks TNF, was created in mice that were genetically modified in embryo;  the animals make antibodies that human bodies think were made by a homo sapiens.  Some of the research on the drug was performed by researchers at the government funded Cambridge Antibody Technology, U.K.   The FDA licensed Humira at the end of 2002.  By 2005 AbbVie, the company that owned it, was selling more than a billion dollars worth a year, and by 2018 it was bringing in close to $20 billion.⁷

Scientists in many of the world’s labs knew how to make monoclonal antibodies to TNF, but they couldn’t market them until they performed placebo control studies that proved their drug was both safe and effective.  And that was costly, ethically questionable, and medically unnecessary.  Then a new law allowed companies to avoid double blind studies if they could prove their “new” antibody worked as well as the current one—that it was “biosimilar.”   A provision of the Affordable Care Act, gave the original antibody maker, in this case AbbVie, the exclusive right to sell the monoclonal antibody in the U.S. for 12 years.  At the time the FDA provided 5 years of exclusivity for new drugs that were not biosimilars.

At the end of the 12 years, as a result of a provision in the act, company lawyers were able to keep biosimilars—the biologic equivalent of generics–off the U.S. market for a few additional years if they claimed that one or several of the original drug’s 126 patents were fundamental.  (All the patents are presumably novel, non-obvious, and useful, but some merely protect a step in production or an inactive ingredient.)

Four companies produced effective biosimilars and wanted to steer clear of years of pointless litigation.  In an attempt to market their Humira-like medications, the manufacturers signed an agreement with AbbVie in 2017 and 2018.  It allowed them to market their medications outside the U.S.  AbbVie will retain their $10 billion a year U.S. Humira monopoly until 2023.⁸

Several cytokine families (including interferons) contain both pro and anti- inflammatory molecules.  Inhibitors are currently available to molecules that belong to one of two cytokine groups:  “TNF—tumor necrosis factor” and “interleukins”.

There are a number of situations where the  immune- system goes rogue.  A marked outpouring of cytokines can destroy joints and organs or bring on fatigue, fever, weight loss and an early death. 

An overly zealous “release” of these proteins, sometimes called “cytokine storm” may explain why

some treatments that rapidly destroy large numbers of malignant cells or severe cases of viral pneumonia kill. 

Huge numbers of cytokines have sickened people who received CAR-T treatment for cancer (discussed later in this chapter) and a four year old hospitalized at UCLA with a bad case of Coccidiomycosis, a soil born fungus infection.

High levels of a number of cytokines were found in the blood of  people with infiltrates in both lungs and low levels of blood oxygen caused by SaRS, MERS, AND COVID 19.³³

Some of the cytokine harm is mediated by one of more than 36 known interleukins–“hormones of the immune system”, and pharmaceutical researchers have developed, tested, and marketed humanized monoclonal antibodies that block some of them.²⁷

TRANSPLANTATION

On more than 34,000 occasions in 2017, organs from donors, dead and alive —livers, kidneys, hearts and lungs–were transplanted into the body of someone in the United States–and the immune system was challenged.

We learned organ transplant was possible in 1954 when an identical twin successfully gave his brother a kidney.  That’s as far as it went for decades because we weren’t very good at keeping a body from rejecting someone else’s organ.  Our original attempt to prevent the immune system from destroying foreign tissue, our “three drug anti rejection regimen”, according to Thomas Starzl, “wasn’t very effective or safe.”  

Starzl, the American who pioneered the effort to replace a failing liver with a healthy one, grew up in a small town in Iowa.  The son of the town’s newspaper editor he spent his teenage free time bulldozing giant rolls of paper into place, draining oil from the presses, and handsetting words letter by letter.  He went to medical school because that’s what his buddies on the football team did.  While attending Northwestern he lived in the Chicago ghetto and earned his keep tending the wounds and illnesses of local employees under the tutelage of a “very competent” physician. After graduating he started his surgical training at Johns Hopkins a programthat “ruthlessly” expected young men and women to be on call 24 hours a day, fifty one weeks a year, and the trainees were not compensated.  At age 26 he married Barbara and needing to earn money he became a surgical fellow at the University of Miami.  The program was new and they paid their doctors.  He cared for “vast numbers of patients”, became a competent surgeon, and on a theory (that he ended up disproving) he started operating on dogs in his garage.  He obtained the poor creatures from the pound and his wife, Barbara, “cared for the animals.”  While he was operating on the animals he figured out how to remove the liver without killing the canine, and he tried to transplant the organ. The blood from the small bowel was a problem.  Before it enters the main circulation it normally flows into the liver and is filtered and cleansed.  Newly transplanted livers couldn’t deal with the flow and they kept failing. Then Starzl learned how to detour the intestinal blood around the liver.  Once that problem was solved his transplanted dogs “were normal for almost a week; then began to reject their new liver.”

In 1961 Starzl became chief of surgery at the Denver VA hospital and used prednisone and immuran to prevent rejection of a few kidneys.  He had a modicum of success, but as late as 1978 “Graft survival was unsatisfactory and patient mortality high.¹⁰   Ambitious, focused, and perhaps a little too preoccupied by the rapid changes in his craft, Starzl remembered the day in 1976 when his wife of 22 years casually drove him to the airport in a snow storm.  He flew to London to present a research paper and while there received “ambiguous phone calls from his family, and he “knew” he could not return home.  After 22 years of marriage his wife Barbara’s “forbearance had run out.”   

Once effective anti rejection drugs were available, transplanted livers survived for many years and Starzl’s group in Denver led the way. 

In 1981 Starzl moved to the University of Pittsburg and brought liver transplantation east.  People who were dying of liver disease came to Pittsburg and were admitted to beds on the medical service where most spent their last days. Three of the first four people Starzl transplanted died and “54 residents and interns in the Department of medicine signed a resolution denouncing liver transplantation as unrealistic and potentially unethical.” In response Starzl admitted his patients with cirrhosis of the liver to the surgery service. (He couldn’t make the patients live longer but he could get the internists off his back.) Nineteen of the next 22 people transplanted survived and Starzl and others turned the Pittsburg transplant program into the largest in the world. 

In 1983 when the FDA approved cyclosporine for use in transplant patients, surgeons who performed the operations had successes and failures.  The procedure was far from standard or easy but some skilled doctors in the community apparently thought: if he can do it, I can too.  In the 1980s a California surgeon attempted a liver transplant and the patient bled to death.  In the process the operating physician transfused the person so aggressively that (hearsay) the blood supply of Southern California was threatened. 

Before the University of California at San Francisco started transplanting livers they agreed to make a major commitment to its support.    In addition to having access to a wide array of subspecialists the hospital needed kidney dialysis capability, respiratory therapy support, and an extensive blood bank.

 Surgeons had to be trained specifically for liver grafting.  A trained team had to be available to recover donor organs, keep them alive, and transport them quickly. Once transplant centers knew a liver was on the way they would call in two potential recipients whose blood type was the same as the liver.  They would start with the person at the top of their list and bring a back up into the hospital, just in case.  People with active infections could not be transplanted until the infection was gone. 

In 1990, having spent most of his life transplanting organs and teaching others, Starzl had a heart attack and wrote a memoir. In it he mused that every person who receives someone else’s organ starts seeing the world in a different way, and that medicine’s ability to save a life by transplanting an organ is a legitimate miracle.

In 1967 Christiaan Barnard in South Africa and Norman Shumway at Stanford each transplanted a human heart.  Neither recipient survived for three weeks.  In 1971 Life Magazine’s story of an “era of medical failure” told readers that subsequent to the first two “166 heart transplants were performed and 143 of the recipients died.²²” 

The son of a pastor and a church organist, Christiaan Barnard, the surgeon who performed the planet’s first heart transplant, was born in a sheep farming region of South Africa.  As a student at the University of Cape Town he was on scholarship, poor, and had to walk five miles to school each day.  After he graduated from medical school Barnard married, had two children, and practiced medicine for 2 years.  Then, deciding he wanted more from life, he accepted a scholarship to the University of Minnesota and spent 30 months (many without his family) working with some of the first surgeons who repaired heart defects in children.  He watched them work, learned techniques, and often operated the machine that oxygenated the bodies of the children whose hearts were not beating. After he returned to South Africa Barnard and his brother who was also a surgeon operated on 48 dogs, and they learned how to transplant a heart.  Then he was introduced to a 53 year old man who was bedridden, had severe heart disease, and was ready to resume his life or die.  The heart Barnard transplanted came from the body of a 25 year old woman who, as the result of a traffic accident, was brain dead.  The man who received the woman’s heart, survived surgery.  The operation became front page news, and the transplanted heart worked for 18 days before the patient developed pneumonia, and died.  Reflecting on the man’s decision Barnard later wrote, “For a dying man it is not a difficult decision because he knows he is at the end. If a lion chases you to the bank of a river filled with crocodiles, you will leap into the water convinced you have a chance to swim to the other side. But you would never accept such odds if there were no lion.”

A second heart transplant recipient lived 18 months.  A few years later effective anti rejection medications hit the market.  By 2001, the year Barnard died, doctors in the U.S. were performing 2,400 transplants each year.  Eighty seven percent lived for at least a year and ¾ more than five years.

Barnard became a celebrity, let his hair grow, started wearing suits made by an Italian tailor, dated movie stars, and ended his first marriage.  During his life he performed 75 more heart transplants, created a tissue heart valve, and was married two more times.  His rheumatoid arthritis eventually crippled his hands, and when he was 61 he stopped operating.

A month after Barnard performed the first heart transplant Norman Shumway, a California surgeon transplanted the second human heart.  At the time Shumway had been transplanting dog hearts for 10 years and knew technically what to do but his patient died within three weeks. 

A member of the high school debate team in Kalamazoo Michigan, Shumway originally planned to go to law school, but he was drafted during the Second World War.  When the government decided they needed more doctors and dentists Shumway was one of the soldiers tested.  He scored high and chose medicine over dentistry.  Assigned to a group of men who received pre-medical training at Baylor University, he was a hospital orderly for 6 months before he went to medical school at Vanderbilt.  During the Korean War he was an air force doctor.  When his service ended he joined the multitude at the University of Minnesota who were learning how to correct congenital heart defects.  Unable to get much hands-on training he decided to go into private practice and joined an older doctor.  It was not a fit.  Someone convinced him to come to Stanford University, an institution that didn’t have any doctors with heart surgery experience.  A modest man who was relieved when someone else performed the first heart transplant, Shumway became the chief of cardiothoracic surgery at Stanford in 1965.

In 1983, after the FDA sanctified the use of Cyclosporine, the first drug that allowed foreign organs to survive for years, the transplant scene changed.  In the subsequent decades over 700,000 people in this country lived part of their lives with someone else’s liver, kidney or heart.  Kidneys, on average, lasted 12 to 15 years; livers had a shorter lifespan.  That’s going to change now that hepatitis C (a frequent cause of liver destruction) can almost always be cured.  When a person with hepatitis C was transplanted, the new liver always became infected, and it had a relatively brief lifespan.

After a person receives a foreign organ, they (almost always) reject the newcomer if they don’t take an immunosuppressant daily for life.  A few anti rejection drugs are currently available.  There’s a marketplace and competition.

Cyclosporine, the first truly effective anti rejection drug, was developed by Sandoz, a Swiss chemical company that, in the 1800s, manufactured dyes and saccharine.  In 1917 the company hired a chemistry professor, and created a pharmaceutical department.  His group isolated ergot from a corn fungus and turned it into a drug used to treat migraine and to induce labor.

In 1958, the company asked employees to take a plastic bag with them when they went on vacation or business trips, and to periodically gather “soil samples that might contain unique microorganisms.”  They knew Penicillin was part of the juice produced by a mold, and they hoped one of their people would find the next great antibiotic.

John Francois-Borel was a company biologist, and reluctant scientist.  He said he originally wanted to make art and he was very gifted.  But, as he put it, “you know how art pays; I am not the Bohemian type.²¹He was the man who discovered cyclosporine, the drug that prevented the body from rejecting foreign tissue and that revolutionized the field of organ transplants.  Borel collected a handful of earth when visiting a desolate highland plateau in Southern Norway.¹⁸ A fungus in his sample of Norwegian dirt produced a metabolite (Cyclosporine) that lowered the immune response of lymphocytes.  It seemed to be relatively safe, and some thought it could potentially become an anti rejection drug.¹⁶

In 1976 Borel presented his findings to the British Society of Immunology.   “A small stocky surgeon with a mop of curly black hair (Starzl’s description) who had been working in transplantation since 1959,²⁰” Sir Roy Calne was one of several who “asked Borel for samples”.  Calne used the fungus juice to try to prevent the destruction of organs transplanted in rats and dogs.  The drug’s effect was dramatic. 

By 1973 the Sandoz supply of fungus derived cyclosporine was largely depleted.  Large sums of money (around $250 million,) would be needed to create more, evaluate its anti-rejection potential, develop a drug, and obtain approval from the FDA.  There wasn’t much of an organ transplant market, and the investment didn’t make much sense. But with Borel’s help, Calne presented his findings to decision makers at Sandoz.  “The pharmaceutical company agreed that the drug looked more promising now that there was evidence of its effectiveness.” Pharma scientists can produce great results.  But to create a truly innovative medication, in addition to money they need a modicum of serendipity, imagination, and stubborn determination.¹¹

Over time, in addition to performing surgery Calne became a well known artist.  He once wrote that art and surgery “Both require careful planning, skill and technique and familiarity.”

          In the early 1980s Starzl used Cyclosporine successfully on liver transplant recipients.  With his results in hand the FDA fast tracked approval of the medication and in 1983 it became available for use in the U.S.  Currently made generically by a number of countries Cyclosporine’s wholesale price is not outrageous.²⁴ $106.50 a month in the developing world– 121.25 pounds per month in the United Kingdom, and about $172.95 per month in the U.S. (if generic drugs are prescribed.)

The second major, now widely used, anti rejection drug was Tacrolimus (Prograf).  Originally isolated from the “fermented broth of a streptomyces bacteria”, it was discovered and developed in the 1980s by Japanese chemists screening “natural substances in the soil for their anti cancer and anti rejection properties.”   They performed their studies at Fujisawa Pharmaceutical, a company located at the foot of the Tsukuba Mountain, a green oasis with hiking trails, Shinto shrines, and a good view of Mt. Fuji.   

English scientists tried Tacrolimus in dogs and “declared it too hazardous to test in humans.”  Dr Starzl’s group kept at it.  They found the drug kept transplanted organs alive in some animals and “rescued some organs that, despite cyclosporine, were being rejected.” Additional clinical trials “suggested that tacrolimus might be safer and better tolerated than cyclosporine.¹²  ”  

In renal transplant recipients the drug led to improved graft and patient survival, and that lead to its routine use in U.S. renal and pancreas transplant recipients.  The FDA made it official in 1994.   Fujisawa later merged with and became Astellas, the world’s 14^th largest.¹⁷ The year before Prograf (its brand name) had a generic competitor, Astrellas sold up to $2.1 billion dollars worth of the medication.

Health care delivery:

There aren’t enough livers for everyone, and the people on the liver transplant list who are closest to death get the first organ of available for a person with the same blood type.  There are rules that limit the use of organs for people who are addicts, alcoholics, or obese.  If someone’s BMI (body mass index) is too high they can’t be transplanted with a “brain death” liver. 

One day I was asked to see a middle aged female who drove a fork lift in a warehouse.  She had never been sick before, was muscular and didn’t drink or have hepatitis.  But her liver was full of fat and she had developed hepato-renal syndrome.  Her liver disease had caused her kidneys to stop working. (I’m not going to explain what happens physiologically.  I’m just going to say it’s a well known, infrequent complication of advanced cirrhosis.)  When renal failure is caused by liver failure, dialysis doesn’t work.  She was in trouble and needed a liver transplant.  I explained the problem to the patient.  She said O.K., and I called the transplant intake doctor at the university.  It was Friday afternoon, time to go home, and the woman’s BMI (weight) was technically over the limit.  The university doctor was sorry but he had to turn the patient down.   I explained that part of the weight was caused by fluid retention. Her dry weight BMI (body mass index) didn’t exceed the threshold.  “No can do”, the University physician explained.

I told the patient.  She cried.  Her sister who was in her room cried, and the sister offered to donate part of her liver.  The patient refused.  She was given infusions of a few drugs and for some reasons during the next few days her kidney function didn’t get worse or better.  I visited her each day and we talked.  When Friday arrived she was still alive, and on a whim I called the hepatologist on call at the medical center.  The University of California had a group of liver specialists who accepted or refused referrals.  Each doctor was on call for a week; then a new physician took over.  I explained the situation to the new intake doctor and she said “no problem.  Send her over.”  The nurse called an ambulance.  The woman got a new liver and she did well. 

Currently, in addition to brain death, patients who have severe brain injuries but who are not “brain dead” can become organ donors if the patient consents by means of an advance directive, or the patient’s family decide that life support should be withdrawn. “To avoid obvious conflicts of interest, neither the surgeon who recovers the organs nor any other personnel involved in transplantation can participate in end-of-life decision or the declaration of death.”

Some countries have a system where an appropriate dead person’s organs can be transplanted in another unless the person explicitly objected while he or she was alive and competent.  The U.S. and a number of other countries require specific consent.

When someone “dies” and donates their organs teams of doctors come to their hospital.  Livers and kidneys are removed without dissection, without traumatizing blood vessels. The organs are cooled, and transported (sometime by plane or helicopter) to a hospital where surgeons and recipients are waiting.   

Once outside the body “The heart is most sensitive to lack of blood flow, “and needs to be planted in a body within 4 hours.   Lungs, with appropriate cooling “remain viable for 6 to 8 hours”, livers 12 hours and kidneys 24 to 36 hours. 

In 2011 an average transplant of one kidney had a price tag of $260,000.  Combined heart and lung transplants were costing $1.2 million dollars.   The first 180 days of post transplant medications was costing $18,000 to $30,000, and a number of generic immunosuppressive drugs have been marketed.  “Cellcept was approved in 1995.  Mycophenolate mofetil became available in 2008, Tacrolimus in 2009, and sirolimus in 2014,”

It’s claimed that the annual cost of U.S. transplant immunosuppressive therapy averages $10,000 to $14,000.  If true then the 33,000 transplant recipients in 2016 are (directly or indirectly) paying $330 million to $462 million a year.  That becomes $3.3 billion to $4.6 billion over ten years if drug prices don’t rise or fall.¹³  

In India (where the culture surrounding pharmaceutical prices is quite different from ours), the amount paid for Tacrolimus was slashed 65 percent in 2016.  The average recipient now pays $235 to $314 a month for anti rejection medications.

On October 30, 1972 chronic dialysis and kidney transplant became a Medicare ‘”right.”  If someone receives a kidney transplant (at a cost of hundreds of thousands of dollars) the operation and three years of anti rejection drugs are fully funded by the federal government.  At the end of those years the patient is removed from Medicare, and they have to pay for their own anti rejection drugs.  About 22 percent of people on anti rejection medications stop taking them because of side effects, high cost, or for other reasons.  When a kidney transplant recipient stops their immunosuppressive drugs they almost always reject their kidney, and they are forced to go back on dialysis or die. This is not theoretical.  It happens.  And it’s a problem.¹⁴

Founded in 1984, a private non- profit organ transplant organization (UNOS) under contract with the government, “oversees all organ procurement and transplant programs in the country and makes the rules about who can do transplants and how organs are to be allocated (given) to patients. “

People on dialysis wait their turn.  Managing the waiting list can be difficult.  My former employer tried to open their own renal transplant center in 2004, but closed the unit and paid a fine because they (allegedly) mismanaged the transfer of their patients’ records.      

A few years back our former neighbor’s son, learning he was a “match”, decided to donate half his liver to an uncle he didn’t know that well.  His mother was a mess.  Donors are screened.  They must be young and healthy.  But the operation is tricky.  The liver has a large and a small lobe.  The small lobe is adequate for a small child whose liver isn’t working.  An adult needs part of the large lobe.  The liver has to be “split” and a significant amount of tissue has to be removed.  Over time some liver will grow back, but it takes months.  If too much of the organ is removed the donor is in trouble.  There are occasional complications, mainly bile leaks.  And one in 200 donors dies.  There are only a few centers in the country that do at least 100 living donor liver transplants a year.  The young man’s mother is pretty cool.  She has a strong social conscience but this was hard.  Bottom line: he donated, and survived.  And mother and son are doing well.²⁵

• In the U.S. in 2019: 23,401 kidneys were transplanted as well as
• 8896 livers, 3551 hearts,  and 2714 lungs
• 11,900 of the donors were brain or heart dead.         
• 7397 of the organs came from living donors.      
• In 2019 over 112,000 Americans were on one of many transplant lists and “The wait for a deceased donor was often 5 or more years.²⁵”

Immunotherapy and Willie Nelson

Discovery consists of seeing what everybody has seen, and thinking what nobody has thought. Albert Szent-Gyorgyi

That’s what Jim Allison had to deal with when he learned how to use the immune system to attack and sometimes cure metastatic cancer.  Allison was from a small town in Texas.  His father was a country physician and his mother died of lymphoma when he was still a boy.  Initially planning to follow in his father’s footsteps, he got interested in research as a high school student. In an interview he said that he was reluctant to become a physician because doctors have to be right almost all the time.  Researchers, on the other hand, develop hypotheses and test them.  If they aren’t wrong most of the time “they’re not on the edge.” 

He wrote poetry, liked to read, loved country music and later in his career played harmonica on the stage with Willie Nelson.  At the University of Texas he studied biochemistry and earned a PhD.   After his post doc year he got a job in a small lab that the University of Texas/M.D. Anderson Cancer Center was operating in Smithville… close to Austin.⁶

While in Texas he worked out the structure of the T cell antigen receptor and gained some notoriety.  T cells, one of the white blood cells that float in our blood, are part of the immune system.  They aren’t very good at recognizing abnormal proteins but they are efficient destroyers.  When a dendritic or other watchdog cell spots a virus it processes the “stranger” and “presents” it to the antigen receptor on the T lymphocyte. Then the T lymphocyte recognizes and deals with it.  At one time early in Allison’s education he recalls a professor who doubted there was such a thing as a T cell.   

After his “receptor” accomplishment Allison took a year sabbatical and became a professor at the University of California, Berkeley.  He considered himself an immunologist and his lab tried to work out the relationship between the T cell and cancer.  In animal studies T cells seemed to recognize and attack cancer cells.  They latched on and released a poison, a protein called CD28 that should have destroyed the malignant cell.  But the cancer somehow survived.

Another protein, CTLA-4, showed up after the CD28 was released.  What was it doing there?  A large pharmaceutical company had concluded it was another cell poison, and the company patented it as a poison.

Allison wasn’t so sure.  So he developed an antibody to CTLA-4 and one of his fellows gave it to a mouse with cancer.  A few days later the cancer was gone.

The results surprised Allison.  Blocking a cancer poison should not have contributed to the death of the tumor.  Allison asked his fellow to repeat the experiment.  Since it was Christmas, the fellow went on vacation.  Allison manned the lab and watched the mouse as the tumors grew for a few days.  Then they faded away. 

Allison immediately realized he might have something big, but he had to be sure.  His group injected antibody that blocked CTLA-4 into the bodies of many different strains of mice.  In the absence of CTLA-4 the poison produced by the T lymphocyte– the CD28– was able to destroy one tumor after another.

Allison realized his success meant our understanding of cancer and the immune system was wrong.  “The biology was backwards”.  T lymphocytes, he hypothesized, recognized cancer cells, and they latched on.  They injected a “poison” (CD-28) that should have killed the malignant cell.  But cancer cells made an “antidote” (CTLA-4).  It stopped the poison from working.   His antibody blocked the antidote and it ALLOWED the poison to keep killing the bad cells.

Bristol Myers Squibb had patented CTLA-4.  Their patent claimed CTLA-4 was the poison not the antidote.  It was wrong—backwards.  But the company had a patent and lawyers and money.

Allison was a valued scientist.  His identification of the T cell antigen receptor was important.  People in the field respected him.  He was a full professor of Immunology at Cal Berkeley.  Bright ambitious students studied with him.  But he wasn’t an M.D.  His only interaction with sick people had occurred when he was a boy in a small Texas town.  He had gone on house calls with his father, the town doctor.  Allison wanted to try his antibody on patients, but he didn’t think he could take the next step without Pharma’s help.  He “spent the following close to two years going around and talking to a number of large and a few small biotech firms trying to interest them in his idea.  There was a lot of skepticism.  And the fact that Bristol Myers Squibb had a patent put people off.  They claimed the intellectual property was ‘dirty.’

Eventually a small firm, Medarex, decided to give his antibody a shot.  It was a big investment.  Niels Lonborg a scientist at GenPharm, a company that was purchased by Medarex (in 1997), had mice that had made fully human antibodies.   Lonborg created the antibody to CTLA-4 that later became the drug, Ipilimumab.

A trial of the drug on patients was arranged.  As Allison explained in an interview, he “was totally committed” to the endeavor.    He moved to New York, to be near Sloan Kettering Cancer Center “to make sure nobody hurt his baby– Nobody screwed up.”   He moved to be a nuisance.  The biology of Ipilimumab (the drugs generic name) was different than that of most cancer drugs.  “Usually you treat patients.  If the tumor grows in the face of treatment the drug is a failure.”  But in the treated mice the cancer grew for a while, then it withered.  The tumor didn’t always regress but “there was overall survival.”

As shown in Breakthrough, a film that documented Allison’s subsequent struggle, his discovery came face to face with the people who ran big Pharma. Immunotherapy had failed in the past and to the conservative corporate money men Allison’s drug seemed like a long shot, not worth the risk.  The doctor coordinating the trials, Rachel Humphrey, believed in the product and she was its chief advocate.   When she faced the Pharma company’s board she emphasized the fact that the drug had been effective in one person.  That made it worth pursuing.  Men yelled at her and she took it. 

A competitor, Pfizer, had an immunotherapy drug trial running at the same time.  They halted their effort when the tumors in the patients they treated didn’t shrink 30 percent in 12 weeks.  That was the FDA Standard. 

With Allison’s drug the tumors kept enlarging but the patients felt well.  Allison explained that’s how the drug works.  The T cell gets into the tumor and starts killing cancer cells.  It takes a long time before the tumor stops getting larger.  As the trial progressed some of the people who were treated, went home and their doctor gave them a drug that had not previously worked—and this time it seemed to work and they got well.  The recent drug got the credit for the improvement.  Allison knew it wasn’t the recent medication.  The T cells had continued to methodically kill the tumor. 

Bristol Myers Squibb, his company, eventually agreed the end point of their study would not be the number of people who were alive at one year or two years.  They agreed to see if there was an improvement in total survival.  The study kept going for years.  After three years the patients who hadn’t received immunotherapy were all dead.  Three, four and five years after they were treated over 20 percent of the people who received Allison’s drug were alive and well.  The company couldn’t call it a cure.  The tumor might someday start to grow. You never know.  But the people who responded stayed well and the treatment sure acted like a cure. 

Allison went to New York in 2004 but the drug wasn’t approved by the FDA until 2011.  During the 7 years Allison lived in an apartment 3 blocks from the hospital.  He gained weight.  He knew the drug cured cancer and was frustrated by his need to keep explaining why the tumor was still present.  At times he became angry. On one occasion he went into a tirade.  He’d come so far and he was afraid they would conclude the drug failed.  He became single minded and obsessed, and it affected his relationship with his wife.  Malinda, the woman he met when he was a college student, the coed who always felt he was the only person she ever loved—the most amazing human she ever saw– left. 

In 1997 Medarex acquired GenPharm.  In 2009 Bristol-Myers Squibb paid Medarex $2.4 billion and the companies merged.  

Squibb charged $30,000 for an ipilimumab injection or $120,000 for a course of therapy.  During Ipilimumab’s first year on the market, Squibb sold $706 million worth; they took in $462 million through the first half of 2013, and they thought they would sell$1.54 billion worth of the drug in 2018.

In 1992 a few Japanese scientists found another poison/antibody combination.  The antidotes were called PD-L1 and PD-1.  Drugs that block them were created by Pharma researchers, tested, approved by the FDA, and sell for about $150,000 a treatment.

In 2017 close to 1300 people with advanced melanoma were assessed 3 years post treatment.  They lived in 21 countries.  All had received a combination of two drugs nivolumab-plus-ipilimumab.  58% were alive and in 39% the disease had not progressed¹.  In another study: 5 years after the trial was started, Dr. James Larkin of the Royal Marsden in England and others assessed 300 plus people who had metastatic melanoma and who had been treated with two drugs that blocked the antidotes produced by melanoma cell.  Marsden reflected that “Historically, 5-year survival rates among patients with metastatic melanoma were dismal.”  The treatment had been hard on the bodies of the sufferers; only 58 percent seemed to have had a favorable response.  But 52 percent of the 300+ were alive at five years. The median progression-free survival was 11.5 months.  More than half the people treated, most of whom would have died without treatment, were still alive at 60 months.² Doctors evaluating new treatments for advanced cancer are reluctant to use the word cure.  It’s always possible that the tumor will, at some point, start growing again. 

In 2018 the PD-1 inhibor, nivolumab “showed a clinically meaningful survival benefit in some people who had advanced lung cancer.³

The clinical trials, hospital days, and advertising cost a lot of money and the companies that tweeked, developed and manufactured the drugs are working hard to recoup their outlay.  In 2015 Dr Saltz of Sloan Kettering estimated the price tag for the two drugs used to treat melanoma was about $300,000 per person, and the copay, the “out-of-pocket charge was usually $60,000.⁴” 

In 2018 Bristol-Myers Squibb sold $7.5 billion worth of Opdivo/nivolumab and Merck sold $7.1 billion worth of Keytruda/pembrolizumab

In 2018 Jim Allison was awarded the Nobel Prize

CAR-T

A T-cell is capable of eradicating a cancerous cell, but it’s not good at identifying the target. B lymphocytes are experts in identifying and marking targets, but they have no killer mechanism. What if the capabilities are combined?   

    Paraphrased words of Zelig Eshar, Weizmann Institute, Israel

The man who envisioned inserting a seeing-eye gene into T-cells, was raised in Rehovot, Israel, at a time when “the fragrance of orange blossoms and the sounds of crowing roosters” filled the air¹.” He was earning a PhD in Boston, when he “decoded” the T cell receptor. That’s the molecule on the “skin” of T-lymphocytes that spots the remnants of viruses that are displayed on the surface of sentry cells.  During his 20 years at the Weizmann institute in Israel, Eshar and his team developed and refined a special gene (a chimeric antigen receptor) that can be planted in the cytoplasm of a T cell. When it’s up and running it gives the T-cell the ability to both recognize and destroy targets. 

In another part of the world a surgeon at the NIH, Steve Rosenberg, led a team that assembled a similar gene.  Their CAR-T, however, specifically targeted cells that have the protein CD 19 on their outer membranes. They targeted B lymphocytes.

          A compulsive researcher Rosenberg once wrote that he enjoyed “working through the night in the lab, drinking thick pasty coffee that had been on the burner for hours, walking out into the sunrise, and watching the city come to life.”   During his 40 NIH years “there were probably only 40 days when I wasn’t in the hospital, checking on research or seeing patients.” Over the years he wrote a book about his efforts, was interviewed on T.V. by Charlie Rose, and was featured in a Siddhartha Mukherjee’s cancer documentary.

When young, Rosenberg was present when his father, a Jewish immigrant from Poland, received one post card after another telling of relatives who had died in the death camps.  The notes evoked a depth of silence and Rosenberg tasted pain.  His desire to stop everyone’s ache was not the sole reason he wanted to become a doctor, but it played a major role. 

Steve was married to Alice, an emergency room nurse who disliked doctors egos and was determined she would not marry one.  They met when he was on call in the ER.  It was a slow night—not much business, and she called him over and led him outside to gaze at the moon filled sky.  They dated for 5 years, then he told her “we can’t see each other anymore.  Otherwise it will be too difficult to break it off.”  She answered “It’s already too late.” After they married and had children he was a surgical resident.  His sleep deprivation was brutal.  He once fell asleep at a patient’s bed side and he routinely dozed when he encountered a red light when driving home. 

In 1968, as surgical resident he recalls admitting a man with a gall bladder attack who, 12 years earlier had undergone a cancer operation.  The primary malignancy in the stomach was cut out but metastatic implants in the liver could not be resected.  The man should have died but he didn’t.  His immune system had apparently overcome the cancer.  It happens rarely, but the event made an impression on Rosenberg

In 1974 Rosenberg started working at the National Cancer Institute and began his search for a way to help the body’s immune defenses fight cancer.

In 2010 he and his colleague James Kochenderfer told their medical colleagues about a patient with lymphoma whose tumors shrank after they gathered his T cells, added a gene that recognized CD 19, an antigen that is only found on the surface of the healthy and malignant B cells.  They allowed the T cells to multiply, then poured them back into the person’s bloodstream.⁸ The patient became and remained cancer free.  Rosenberg subsequently treated an occasional patient, and sent a copy of their gene in a plasmid to Addgene, the non-profit that makes genes available to interested researchers.  By 2010 researchers could buy one of their plasmids for $75.

The following year Dr. June, at the University of Pennsylvania, wrote about two of three patients with chronic lymphocytic leukemia who were similarly treated and went into complete remission.  At the time he didn’t think the infusion was ready for general use.  As Dr June explained “Some of these responses don’t last—there’s resistance. “We still have to run rigorous randomized studies to determine if the therapies are effective, and whether they are cost-effective, and whether they can be delivered at scale.”

          CAR-T infusion often takes a month to prepare.  After an appropriate person, someone with a difficult to treat lymphoma, is identified, a large bore needle is inserted into a vein.  It’s hooked to an apheresis machine and blood is drawn into a sophisticated instrument.  The gadget’s centrifuge spins the blood, picks out the T cells, and returns the rest of the blood to the patient.  The collection is then “prepared, frozen, and sent to the facility where CAR-T genes had already been inserted into a number of harmless viruses.  The viruses are co-mingled with the T cells. They enter the “killers” and deposit the Car-T gene in its cytoplasm. The T cells are given time to reproduce, to increase in number.  A month later, now modified, the T lymphocytes are returned to the treating facility.  The patient is sometimes given intensive chemotherapy before the modified T-cells are dripped into his or her body.  The person who received the modified T cells is watched carefully for up to 35 days because killing a large numbers of tumor cells can cause the body to release a large number of cytokines and they can make the patient very sick.

In the years after the process was developed a few desperate individuals without other options were treated.  The approach was a new, expensive, and time consuming process.  There were risks.  Short term improvements might or might not mean a person’s life would be prolonged.  

In 2009 a UCLA urologist and a businessman, Arie Bellgundrun, founded Kite pharmaceuticals in Santa Monica.  He searched the academic market to see who if anyone knew how to use the immune system to fight cancer.  Years before, as a young doctor he had briefly worked with Steve Rosenberg on cancer immunology. He eventually contacted his old boss, Dr. Rosenberg.  Rosenberg showed him the x-rays of several patients he had successfully treated with CAR-T. It’s a onetime treatment.  When gene therapy works the cancer is gone in three to four weeks. Rosenberg had tried to get interest from J and J and other companies but the approach was too new and different. 

          Bellgundrun was impressed. 

          “In 2012, Kite pharmaceuticals, partnered with Dr. Rosenberg and the NCI (National Cancer Institute) to further the research and development of multiple chimeric antigen receptor (CAR) and T cell receptor (TCR) based products.”

In 2018 the FDA gave two companies permission to sell a new, unproven, type of immunotherapy that treats lymphoma.  When used in kids, in one study, CAR-T cells had eliminated malignant cells 83% of the time for at least three months.

To get FDA approval Kite had to prove their approach was effective.  Since it’s unethical to run a study where some qualified patients are not treated—where they are “controls”, the FDA is allowing the company to use historical controls to prove their approach is effective.   By the end of February 2020, 108 people had been followed long enough.  Kite can now say they have proven the approach prolongs the life of at least some people.

Kite, Novartis, and other companies are starting to offer CAR-T treatment.  Kite charges $375,000 for processing a person’s lymphocytes.  In 2017 Gilead purchased Kite for $11.9 billion. Three years thereafter Medicare said they would pay for the process.

Chapter 8- GENE THERAPY

Each year 100 American babies are born with Pompe’s disease.  The children are floppy, their muscles barely work, and their heart is enlarged.  Few survive infancy.   A genetic, recessive condition, the disease is only seen when both parents carry the defective gene.

The malady is the result of an enzyme deficiency.  The kids’ cells don’t make enough lysosomal acid alpha-glucosidase, a protein that’s used to convert stored glycogen into glucose—energy.

We eat carbohydrates and sugar, and we turn what we don’t use into a storage polysaccharide called glycogen.  We stockpile the excess fuel in our muscles and liver.   Between meals, when we need sugar to keep going, we use enzymes like lysosomal acid alpha glucosidase to turn glycogen back into glucose.  Babies who lack the enzyme won’t stay alive long.

The condition was “characterized” by and named for a Dutch pathologist–Joannes Pompe.  A member of the Dutch resistance, he was executed by the Nazis in 1945.   The absent enzyme, Lysozyme, was isolated in Belgium in 1955, and the responsible GAA gene was identified in 1979.  (It differs a little from one family to another.)

The needed enzyme was first made at Duke University by a dedicated team of researchers.  Their leader, Dr. Chen, the chief pediatrician, started his quest after he went to the funeral of an infant who died of the disease.  The pastor said God must have given the child life for some reason.  Chen took the message to heart, and decided to assemble a team of Duke University researchers and go to work.

The inspiration came at the right time.  Researchers knew how to clone genes, how to isolate the fragment of DNA that is the gene and make large numbers of identical copies.  Scientists with genetic engineering training could plant the genes into plasmids, collections of DNA in the cytoplasm.  After an investigator spends years isolating and characterizing a gene and is ready to move on, most want to preserve and immortalize the fruits of his or her endeavor. So they send one of the genes in a plasmid to Addgene.  It’s a Massachusetts based non-profit that, since 2004, has collected and stored genes in plasmids from investigators all over the world.  For a nominal fee (like $75) they then supply genes to researchers.    

In mammals most of the DNA is found in the nucleus.  Bacteria and certain other microscopic organisms also have circular fragments of DNA—plasmids—floating in their cytoplasm.  Scientists have learned how to plant plasmids into viruses.  They, in turn, infect cells and deposit the DNA.  If everything goes well the deposited genes start telling the “infected” cell to make the desired protein. 

 I don’t know where or how the Duke researchers acquired the plasmid that contained the gene that’s defective in Pompe’s disease, but they made their therapeutic protein by inserting the gene/DNA into cells derived from the ovaries of Chinese Hamsters.  They are currently the most common mammalian cell line that is used for mass production of therapeutic proteins¹.  It took the researchers three years to make enough Lysozyme for their early tests.  The produced enzyme was injected into a quail that had been bred to be Lysozyme deficient.   The poor bird was in bad shape.  It couldn’t get off its back, much less fly.  Post injection the creature stood and even flew a little.

After 6 years of successful research, the Duke scientists got some manufacturing help.  Production rights were licensed to Synpac, a British/Taiwanese company with a presence in Durham, Dukes home.  Synpac, in turn “used experienced contractors to manufacture the enzyme.”  (Having done the heavy lifting, the Duke scientists gave a lot away, but they retained some royalty rights.)

Once the company had produced enough enzyme, physicians at Duke infused the protein into three kids with Pompe’s disease.  Lysozyme replacement worked.

In 2006 Synpac made a deal with Genzyme.  It was a “15 year royalty sharing agreement that was potentially worth $821 million.”  At the time Genzyme was huge.  Based in Boston, their 2010 revenue was $4 billion.  The company planned to spend more than $500 million dollars creating production facilities for Myozyme (their name for the enzyme).

The following year Genzyme was acquired by the French Pharmaceutical giant, Sanofi for $20 billion.  As part of the process Duke University was paid $90 million, and relinquished its royalty rights.   In 2016 Sanofi sold $800 million worth of the needed enzyme.

Now called Lumizyme, the enzyme is currently made in large sterile factories.  Babies with the disorder get an injection of the protein every two weeks.

In this country “according to Sanofi, the average annual cost of treatment is $298,000.”   If it works the first year it’s needed the second and third year.  By the time a child is 10 years old—if no one develops a less expensive generic product, the system (insurance companies and Medicaid) will have shelled out $1 to $3 million dollars per child and big Pharma will have been handsomely compensated.

For those who feel health care is too expensive:  You ain’t seen nothin’ yet. Scientists have started to effectively attack and control an increasing number of genetic diseases.  Insurers are not allowed to deny coverage to anyone who has a pre existing condition nor can they impose lifetime or annual coverage limits.  Given the way our economy works, the treatments and cures that create so much hope, will cost a bundle.  And no one knows how we’re going to control their price tags.   

During my early days as a Kaiser doc a young neurologist named Frank asked me to care for ”B” a 40 year old woman who had uncontrollable diarrhea and incontinence.  She was in a wheel chair, couldn’t walk due to weakness and numbness caused by nerve damage, and she was slowly getting worse.  A close cousin of hers had the same problem and would develop each new disability a few weeks before “B” did.  The “disease”, amyloidosis was genetic and affected many of B’s relatives. The afflicted started deteriorating in their 30s and died young.  The defective gene told the liver to make a mutant protein.  It accumulated in parts of the body and damaged hearts, kidneys, and a person’s small bowel. 

Frank (the doctor) contacted a researcher at Boston University who had an unproven diagnostic tool that could predict who among the offspring had the gene.  Carriers could choose to avoid bearing children. 

B’s relatives from all over the country were invited to a funded reunion.  I didn’t go, but I was told the get-together was a disaster.  Family members met distant cousins who were in different stages of deterioration.  Everyone was visibly shaken by the clear picture of what was going to happen to their bodies. 

B’s daughter didn’t want to be tested and I didn’t see her for more than a decade.  Then one day she called.  She had chronic diarrhea and knew what that meant.  Liver transplants had stopped the progression of familial amyloidosis in some and she wanted to give it a shot.  Getting her transplanted was tricky.  She was initially disqualified on the basis of a technicality.  Then a 32- year-old ex-convict died in an accident.  My patient was given his liver and a 67 year old grandmother with liver cancer received my patient’s liver. The organ was normal except for its secretion of one toxic protein and it helped the grandmother a period of time.   It was San Francisco’s first domino transplant.³⁰

The daughter was stable for a while, then became septic.  The drugs that kept her from rejecting the new liver were suppressing her immune system, and the disease had weakened her. She somehow accepted what was happening to her body but she worried about her sons.  She wanted the boys to be tested before they considered having children, but she knew that if they had the genetic disease they wouldn’t be able to get health insurance. 

Decades later the FDA approved two therapies that keep cells from making the harmful protein by interfering with the expression of the gene. (The DNA sends the instructions to make a protein to the cell’s factory, the ribosome.   The directions are carried by RNA.  If the RNA, the courier, is blocked the instructions won’t reach the factory– the protein won’t be made– and the gene will not be “expressed.”)

In 1983 researchers found the first genetic disease marker.  It was linked to Huntington’s, a dominant malady that strikes in midlife.  The disorder became well known after Woody Guthrie, the Oklahoma folksinger who wrote “This Land is Your Land” learned, at age 40, that his jerky movements, rigidity, clumsiness and inability to think clearly were caused by the disease.  Abandoned at age 14 by his mother, who was hospitalized with Huntington’s, and his father, who moved to nearby town for a job, Guthrie spent his teenage years sleeping at various friend’s homes.  He was able to rejoin his dad after a few years, but was more interested in his guitar than he was in high school.  Guthrie married when he was 19 and the couple had three children.  During the dust bowl Woody was living near the Oklahoma panhandle.  Giant clouds of dust periodically blew in, filled lungs and killed cattle and a few children.  The clouds were the result of decades of farming in an ecosystem that had adapted to long droughts. Farmers had pulled out the deep seeded grass that had covered and protected the dirt for over a century. During the wet years the crops were bountiful.  Then came years when it barely rained.    The soil became hard and the winds were fierce.  Called the center of the dust bowl the area was now barely habitable and people were pulling up stakes and heading to California.  Woody decided to join them.  He left his family and headed west.  Two of Woody’s first three children developed Huntington’s in their early 40s. During his 55 years Woody served in the merchant marines, lived in California and New York and was a popular entertainer.  He married two more times and wrote 1000 songs, one of which turned out to be his final message: “so long it’s been good to know yuh.”

The nucleus of each cell in our body contains 23 strands of DNA, 23 chromosomes.  That’s where the 20,000 genes that are unique to each person are found.  These genes account, at most, for 3 percent of the DNA in each nucleus.

Over several decades researchers identified the nucleotide sequences responsible for one genetic disease, then another.  In 1990 scientists started mapping the entire human genome; the task was “declared completed” in April 1993, and genetic research got a huge boost. . We also learned that after a cell makes a protein it has to coil and fold into a specific three-dimensional shape.  Misfolding produces inactive or toxic proteins and causes a number of genetic diseases.²⁴

Every so often a child is born with one of over 2000 really bad genetic diseases, and his or her family has to raise a disabled infant who will die young. Researchers working on the problems, have developed treatments that supply or teach the body to replace a vital protein. The number of children currently alive with each disorder is increasing.    

Companies that market these life saving products charge a lot, too much for most people.  It’s estimated that “orphan drugs will make up one fifth of worldwide prescription sales, amounting to $242 billion in 2024.  Much of the money will go to either big Pharma or big biotech.²”  “The cost per patient per year of the top 100 orphan products was $150,854 in 2018.”  Insurance companies that stay in the market and ultimately the taxpayers will have a new flood of costs they will increasingly have to deal with.   

In the U.S. 49% of our health dollar is spent on 5% of the people.  The total cost of care in the 2 ½ decades between 1980 and 2004,”has gone from $1,106 per person ($255 billion overall) to $6,280 per person ($1900 billion overall).” 

There was a time when drugs for uncommon diseases had a difficult time getting FDA approval.  Tests involving large numbers of affected people were needed before the FDA would conclude a new drug was safe and effective.  That usually wasn’t possible when relatively few people were afflicted. 

Then parents got together, pressured members of Congress, and the legislature acted.  In 1983 Congress passed and the president signed the Orphan Drug Act. 

          Companies that manufactured drugs for less than 200,000 Americans got a lot of rewards:  Their FDA monopoly lasted seven, not five years.  Companies got tax credits—they could write off half of the development costs.  If the disease was rare, developers skipped the usual wait and joined the “fast-track” line.

 The law worked better than anyone could have predicted.  There are 7,000 rare diseases affecting 25 million to 30 million Americans. In the first 20 years 249 orphan drugs were marketed. 

The FDA has approved 3 drugs that help people with cystic fibrosis.  They were developed using seed funding from the Cystic Fibrosis Foundation –$47 million over 5 years; and $20 million from the Gates foundation. 

The condition is genetic, and recessive.  If both parents are carriers, one of four offspring is afflicted. 

In kids with Cystic Fibrosis the mucous that collects bacteria and foreign particles is not watery, not easily swept out of the lungs and swallowed or coughed up.  It’s thick, “glue like”, and people with the disease have a hard time getting rid of it.  They periodically develop pneumonia, and over time they lose lung function.  A century ago most of the afflicted weren’t aggressively treated like they are now with inhaled bronchodilators, physical therapy, postural drainage, and appropriate antibiotics.  Few survived childhood.

The Cystic Fibrosis Foundation has established 117 centers of excellence.  They are manned by experienced health care professionals and have guidelines, “best” practices, and public monitoring. As a result of their aggressive approach the average person with Cystic Fibrosis now lives an average of 35 years, though getting kids through their teen age years is typically tough. 

Vertex, a biochemical startup that spent $4 billion during its first 22 years without developing an approved drug was not anxious to get into the Cystic Fibrosis business.  There were only 30,000 Americans with the condition.  If the company found a potential drug, it would cost $100,000 to test each person. In the company’s mind the expense of getting involved was “prohibitive.” Richard Aldrich, a deal maker and advisor, thought Vertex should only work with the CF (cystic fibrosis) foundation “if the foundation agreed to fund some of the early stage (drug) development.”

The Vertex research team was headed by Eric Olson.  An experienced research biologist, he was interested in CF.  A Colleague/friend’s daughter had the disease.

The faulty piece of DNA, the cause of the disease, was located in 1989 by a group of Canadians geneticists working with Hong Kong born Lap-Chee Tsui.  The mutation responsible for most cases of cystic fibrosis occurs when three nucleotides are deleted from a gene on chromosome 7.  Called Cystic fibrosis trans-membrane conductance regulator (CFTR), the abnormal gene causes the cell to make a defective membrane protein, one that doesn’t “fold” normally.  Appropriately folded protein regulates the amount of chloride, salt and water that flows in and out, of the cell. The fluid travels through “channels” in the cell’s outer wall or membrane.  In people with Cystic Fibrosis, salt accumulates outside the cell and secretions are thick. Sweat is salty. 

By June of 2011 Vertex had two drugs that dropped the salt content of sweat.  They decreased the exacerbation rate, and the unexplained “worsenings” that contributed to a more rapid decline in pulmonary function.  In the early 2000s Vertex added a third drug.  It had an effect on people who have a “Phe508del  CFTR  mutation.” It’s the most common abnormal gene.  90 percent of people with cystic fibrosis have at least one copy of the mutated DNA.  One analyst felt the triple-drug combo will rake in close to $4.3 billion by 2024.

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The most common genetic cause of death in infancy, Spinal Muscular Atrophy “causes severe weakness by 6 months of age and inability to breathe by the age of two.”  The gene that directs cells to make an essential protein is deleted or mutated.  In the absence of the protein the nerves that send signals to muscles die. 

We all have a second gene that’s similar and that isn’t genetically affected.  But it doesn’t work.  It’s not able to make the needed protein.  And that’s OK because most of us don’t need it.

Scientists at Cold Harbor Laboratory, a large nonprofit research center on the north shore of Long Island developed a “segment of RNA” that, when injected into the spinal fluid, allowed the second gene to make the needed protein. It was a great accomplishment and it wasn’t easy.  The chemical, called nusinersen, was developed with the assistance of researchers at Isis pharmaceuticals, and another for-profit firm, Biogen Pharma paid for the testing.  Once nusinersen was shown to be effective Biogen paid millions and bought everyone else out. When the drug was approved by the FDA its owner decided to charge $125,000 for each dose, or $750,000 the first year and half as much each subsequent year.²⁷ 

          A physician at the University of Utah who cares for “about 150 patients with the disease, complained in an article that if each child was treated with nusinersen, the cost would be $113 million the first year and $56 million thereafter.¹⁰”

In November 2017 an Ohio company developed alternative approach to the problem.  Their therapy was based on research performed at Nationwide, a Columbus Ohio children’s hospital, by Brian Kasper.  An employed researcher, he studied adeno-associated viruses (AAVs).  One day his team discovered a serotype that penetrated the blood brain barrier. There are 50 serotypes of adenoviruses, they don’t usually make people sick, and most can’t get into the brain.

Kaspar and team believed they had “a new way of delivering genes to widespread regions of the central nervous system.  The drug companies they approached allegedly weren’t interested.  So in 2013 with the help of a biotech entrepreneur, Kasper formed a startup, AveXis.  They raised $75 million and licensed the therapy from the Columbus hospital.  To this point all research and development was paid for by the U.S. government and charitable funds.

Researchers placed a gene that promoted the production of the needed protein into an adenovirus.  Then they infused a high dose of the virus that contained the gene into the bloodstream of 12 affected children who were about 6 months old. 

After 1 ½ to two years 11 of the children were able to speak, 9 could sit unassisted for at least 30 seconds, 11 achieved head control, 9 could roll over, and 2 were able to crawl, pull to stand, stand independently, and walk independently.

The startup spent some of its $75 million. I presume that the SMN1 and SMN2 genes in plasmid form were available and could be purchased from Addgene for $65. The scientists had spent years learning how to use a modified virus to transfer genes. Injecting infants and running trials, they gave kids very high doses of a virus that contained the gene they lacked; and the results were pretty good. ¹²

In April 2018 Novartis bought Avexis for $8.7 billion.  After the FDA approved the therapeutic approach, Novartis named the treatment Zolgensma.  In an attempt to recover their multi-billion dollar investment, and make a profit the Swiss set a high price for a treatment.  Novartis will charge each child or their insurer $2.125 Million.²⁹

Currently “there are more than 800 cell- and gene-therapy programs in clinical development; several of these therapies have been approved by the FDA.”—And the science is in its infancy. Some of the treatments on the market are owned by Biomarin, a company headquartered in San Rafael California.  Founded in 1997 the company has acquired 6 biomedical startups in the last 15 years.  In 2016 were marketing 5 orphan drugs.

When a tear occurs in a blood vessel people bleed, platelets plug the hole, and a sequence of proteins pile on.   A clot won’t form if the person’s serum doesn’t contain enough clotting factor 8 or clotting factor 9.  People who genetically don’t make sufficient amounts of either of these proteins have hemophilia.

A genetic condition that “occurs in approximately one in 5000 live births,” hemophilia is sex linked– which means that women carry the gene and their sons get the disease.  When an affected male is injured and his factor 8 or 9 is low he won’t stop bleeding. Joints of men with hemophilia periodically and very painfully fill up with blood.  Over time they develop deformities. 

Victoria, the queen of Great Britain from 1837 to 2001 was a carrier of the hemophilia B gene.  She passed the condition through her daughter Alexandra to her grandson Alexei, the only son of Russian Tsar Nicholas. The couple had 4 daughters.  The boy’s painful and frightening bleeds seemed to be helped by a self proclaimed holy man named Rasputin. 

During the First World War Tsar Nicholas spent a lot of time at the front, and his wife was in charge.  Much to the chagrin of the Moscow elite she seemed to be “under the spell” of the holy man. 

The war went badly for Russia.  Over 5 million soldiers were killed or wounded.  After two years the Russian people had enough and they rebelled.  They deposed the Tsar and Russia withdrew from the conflict.  Hemophilia and the power of the mystic played a big role in 1917 fall of the empire.²⁶

Men whose blood level of clotting factor 8 is at or below one percent have a severe condition.  Those whose blood levels are 5 to 40 percent have a moderate problem and mainly receive factor 8 infusions before surgery or if there is a need.¹⁴ 

In the 1980s, during the height of the AIDS epidemic, small amounts of the factor that stopped hemophiliacs from bleeding was collected from each of hundreds of units of plasma that was obtained from donors.   One usually came from a person who had HIV but didn’t know it.  The young men who received the contaminated factor were given AIDS, a disease that, at the time, was lethal.

Researchers at the University College London recently put a portion of the factor 8 gene into an adeno-associated virus and “infected” a number of men.  With the gene floating in their cytoplasm, cells in the liver made the missing protein. In 6 of 7 patients receiving high doses of genes “factor 8 increased to a normal level and stayed there for a year.  None of the 7 bled during that time.”   After 2 to 3 years the treatment was still providing a clinically relevant benefit.²¹”

10 men with hemophilia B whose blood had less than 2% of the needed clotting factor were infused with an adeno virus containing a replacement gene.  During the subsequent year their clotting levels rose and stayed at a mean level of 33 %, bleeding virtually stopped, and only 2 patients needed a factor infusion. 

Scientists seem to be getting close to solving hemophilia.

As explained on 60 minutes, “Francis Collins of the NIH thinks we can cure sickle cell anemia by using CRISPR gene editing to increase blood levels of fetal hemoglobin (HbF).   Hemoglobin F is the form of hemoglobin that fetuses use to efficiently extract oxygen from the placenta and deliver it to their bodies. Shortly after birth a gene causes most children stop producing Hemoblobin F, and adult hemoglobin takes over.   

People with Sickle Cell disease have a genetic abnormality that affects adult hemoglobin. Red cells that should be round and flexible start looking like sickles or crescent moons.  They clump, stick in small blood vessels, and cause severe pain, anemia, stroke, pulmonary hypertension, organ failure, and far too often, early death.

Researchers at Vertex and CRISPR Therapeutics collected stem cells from a person with severe Sickle cell disease.  In the lab they used CRISPR to destroy the gene in the stem cells that shuts down production of fetal hemoglobin. Then they destroyed the remaining bone marrow with chemotherapy and infused the edited cells into the patient.²³ It seemed to be working.¹⁸

CRISPR derived gene therapy is new, exciting and not fully developed, but will be widely used in future gene editing. The investigators who did many of the studies and developed the concept were publically funded and were—at the time—trying to learn how bacteria defend themselves from assaulting viruses.  They were trying to understand CRISPR.

The following theory of how CRISPR came into being helped me understand the process: When a virus assaults a bacteria, the invader enters the cell, takes over its DNA, and directs the bacteria to make billions of viral particles. 

Most bacteria are enslaved, then destroyed.  A few mount a defense and survive.  Some of the survivors create a “DNA memory file.”  The identifying characteristics of the bad viruses are stored in the DNA’s CRISPR area, and the memory–sequence becomes a “gene” that is passed on to future bacteria.  In subsequent generations the memory DNA creates strands of RNA that float around inside the bacteria.  When a segment of RNA recognizes an invading virus it latches on.  Then it cuts the virus apart with an enzyme called Cas9.

After they understood how bacteria identify and destroy unwanted viruses, researchers tried to use the system to edit genes.  They chose a target–a “twenty-letter DNA sequence” that was part of the gene they wanted to delete. Then they “converted” a collection of nucleotides “into a matching 20 letter strand of RNA.”  This RNA was an exact replica of the DNA.  It would guide the Cas-9 past the cell’s 3 billion pairs of DNA nucleotides and it would eventually identify the desired strand of DNA.

They planted the genetic instructions for making Cas9—the knife—into one plasmid. They put the genetic instructions for “guide RNA” –-into a second plasmid.

Their concoction was able to search a cell’s DNA—3 Billion pairs of nucleotides—and find the desired segment of DNA–and unwind the DNA –and use Cas 9 to cut apart the strand of nucleotides.  In other words they could irreparably damage a chosen gene. 

Cells know how to repair a break in their DNA. The cut ends either come together on their own.– Or the gap can be bridged by a segment of DNA.

The study group was led by UC Professor Jennifer Doudna and Emmanuelle Charpentier.  Doudna, the daughter of a professor of English, grew up in Hawaii and spent the summer that followed her college freshman year in a lab studying a fungus that was invading papayas.  “It turned out to be a lot of fun”, she hungered for more, worked in a few labs, made a few discoveries.  After a decade she became the head of a research lab at the University of California in Berkeley, a campus that was often blanketed in fog, but on clear days provided a spectacular view of the Golden Gate Bridge and the Pacific.

Doudna met Emmanuel Charpenier, a French professor who was working in Sweden, at a 2011 conference in Puerto Rico.  Described as “Small and slight, with eyes so dark that they seem black” Charpentier was a PhD student at the Pasteur Institute in Paris when she “realized she had found her environment.” Once hooked on the life she spent more than 20 years performing research in 9 different institutes in 5 different countries.²⁹

The two women discussed possibly collaborating while they explored the narrow cobbled lanes of old San Juan.   

In June 2012 Doudna and Charpentier published a study that showed how RNA and Cas9 could be used for “site-specific DNA cleavage and RNA-programmable genome editing”.  Investigators around the world took notice and got busy.

Scientists already knew how to add a new, good gene.  Mario Capecchi, years back learned that genes intuitively know where, amidst the 3 billion pairs of DNA nucleotides, they belong.  If good genes are developed and put into cells, they migrate and attach to “their place.²².” 

After Doudna’s paper was published Kevin Esvelt (currently at MIT) explained how using CRISPR + selfish genes in the germ line can create changes that will be inherited by future generations of cells.

Sensing that there wasn’t time to write grants and get government funding, Doudna and other scientists formed a venture capital company– Editas Medicine.  Charpentier and others founded CRISPR therapeutics.  Both firms, according to their web sites, are trying to cure Sickle Cell disease, cystic fibrosis, and a few other genetic conditions.¹⁹

Medical thinkers have argued that:  given our current experience with the price of drugs we should start thinking about how we’re going to pay for “future success in gene therapy.”²⁰