The Resilience of Monoclonal Antibodies and their Makers

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In 1975, Georges Köhler and César Milstein, biochemists from the Medical Research Council Laboratory of Molecular Biology, published a paper in Nature about a hybridoma technology that enabled the production of large numbers of monoclonal antibodies.¹ Their discovery ushered in a new era in the field of antibody research.

For decades, researchers fixated on the extraordinary potential nestled within the human immune system, particularly the formidable prowess of antibodies to combat cancer. Since monoclonal antibodies are highly specific for a single antigen, researchers wanted to leverage this ability to specifically target cancer cells. However, at the time, their pursuits to use these small but mighty proteins ran into major limitations: supply and shelf life.

Typically, researchers injected mice with an antigen to provoke an immune response from B cells that secreted antibodies against the target antigen. However, researchers struggled to make large quantities of antibodies and maintain their stability due to their inherent short half-lives. Fusion unlocked the key to overcoming these limitations. Köhler and Milstein fused the antibody-producing plasma cells with immortal myeloma cancer cells. This hybrid cell line had the antibody-producing ability of a B cell and the immortality of myeloma cells.

This discovery inspired researchers to embark on a quest to harness antibodies to fight cancer. It laid the foundation for monoclonal antibody research but posed a daunting challenge: How can monoclonal antibodies be developed as cancer therapies? 

Laying the first monoclonal brick

Just as Rome wasn’t built in a day, the first monoclonal antibody treatment for cancer developed over the decades that followed, one brick at a time. 

“Everybody’s running to put bricks down. But we didn’t know where to put the bricks, so we’d put up a wall here and then a wall there,” recalled Lee Nadler, now a physician at Dana-Farber Cancer Institute. “I believe I put some good bricks down.”

I told [my boss], I was going to make monoclonal antibodies. He laughed at me.

 —Lee Nadler, Dana-Farber Cancer Institute

Nadler recalled a moment in the late 1970s that changed the trajectory of his career in medicine. He and a friend sat in a rowboat talking about a hybridoma paper they had just read. The discovery of monoclonal antibodies and their potential to change medicine greatly excited them. This inspired Nadler to try making an anticancer antibody.

When he started as a clinical fellow at Dana-Farber Cancer Institute, he aimed to turn his vision into reality and make a targeted therapy for lymphoma. “I told [my boss], I was going to make monoclonal antibodies,” said Nadler. “He laughed at me.” Undeterred, Nadler pressed forward. 

Initially, Nadler struggled to get lymphoma samples for monoclonal antibody production. He recalled standing around in the pathology suites begging for samples. One afternoon, Nadler received a call that changed everything. Five bottles of abdominal fluid, called ascites, from a young boy with Burkitt Lymphoma sat in the garbage; if he could get there before the garbage collectors picked it up, it was his.

Nadler rushed to the suite and put away enough material to make monoclonal antibodies. Although the boy’s cells did not lead to the first antibody for treatment, they helped kickstart Nadler and his colleagues’ journey toward making monoclonal antibodies.

Nadler received another call that a lymphoma patient had exhausted treatment options. Nadler and his team agreed to generate an antibody directed against a human lymphoma-associated antigen for this patient as a last resort. Eight months after that phone call, they treated the patient. “I’m not exactly sure how we ended up with a fairly specific antibody, but we did,” said Nadler. They designated this monoclonal antibody as antibody (Ab) 89.

         

Once Nadler wrote a protocol, all it took was his physician chief’s signature. Nadler became the first person to administer monoclonal antibodies to a patient.² “That was the most shocking thing of my life. I understood how to make monoclonal antibodies, but now I was treating somebody,” recalled Nadler.

The patient had the tumor circulating in his blood, and once injected with the antibodies, the number of tumor cells only dropped marginally. Nadler was perplexed. He increased the dose, expecting to find the antibodies on the surface of the patient’s cells but found nothing. He drew out some of the tumor cells, radioactively labeled them, put the antibodies on the cells, and injected them back into the patient. Only some of the tumor cells died. It turned out that the patient’s serum contained a circulating antigen that specifically blocked the reactivity of Ab 89.³

While the trial didn’t produce significant clinical results, it demonstrated the safety of laboratory-made monoclonal antibodies and their capacity to decrease the amount of antigen in the patient’s blood. Optimistic about the results, Nadler went on to identify several antigens from that initial trial. At a workshop, he designated the antigens as clusters of differentiation (CD) 19-23, to characterize malignant B cells. He was ecstatic to have antigen targets to develop an array of antibodies. Of these, CD20, originally named B1, became a promising target as a specific B cell marker.^4-6 In the years that followed, Nadler and other researchers continued to lay down bricks that brought them closer to a medical breakthrough with CD20. 

The early success of monoclonal antibody therapy

Over 3,000 miles away, Ronald Levy, a physician and researcher at Stanford University, wondered how he could take his own research into the clinic. He generated monoclonal antibodies by adopting a technique that used mouse spleens to develop specific antibodies. However, he ran into the same challenges as other researchers with the limited supply and short shelf life of antibodies. So, he was ecstatic when the landmark hybridoma technology paper was published in 1975. “There was a land rush for finding the best monoclonal antibody,” recalled Levy. “The world opened up for us at that point.”

         

Instead of focusing on finding antibodies that target the tumor specifically and don’t cross react with anything else, Levy went a completely different route from Nadler. Together with his team, including Richard Miller, then a fellow in oncology, and David Maloney, then an MD-PhD student in Levy’s laboratory at Stanford University, he set out to create anti-idiotype, or patient-specific antibodies.⁷ 

“I wanted to learn [monoclonal antibody] technology and to use it. I was really interested in the therapeutic angle,” recalled Miller. Idiotypes, a part of the immunoglobulin that distinguishes it from one person to another, presented a promising target for lymphoma. Maloney and Miller spent much of their time in the laboratory generating large quantities of antibodies while trying to outpace their patients’ cancers. It was a waiting game for the perfect conditions to maximize therapeutic benefits.

         

“No one thought it would work. In fact, it was common knowledge that it wouldn’t work. Antibodies would never be therapeutic agents,” said Levy. However, one of the most memorable moments happened with Phillip Karr, a patient who suffered from B cell lymphoma.⁸ 

On that day, Miller recalled that things looked perfectly aligned for treatment. Karr’s cancer was progressing and was no longer responding to the chemotherapy treatments. Sensing the urgency of the situation, Miller went to the chief of the division and made a case for this experimental trial. 

“He said to me, ‘Look, you can do what you want, but I wouldn’t do it,’ recalled Miller. But Miller walked out of the office with a steely resolve. Miller treated the patient with a very low dose of anti-idiotype monoclonal antibodies, and slowly increased it over time. Soon, the patient got better; his lymph nodes shrunk; and his platelets and blood counts improved. The patient remained in remission for 30 years after this trial. This was a huge success for monoclonal antibody therapy. “It was a proof of concept for the problem. With this approach, it was one antibody that was only good for one patient. You had to make an antibody for essentially every patient,” explained Maloney. 

No one thought it would work. In fact, it was common knowledge that it wouldn't work. Antibodies would never be therapeutic agents.

 —Ronald Levy, Stanford University

Trials and tribulations of rituximab

Following the initial success of the therapy, Levy and Miller started IDEC Pharmaceuticals to continue making their custom antibodies. Scalability became a challenge as the manufacturing process was technically challenging, slow, and expensive. They hunkered down to rethink their strategy. Instead of a patient-specific antigen, they needed to find a more generic antigen. 

When Nadler ran into Levy and Miller at a conference, he recalled suggesting, “You’re going about this all wrong. Take this antibody that I had made called B1, which is CD20, and make it, and use it as a drug.” 

CD20 was widely expressed on both healthy and cancerous B cells, but not on immature, developing B cells. Thus, targeting CD20 would eliminate healthy and cancerous cells, and the remaining immature B cells would replenish the supply of healthy B cells. 

Initially, Levy had his doubts. He worried about the safety of using a pan-B-cell-depleting agent to treat cancer, as it might kill all B cells and leave no chance for B cells to grow back. He passed on the idea. Later, the rights to CD20 were sold to Coulter Pharmaceuticals. 

Eventually, Levy and his team reconsidered CD20 as a potential antitumor target. IDEC Pharmaceuticals’s initial customized approach was circumvented by the work to develop anti-CD20 antibodies. Like other researchers, they derived their monoclonal antibodies from mice. So, to reduce the immunogenicity of mouse antibodies in human patients, they needed to find a method for humanizing the antibodies. 

The magic moment was a breakthrough in the mid-1980s with recombinant DNA technology.⁹ This method generated new antibody molecules with the specificity of mouse antigen binding domains and reduced the likelihood of triggering an unwanted immune effect by including human constant domains.

This drug could have died on the vine. That really shows you that sometimes things can happen with a lot of good results but also a lot of luck. 

 —Ronald Levy, Stanford University

With this technology, IDEC Pharmaceuticals researchers identified a hybridoma (2B8) that recognized CD20 and then developed a chimeric antibody (C2B8). This chimeric antibody was the combination of the mouse antibody fused to a human constant region and was a promising drug. Before clinical trials, they conducted studies in macaques and found B cell depletion.¹⁰ The C2B8 antibodies targeted CD20 expressing B cells in the blood and lymph nodes.

Then they conducted the first clinical trial of rituximab, initially named IDEC-C2B8, for 15 non-Hodgkin’s lymphoma patients.¹¹ The results surprised the team because they observed some patients responding to a single infusion of the antibody. Even when they scaled the dosage from ten to 500 mg/m² of antibody administered intravenously, there was minimal toxicity and clear tumor regression in six of the 15 patients.

The data excited Levy, and he wanted to get rituximab onto the market. There was one problem. The company needed more funding, and Levy wanted to find someone to invest in this product. Despite initially passing on the proposal, IDEC Pharmaceuticals partnered with Genentech to develop the drug together. “This drug could have died on the vine,” remarked Levy. “That really shows you that sometimes things can happen with a lot of good results but also a lot of luck.”

The fortuitous collaboration continued to yield benefits. In a second trial, they established a dose that worked for patients; nearly half of the patients entered clinical remission.¹² The antitumor effects became evident, leading to the approval of rituximab for medical use by the Food and Drug Administration in 1997.

Monoclonal antibodies for cancer and beyond

Despite years of naysayers and skepticism, researchers’ seemingly radical vision of developing monoclonal antibodies for therapy became a reality. Rituximab was the first monoclonal antibody treatment for non-Hodgkin’s lymphoma. “Rituximab was the first sort of protein recombinant product that was really, really successful in medicine,” said Miller. Because it didn’t affect other normal cells in the body, rituximab could be combined with other therapies and paved the way for new discoveries for targeted therapies using antibodies.¹³ 

Since then, the techniques for developing monoclonal antibodies evolved. The use of recombinant DNA led to the development of more chimeric monoclonal antibodies and later extended this strategy to humanize monoclonal antibodies. In tandem with hybridoma technology, phage display techniques also propelled monoclonal antibodies such as adalimumab to the therapeutic forefront.

These continuous improvements enabled researchers to finetune immunologic effects and increase specificity for patients with a wide range of ailments, from cancer to autoimmune disorders. Monoclonal antibodies have a staying power in therapeutic treatments and their versatility is evident; they work well as standalone treatments or in combination with other treatments, such as chemotherapy and biologic agents. 

The pursuit of making monoclonal antibodies continues to evolve, ushering in a new era of research and therapeutic possibilities. “Everything in science is chance, luck, and passion,” said Nadler. “People told me the work I was doing was a waste of time, but we got the inkling that we could make monoclonal antibodies work, and we did.”