Structural Biochemistry/Rituximab

Brief History
Finding a cure for cancer has always been a goal for many health care professionals. Many have tried, but few have made as much of a stride as Dr. Antonio Grillo-López. He, along with several colleagues, pioneered a new drug named rituximab that serves as the first FDA approved antibody to treat cancer. Depending on the severity of the cancer, this drug could either completely treat or extend the lifetime of the patient. This extraordinary feat had very humble beginnings.

The Man Behind the Drug
Dr. Grillo-López has his roots in Puerto Rico. Growing up, he received both his Bachelor of Science and Doctor of Medicine degrees at the University of Puerto Rico, San Juan. He served as an associate professor at the University of Michigan from 1980 to 1990. He was also a part of DuPont-Merck Pharmaceuticals Parke Davis. He was later offered a position at, what was then, a start-up company named IDEC. Dr. Grillo-López acted as Chief Medical Officer from November 1992 to January 2001 at the company. Later, he acted as Chief Medical Officer Emeritus from January 2001 to November 2003. It was at this company where rituximab came to fruition.

About the Drug
Rituximab is a liquid drug that is administered into patients’ veins. It is used in the treatment and care of patients with non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis. Dosage can vary depending on the desired treatment. For example, in rheumatoid arthritis patients, it is given as two doses two weeks apart. But in non-Hodgkin’s lymphoma, it is given once a week for four to eight weeks. In the United States, it carries a trade name of Rituxan. In Europe, it is offered as MabThera. The main focus will be on non-Hodgkin’s lymphoma, or NHL.

NHL is a term that encompasses all lymphomas except for Hodgkin’s lymphoma. Hodgkin’s lymphoma is distinguished by the presence of binucleate giant cells known as Reed-Sternberg cells. Lymphoma is a cancer of the lymphocytes in blood. Lymphocytes are a type of white blood cells that serve an integral part of the human immune system. They are classified into B cells, T cells, and natural killer cells. In NHL patients, an antigen known as CD20 is found on B cells. The rituximab antibody binds to the CD20 antigen and destroys excess, overactive, or dysfunctional B cells as a result of the disease.

Attacking the Cell
Rituximab kills cancerous B cells via three main mechanisms: complement-dependent cytoxicity (CDC), antibody-dependent cell-mediated cytoxcity (ADCC), and apoptosis. CDC occurs when a large group of plasma proteins (complement) work together to destroy invading pathogens and malignant cells. Antibody-antigen complexes, such as the one between rituximab and CD20, activate the complement. The complement protein C1 binds to the tail of the rituximab antibody in a “lock and key” fashion and starts a series of reactions that creates a membrane attack complex lining the B cell membrane and then creating a pore to allow the cellular contents to escape and eventually die. ADCC is a process where the antibody-antigen complex forms and then attracts other components of the immune system, including natural killer cells. The receptors on these cells recognize and bind to the tail of the rituximab antibody. The natural killer cells also carry granules filled with cytotoxic molecules. When the granules are released after the natural killer cells bind with rituximab, they penetrate the cellular membrane of the B cell and cause pores that facilitate the release of cellular content leading to the cell’s death. The granules can also destroy the cells by attacking the nucleus. The final mechanism is known as apoptosis, or programmed cell death. Apoptosis is defined the death of cells that occurs as a normal and controlled part of an organism’s growth or development. When rituximab bonds to CD20 and forms the antibody receptor complex, it signals the cell to start the process. The cytoskeleton collapses upon itself, the nucleus condenses, and the DNA fragments into small pieces via enzymes. Membrane-bound vesicles are also shredded. At the end of apoptosis, the cell has effectively destroyed itself. It is still unclear, however, whether these mechanism act independently or in concert. Despite this, rituximab still proves to be an effective cure.

Experiments Involved
The rituximab antibody was created after several experiments involving mice. Three mice were injected with cells from a lymphoma tumor. These malignant cells carried the CD20 antigen that is found only on B cells. Observations revealed that the mice’s immune system had produced antibodies to fight these foreign substances. The tail of the antibody is humanized. Mice and human gene sequences were used together to create the chimeric antibody that is rituximab.

Pathway to Success
Dr. Grillo-López accredits much of his success to the 1908 Nobel Prize winner Paul Ehrlich. The late Paul Ehrlich was a German scientist who had experience in the fields of hematology, the study of the physiology of blood, and immunology. His work in the latter field gained him the joint Nobel Prize in Physiology or Medicine in 1908 with Ilya Ilyich Mechnikov. Ehrlich popularized the concept of a drug that would act as a “magic bullet.” This meant that a drug would be able to selectively target a disease-causing organism. This way a toxin could be made to be delivered to only that organism. This is the basis of Dr. Grillo-López’s work. The rituximab works by selectively binding to the CD20 antigen and forming the antibody receptor complex that is a vital part of the mechanisms used to destroy infected B cells. Ehrlich had said that success in research is only attainable with several big “G’s”. In German, these are Geduld, Geschicklichkeit, Glück, Geld, and Geräte. These translate to persistence and determination, skill, luck, money, and tools, respectively. The story behind the approval process for this cure shows the influence Ehrlich had on Dr. Grillo-López.

Speed Bumps along the Road
The U.S. Food and Drug Administration, or FDA, is an agency that is responsible for protecting public health by passing and enforcing laws and regulations regarding products such as foods and drugs. All drugs must go through their approval process. Dr. Antonio Grillo-López likens them to the pace of snails and considers them to be “the single most important obstacle to the timely development and approval of anti-cancer agents.” He cites examples such as the five-month-review to simply approve the proposed name for rituximab. PanThera was not approved and had to be revised to Rituxan for the U.S. markets and MabThera for European markets. During the pre-digital age, where digital books and social networking site were unavailable, everything had to be done manually. Dr. Grillo-López and his IDEC colleagues, Chet Varns, Alice Wei, and John Leonard, had to send in boxes upon boxes to the FDA for them to review and approve the use of rituximab. In the data set, there was one patient who the group at IDEC considered to not be evaluable due to some minor errors. To present an obstacle to the approval process, an FDA medical reviewer asked them to consider that patient as evaluable now. This only worked in favor of the drug creators because that particular patient had a complete response to the drug. This increased both the overall response rate (ORR) and complete response (CR) rate. The team sent in their biologics license application and it was finally approved after a slow nine months on November 26, 1997. After this, Dr. Grillo-López and his colleagues still faced some obstacles such doubts that a proposed study of using rituximab as part of a combination regimen known as R+CHOP instead of the traditional method of using antibodies following chemotherapy, not in conjunction, would be effective. Despite these doubts, the combination regimen proved to be a cure for NHL. Dr. Grillo-López was persistent and determined according to the philosophies of Paul Ehrlich, leading to his success in finding an effective cure for cancer.

End Result
Several milestones were achieved during its development. Clinical trials were completed in record time by patients who would volunteer and even dress up as a mouse, harkening back to the chimeric nature of the antibody. Ritxuimab became the first FDA approved antibody to treat cancer. Dr. Grillo-López even paved the way for quicker FDA approvals. Before, the FDA would take an average of seven years to approve a drug that many patients needed sooner. He was able to reduce that approval timeframe in half. The 2004 Discovery Health Channel’s Medical Honors recognized some of these achievements. Rituximab’s development did not rely on government support. Investors and partners funded the entire project. No federal or state grants or incentives or research funding were involved either.

Impact
The impact that rituximab had is unprecedented. It provides a cure for curable lymphomas such as diffused large cell lymphoma. The lifetime of patients with incurable lymphomas have been extended. Recent numbers further demonstrate the success of rituximab. It has been considered the top anticancer drug in the world since 2001. Sales in 2010 alone totaled $6.7 billion. Each year, around 50,000 lymphoma patients are cured. Since its introduction in 1997 until 2010, over two million patients have been treated. Prior to this discovery, there has been a long of stagnation in finding cures or ways to extend lifetimes. Hopefully the success of Dr. Antonio Grillo-López will inspire others to follow in his footsteps. He, himself, has said that he is neither a saint nor a magician. Geduld led to his success, something that can be replicated by any ordinary person with persistence and determination.