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Building a drug

Ovarian tumor antibodies developed by a Brazilian company and Brazilian research institutions are ready to be tested on humans

Self-guided missiles: artistic representation of B lymphocyte, cell of the immune system that produces antibodies (Y-shaped molecules)

TIM VERNON / SCIENCE PHOTO LIBRARYSelf-guided missiles: artistic representation of B lymphocyte, cell of the immune system that produces antibodies (Y-shaped molecules) TIM VERNON / SCIENCE PHOTO LIBRARY

By the end of 2014 six women who have had surgery or undergone chemotherapy for ovarian cancer at Gothenburg University Hospital, located in Sweden, will have received a treatment booster using an experimental compound developed by Brazilian research institutions and a Brazilian company. It will be the first time a version of this compound, a monoclonal antibody with high affinity for ovarian tumor cells, specifically produced for use in humans, will be used in a clinical trial. Dr. Ragnar Hultborn, an oncologist, and his team in Gothenburg have already obtained approval from the Swedish health authorities to give patients low doses of the antibody, to which a radioactive chemical element will be added. With this support therapy, they hope to eliminate tumor cells that might have escaped initial treatment or had already spread even before the problem was diagnosed.

If it works, this strategy can prevent malignant cells in the ovaries from reestablishing or migrating to other organs. Ovarian cancer is about 10 times less common than breast cancer—the most common cancer among women and the second most prevalent of all cancers—but it is proportionally more deadly because it is often identified late. “We believe that cancers are usually deadly because at the time of the initial diagnosis tumor cells have already spread throughout the body,” Dr. Hultborn said in an interview with Pesquisa FAPESP at the end of August 2014. “By attaching a radioactive element to specific antibodies for a particular tumor, our goal is to find and eliminate malignant cells wherever they are.”

In its first test in humans, this monoclonal antibody, now known as RebmAb200, will work primarily as a means of transport. It will be a kind of microscopic self-guided missile carrying a nuclear warhead—in this case the chemical element astatine—to the cells targeted for destruction. But we know that’s not all it can do. Experiments on animals and human cells conducted in Brazil have shown that this antibody kills tumor cells even without radiation. As it binds to cancer cells, the antibody triggers lymphocyte exterminators, also known as natural killers, which are cells of the immune system that bathe the tumor in toxic substances (see Infographic). According to Dr. Maria Carolina Tuma, director of research and development at Recepta and responsible for coordinating the RebmAb200 development project, preliminary results obtained by the company’s researchers using animal models suggest that this activity reduces the rate of tumor growth.

The clinical trial now under way in Gothenburg is another important step in the long approval process for a candidate compound before it becomes a drug. After a long battery of tests with human cells and tissues and rodent experiments conducted in Brazil and Sweden, this is the first clinical phase assessment of whether the antibody is safe to be administered to humans as a therapeutic agent. It is also at this stage that potential unwanted side effects begin to be identified. These measures are necessary before proceeding to the next stages of evaluation, with more patients, and, if all goes well, placement on the market. If the results are good, Dr. Hultborn plans to carry out a larger study in 2015 or 2016 with about 100 women to evaluate the efficacy of the procedure.

Regardless of the clinical results, there are reasons to celebrate. Developing RebmAb200 allowed Recepta to master from start to finish one of the most complex and laborious stages of monoclonal antibody production: obtaining cell lines with the ability to produce, in large quantities and with the same degree of quality and stability, antibodies to be used in human beings—the so-called humanized antibodies, which have a lower risk of causing allergic reactions. This result, described in a 2013 article published in the journal PLOS One, was recognized in August 2014 by the Octavio Frias de Oliveira Award in the technological innovation category. The award is sponsored by the São Paulo State Cancer Institute (Icesp) and the Folha Group, which publishes the newspaper Folha de São Paulo.

“Mastering how to generate a stable and efficient cell line that produces monoclonal antibodies eliminates a technological hurdle in Brazil,” says Dr. Roger Chammas, a professor of oncology at the University of São Paulo School of Medicine (USP) and a researcher at Icesp. Dr. Chammas and his team recently participated in one of the RebmAb200 testing stages. Using another radioactive material, technetium 99, Luciana Sousa Andrade, a biologist, examined how the antibody is distributed in the body after being injected into the bloodstream of rodents. “We saw that it focuses on ovarian tumor cells but not on the healthy ones,” says Andrade, a former member of the Recepta research team.

016-021_anticorpos monoclonais_223-01Some oncologists believe RebmAb200 is a candidate drug for treating certain ovarian tumors and also certain types of lung, kidney and breast cancer, as it is an innovative molecule and not biosimilar to an existing drug.

“Development of this innovative molecule is a landmark step in the field of biological products in Brazil,” says Carlos Gadelha, an economist and the Secretary of Science, Technology and Strategic Inputs for the Ministry of Health. Brazil long ago mastered the technology of vaccine production, but it has yet to develop a completely innovative biological medicine (biopharmaceutical) that could reach the commercial development stage. According to Gadelha, expanding the capacity for technological innovation to produce biopharmaceuticals across the healthcare field is a strategic issue for Brazil. “Health is one of the three or four areas that will determine whether Brazil is to be one of the strategic players in the global economic scenario or just play a subordinate role,” he says.

A long journey
The fact that RebmAb200 has reached the clinical trials stage can be considered a significant victory in biotechnology development. It is the result of an unusual partnership model between the public and the private sectors. Its development involved the interaction of teams from public institutions engaged in research, such as the Butantan Institute and USP, and that of a private technology company, Recepta Biopharma. The company, by combining its in-house know-how with that of academic centers of excellence, was able to coordinate the effort and achieve this result. Funding for the purchase of equipment and the work of these groups was also provided by two sources: public, through FAPESP (approximately R$3 million) and the Brazilian Innovation Agency (R$6.2 million), and private, capital investments by Recepta (R$5.8 million).

The long journey leading to the antibody being tested in Sweden began nearly 10 years ago, when José Fernando Perez, a physicist/engineer and former scientific director of FAPESP, left his position as head of the Foundation and created Recepta with two private investors; their goal was to develop innovative biopharmaceuticals. As the company’s head, Perez put into practice his ability to act as an organizer, by identifying professionals of great technical ability and creating partnerships.

016-021_anticorpos monoclonais_223-02He recruited Dr. Ana Maria Moro, an immunologist of the Butantan Institute, who worked with monoclonal antibodies and had already produced at the institute murine antibodies used in the clinic to reduce the risk of acute rejection in kidney transplants. “I received a call from Perez in February 2005 asking if we could arrange a meeting, if possible, for that same day,” recalls Dr. Moro. “In April I was already in New York attending meetings at the Ludwig Institute for Cancer Research (LICR).”

She helped Perez to analyze the murine antibody portfolio that had already been obtained by the Institute. Along with Dr. Oswaldo Keith Okamoto, a biologist, and Dr. Oren Smaletz, an oncologist, they staked a claim to four antibodies that appeared to have the most potential to become cancer drugs; Recepta obtained an exclusive international license to them for research, development, clinical trials and, if they become drugs, their commercialization (see Pesquisa FAPESP Issue No. 137). In 2009, Dr. Tuma, with her experience working on antibodies at an American company, took over leadership of the project, coordinating multidisciplinary and multi-institutional research, which, she said, was a big challenge.

Although murine antibodies have been around for quite some time, it is not easy to get the humanized version. Obtained by injecting human tumor cells into mice, murine antibodies generally cannot be used in a patient more than once. Because these antibodies are foreign to the human body, they carry the risk of activating a severe immune response, which in extreme cases can lead to death. The technical and technological advances that have enabled the development of humanized antibodies are relatively recent, begun less than 40 years ago, and the first monoclonal antibody approved for human cancer treatment was generated only 15 years ago.

The first antibodies were discovered in the late 19th century by the German physician Paul Ehrlich, who identified their characteristic property: the ability to bind to a specific target. This property, which led Ehrlich to dub them the “magic bullet,” would be successfully used to fight tumor cells.

But decades of advances and setbacks were needed before antibodies that fight tumors could be produced in a controlled manner. Antibodies are proteins that cells of the immune system produce to neutralize molecules that are foreign to the body. They are much larger, more complex and difficult to produce than drug molecules, which are generally made ​​by chemical synthesis. To produce antibodies, it is necessary to use cells as bio-factories. And the antibody produced by a cell may differ from one generated by another of the same variety. Antibody-producing strains, which are difficult to obtain, need to be able to reproduce indefinitely—a characteristic they share with tumor cells.

Almost identical: antibody-producing cells, seen through a microscope

INSTITUTO BUTANTANAlmost identical: antibody-producing cells, seen through a microscope INSTITUTO BUTANTAN

In the 1970s two immunologists, Georges Köhler of Germany and César Milstein of Argentina, created the first successful strategy to generate antibody biofactories capable of living for many generations by fusing murine tumor cells with the antibody-producing cells (B lymphocytes) of rodents. This strategy, which won them the Nobel Prize in Physiology or Medicine, has led to genetically identical cells or clones able to produce large numbers of antibodies.

But they did not always work as expected. Only a decade later would advances in genetic engineering improve the likelihood of obtaining antibodies that preserve the desired affinity for specific cells and are not recognized as foreign by the human immune system. “The development of therapeutic antibodies involves an in-depth knowledge of cancer serology, protein engineering techniques, mechanisms of action and resistance and the interaction between the immune system and cancer cells,” wrote Andrew Scott, Jedd Wolchok and Lloyd Old, Ludwig Institute researchers, in a review of cancer therapy with antibodies, published in 2012 in Nature Reviews Cancer.

The first monoclonal antibody to become a drug to treat human cancers was rituximab, used against certain types of blood cancers, such as lymphoma and leukemia; it binds to a protein found in large amounts on the surface of immune cells called B lymphocytes, which are continuously multiplying. The U.S. Food and Drug Administration, the agency that regulates drugs in the United States, approved its use in humans in 1997. Since then, just over a dozen monoclonal antibodies have received the green light from the agency for use against cancer.

They are expensive drugs, which are usually paid for in Brazil by the public health system. The price of a single dose of a monoclonal antibody ranges from about $1,000 in the case of older drugs, to $26,000 for the more recent generation of drugs. Their use is generally justified because they act on tumor cells in a more targeted way than chemotherapy drugs, which usually not only eliminate cancer cells but also healthy cells, causing severe side effects.

The therapeutic performance of these antibodies against specific groups of tumors—in addition to, of course, their cost—justifies Brazil’s effort to assume at least part of their production, even if Brazil is a little more than a decade behind richer and more technologically developed countries.

Clone holders: containers with the monoclonal antibody-producing cell cultures

Léo RamosClone holders: containers with the monoclonal antibody-producing cell culturesLéo Ramos

Combining know-how
With the legal transfer of these antibodies to Recepta, Perez set up a collaboration with Dr. Moro, through an agreement with the Butantan Foundation, to develop a cell line to produce a humanized version of the antibody now known as RebmAb200. The murine version of this antibody, called MX35, had been obtained by Ludwig Institute researchers in the 1980s. The Institute itself provided the information obtained through computer modeling and needed to humanize the antibody; it had to meet two characteristics: maintain the affinity for ovarian tumor cells typical of the murine version and be compatible with human antibodies, which reduces the risk of allergic reactions. “The first characteristic can be evaluated in trials in the laboratory, but the second can only be known after testing in humans,” says Dr. Moro, who is the coordinator of Butantan’s Monoclonal Antibodies Laboratory.

Lilian Tsuruta, a pharmaceutical chemist and former member of Recepta’s team working in this laboratory, had a seemingly simple mission: to humanize antibodies. Based on the sequence, she drew the genes and commissioned the German company Geneart to do the synthesis. The two synthetic genes contained the recipe for the humanized version of the antibody, a molecule in the form of the letter Y which recognizes, and binds to, specific proteins abundant on the surface of tumor cells. Tsuruta’s work was to cut these genes from the mold sent by Geneart and paste them into a DNA framework that could be incorporated and expressed by cells chosen to produce antibodies. “It took nearly six months of laboratory work, through trial and error, until I found the optimal situation,” says Tsuruta.

The next step, which fell to Mariana Lopes dos Santos, a biologist and former Recepta researcher working on Dr. Moro’s team, was to make this strand of DNA reach the nuclei of such cells. A line of human cells, which preserve the ability to reproduce for many, many generations, was selected. It is a long and slow process, which, in addition to great skill, requires a certain amount of luck. Santos placed a mixture containing tens of thousands of cells and millions of copies of the antibody genes into containers of just one cubic centimeter, less than the size of dice used in many games. She then gave them a slight electrical charge and waited almost a week to find out if they had been incorporated into the genetic material of the cells. It is an inefficient process based on chance.

“When everything goes right, only about 30% of the cells incorporate the genes,” says Santos, who now works for the Butantan Institute and is the lead author of the article in PLOS One. The genes often do not fit correctly into the genetic material of the cells, which thus cannot produce the antibody. In general the optimum is to select only a very few cells that incorporate many copies of the gene. Once identified, Santos separates them into different containers. Each is a clone, which will be multiplied over several generations, as the researchers evaluate, through a series of tests, their ability to produce antibodies effective in high quantity and uninterruptedly.

Of the 210 clones Santos obtained, less than a dozen were efficient enough, able to produce antibodies against tumor cells. The most effective of all was clone number 105, which she happened to generate on Independence Day, Monday, September 7, 2009, when despite the holiday, she was in the laboratory. As they were produced and purified, the antibodies began in vitro testing to see if they preserved the ability to bind only to tumor cells and kill them.

Strategic vision
While the production stage of clones was being mastered at Butantan, Dr. Venancio Alves Ferreira, a pathologist, and his team worked at USP’s School of Medicine to help define against which types of cancer the antibodies licensed by the Ludwig Institute could work. Through the Medical School Foundation, Recepta engaged the group led by Dr. Ferreira to set up a biobank. It contained samples of normal and tumor cells from 33 organs extracted from over 2,000 patients arranged into 347 microarrays. This allows simultaneous testing of up to 200 samples. This data, partially published in 2012 in the journal Applied Immunohistochemistry and Molecular Morphology, reinforces the robustness of the evidence for clinical use of antibodies.

Tests done with the MX35 murine antibody and the RebmAb200 humanized version obtained by Recepta researchers at Butantan confirmed that both have a high affinity for ovarian tumor cells. Ferreira tested these antibodies on samples of 38 ovarian tumors. In 30 of them (about 80%), more than half of the cells expressed high amounts of the protein recognized by the antibody. “If RebmAb200 proves to be safe and effective in clinical trials, it may become easier to get international registration of the drug for treatment of an orphan tumor,” Ferreira says. There are currently no monoclonal antibodies approved for use against ovarian tumors, which are of less interest to pharmaceutical companies since ovarian cancer is a less common form of cancer. Unpublished results obtained by Recepta’s researchers and Ferreira’s team indicate the potential for using RebmAb200 in a subgroup of patients with breast cancer who currently have no effective therapeutic alternative.

These 10 years of coordinated work culminated in mastering one of the stages of monoclonal antibody production, but there are still obstacles to overcome, if Brazil opts to develop a national policy and cease being just an importer of technology. For example, the production of monoclonal antibodies requires help from abroad. That is why Perez decided to engage a company in the Netherlands to manufacture a pilot-scale batch of RebmAb200 and another company, also located abroad, to package it.

“The conditions required to produce it in Brazil do not exist,” says Perez, the president of Recepta, who recently partnered with the Ludwig Institute, the Swiss company 4 Antibody AG and the Brazilian pharmaceutical laboratory Cristália to test two second-generation monoclonal antibodies that act differently from RebmAb200. From a business standpoint, there may not be a need to master every technological stage of production of antibodies. “In today’s world, the view that one has to develop the whole production chain in a single country is not tenable; even companies of developed countries like the United States manufacture many of their products in China.” From a strategic point of view, however, it may be necessary to meet this challenge.  “Knowing how to do biotechnology requires a national policy to be developed over decades,” says Gadelha of the Ministry of Health. “When it comes to the fields of health, energy, agriculture and defense it would take a great effort on the part of the government for Brazil to break the logjam and become an international player. Unless Brazil is able to absorb and develop technology, it will be unable to expand access to health care,” says Gadelha.

Over the past two years, supported by a national program—partnerships for productive development (PDPs)—the federal government has encouraged the pharmaceutical industry to become more active in incremental innovation. Brazilian research institutions and pharmaceutical companies, in partnership, have already concluded some agreements with foreign companies to bring the technology to produce 14 biopharmaceuticals available in the market (vaccines, blood products and monoclonal antibodies for cancer and autoimmune diseases) to Brazil.

By stimulating the domestic production of the technology for these compounds, the Ministry is attempting to reduce the costs of the public health system. There are plenty of reasons to do this. After all, biopharmaceuticals account for only 4% of the drugs purchased by the Ministry of Health, but consume 51% of the procurement budget. “Since 2012, 27 partnerships have been concluded to produce 14 biopharmaceuticals of the current generation in Brazil,” says Gadelha. “Now Brazil has the capacity and need to move into the era of radical innovation,” he says. The Recepta antibody ready to be tested in Sweden seems to be a step in that direction.

“The development of RebmAb200 shows how an innovative project of a Brazilian company can serve as a catalyst for translational research coordinated at the frontier of scientific knowledge and conducted collaboratively among academic institutions of excellence,” says Perez.

Projects
1. Cell lines of high productivity and stability of humanized monoclonal antibodies for cancer therapy (nº 05/60816-8); Grant mechanism Program to Support Research in Partnership for Technological Innovation (PITE); Coordinator Ana Maria Moro (Butantan Institute); Investment R$377,708.00 and $810,616.85 (FAPESP) R$1,793,198.00 (Recepta).
2. Monoclonal antibodies for cancer treatment: development and clinical trials (No. 01.06.0759); Grant mechanism Cross action – Science and technology institutions cooperation – Business – Innovation in therapeutics and diagnostics; Coordinator Ana Maria Moro (Butantan Institute); Investment R$6,197,136.62 (Finep) and R$4,006,802.00 (Recepta).

Scientific article
LOPES DOS SANTOS, M. et al. RebmAb200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: a novel reagent for targeted antibody therapy of cancer. PLoS One. 31 jul. 2013.

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