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SPEAKER 1: This is a production of Cornell University.
KELLY LIU: Welcome to the Ray Wu Memorial Symposium. Welcome, all of you from all over the world, so that we can come together to celebrate Ray Wu's life. Before Dean Henry opens the symposium, I want to make several announcements.
First of all, if you have registered, please pick up your name tag and the program book from outside. And they're going to be there during the break. So if you don't have time now, please pick them up during break, because your name tags are going to be used as your dinner ticket.
The other announcement is that, for people who are going to set up posters, so the posters are going to be set up in a small seminar room on the other side of the revolving door during a lunch break. So during lunch break, everything will be provided there, and you can set up your posters.
For the speakers who are going to be speaking after the coffee break, during the coffee break, would you please come up and load your computer so that we can get everything set up. And the afternoon session will not be here in this building, will be in Rockefeller Hall.
So at the end of your program book there is a map that shows where that building is. So we're also going to put a sign next to the building. And please arrive there before 2. We are going to start the session at 2. All right, so now Dr. Susan Henry, the Ronald Lynch Dean of College of Agriculture and Life Sciences of Cornell University, will give the opening remarks.
[APPLAUSE]
SUSAN HENRY: Thank you, Kelly. And I'm really delighted to be here today. And I want to thank all of you for coming here, many from so far, away to join us and to help in celebrating the life and science and accomplishments of our dear friend and colleague, Professor Ray Wu.
I want to start by recognizing that Christina Wu is here with us today. And it's wonderful to have you here, given the many things that you shared over so many years with our dear friend. I also want to especially welcome members of the Chinese Biological Investigators Society, who are co-sponsoring this Memorial Symposium in honor of Dr. Wu. Please join me in thanking them for their support.
[APPLAUSE]
Perhaps the members from that organization who are here could raise their hands so we can see where you are. Thank you so much. It's wonderful to see so many of you here joining us at Cornell for this occasion. This symposium is our opportunity to honor Ray Wu, our esteemed colleague, eminent scientist, educator, and dear friend.
Ray Wu was one of our college's most distinguished professors. He's widely considered to be one of the fathers of genetic engineering. He's a shining example of all the best that Cornell has to offer in scholarship, in teaching, and in the work that serves humanity.
Ray joined the faculty in 1996 as an Associate Professor in Biochemistry and Molecular Biology. He became a full Professor in 1972. In 2004, the College of Agriculture and Life Sciences honored him by naming him Liberty High Bailey Professor of Molecular Biology and Genetics. And in 2005 he won our Outstanding Faculty Award from the College of Agriculture and Life Sciences.
He was held in high esteem throughout all of Cornell. But the beyond Cornell, the respect for his contributions is shown by the fact that Ray Wu was also named Honorary Professor in more than a dozen Chinese Universities and Research Institutes.
The College of Agriculture and Life Sciences, Ray's college, considers itself to be a standard bearer for the land-grant mission at Cornell. This is an ethic that Ray Wu espoused and lived. CALS faculty-- CALS is the acronym for the College of Agriculture and Life Sciences, for those of you who don't recognize it. I may slip into using that.
CALS faculty and students are firmly committed to research and extension that benefits citizens and communities of New York, the nation, and the world. And that is truly the land-grant mission, to take your work beyond the reaches of your own academic laboratory and classroom.
And Ray's approach to science epitomized all of that. He managed to take his expertise in Molecular Biology and Biochemistry and extend it into a merger with Cornell's land-grant mission to the world. He was committed to helping people by boosting food production in areas of the world where food security is at the highest risk.
Ray's work has had lasting an international impact in many areas, but I'd like to mention three of them. First, he was a person who was a pioneer, perhaps the first in developing a method for sequencing DNA. He created transgenic rice strains that could be grown in hostile climates.
And he created a system for-- this is on the educational front-- a system for bringing the most promising students from his native China to the US for training and collaboration. He has influenced generations of researchers. Ray Wu develop the first method for sequencing DNA and some of the fundamental tools for DNA cloning. The strategy he developed in 1970 for determining the sequence of DNA was the location-specific primer extension method.
After several innovative modifications by other scientists that sped up the process, the same strategy is basically still being used today. Ray was most widely known for his innovative work in rice. In the mid-1990s, he and his group genetically engineered and successfully field tested pest-resistant rice plants.
And in 2002, he demonstrated another strategy to genetically engineer rice and other crops to make them more tolerant for drought, salt, and temperature stresses while boosting yields. You'll hear in much greater detail later on about Ray's work from others here today.
Ray Wu was also a longtime scientific advisor to governments in Asia, and one of the few people to be able to bridge the divide between China and Taiwan. And he was perhaps at his most persuasive and influential in developing US-Chinese cooperation in biological science and education.
I want to just take a moment for a personal perspective. That is actually how I first came to know Ray Wu's work. When I was still a fairly young professor at the Albert Einstein College of Medicine in New York in the 1980s, we participated strongly in the exchange program, the so-called CUSBEA program.
And there were many, many talented Chinese students who came to the institution, that I was actually director of the graduate school there at that time. And I had the pleasure and honor of meeting many of these very talented Chinese students who would not otherwise have had the chance to come to the United States.
So I want to mention that you'll be hearing much more about Ray's work on that regard from Hunter Rawlings in a few minutes. And right now I want to move on and present something that I think is a very important announcement, how we're going to go forward and how we're going to continue to Ray Wu's work in the future.
Since Dr. Wu's death, the College and the University leaders have been considering how to extend Dr. Ray Wu's research program and promote his innovations. From the very beginning, the college took steps to ensure that the members of his laboratory could continue their work, and finish the projects they were on, and find ways that we might continue what was going on in the laboratory so that his many innovations and valuable strains of rice would not be lost. We wanted to ensure that his research would continue into the future.
And we wanted to be able to honor his commitment to society at large. After much debate and planning, Cornell has decided to establish a start up company based on Professor Wu's research and innovations. Cornell's Center for Technology, Enterprise, and Commercialization-- when I speak about this from now on, I'm going to call it CCTEC, its acronym-- is taking the lead in this endeavor, and will be responsible for the management of Cornell's intellectual property coming out of Dr. Ray Wu's work.
CCTEC supports Cornell's land-grant mission by leveraging innovations that were produced here to promote economic development and to benefit the public. CCTEC is currently recruiting a management team, and forming a business plan, and finding partners, and raising capital for this company.
Dr. Wu believed in using technologies and inventions to improve people's lives. His earlier inventions at Cornell, including the rice actin promoter, have been widely used in engineering plants, many of which are very important food staples in the world.
His recent inventions and technologies, if further developed, will have the potential to contribute significantly to the world's food supply. And we all know how important that is in this day and age. To help bring these inventions to fruition and to fulfill Dr. Wu's dream, CCTEC is forming a new business enterprise in Ithaca to continue developing these inventions and technologies.
Those of you here today who share the vision of Dr. Wu and are interested in supporting this endeavor should contact CCTEC. I will mention that Dr. Alan Paau, the Executive Director and Vice Provost for Technology Transfer and Economic Development at CCTEC, will join us at dinner tonight for those of you might care to meet him and talk further about these exciting plans.
This is really wonderful news for all of us. I'm very pleased and proud to be able to announce it here today on this very important occasion when we're honoring Dr. Ray Wu and his accomplishments, and to be able to say that these accomplishments will be taken forward and continue to be able to help people in the world, thank you.
[APPLAUSE]
KELLY LIU: So our President Emeritus Dr. Hunter Rawlings will talk about his fond memories of Ray Wu.
HUNTER RAWLINGS: Thank you very much, and welcome everyone this morning for this symposium in honor of a great man, a great Cornellian, and a great scientist, Dr. Ray Wu. I want to start by congratulating the College of Agriculture and Life Sciences, and also all of the departments that are involved in the new venture that Dean Henry just described.
I think this is an exciting and appropriate way to honor the memory of Ray Wu, by taking the science that he developed and taking it further in order to help humanity on any continent. That was always Ray Wu's vision. And I think this new company is going to be a great way to take that vision further, and to enable Cornell and faculty members here at Cornell to work to take these ideas into the marketplace and make them successful.
This was originally planned as a symposium on Ray Wu's 80th birthday. It is now a Memorial Symposium, which is a bittersweet occasion, but nonetheless gives us an opportunity to reflect on the remarkable career of a great scientist and a great humanitarian.
You will be hearing from many others during the course of the day on the work that Ray did scientifically, and the ongoing scientific work that he inspired in so many students. I'd like to use my time this morning to reflect on Dr. Wu as a great Cornellian.
He came to Cornell in 1966 as an Associate Professor, having earned his BS degree in Chemistry at the University of Alabama, a PhD in Biochemistry from the University of Pennsylvania. And after working in cancer research with senior colleague, Efraim Racker, who would also become a distinguished Cornell professor at the Public Health Research Institute in New York City.
So he had four decades as a Professor at Cornell, and played a pivotal role in the life of the University generally, and helped to extend Cornell's influence around the globe. He was truly an ambassador for Cornell as well as for science. He was a great teacher. He was someone who served as a mentor to many, many individuals who have now become important scientists in their own right.
He was at the forefront of his discipline, and the application of that discipline to improve the human condition in the land-grant tradition of Cornell. He also contributed to international understanding. And in many ways, that might turn out to be Ray Wu's most significant contribution to humanity, the way in which he drew people together from widely different parts of the world, always with the smile that you see here in these pictures, which I shall never forget.
As a scientist, he developed the first method for determining the nucleotide sequence of DNA, as you have just heard from Dean Henry. It was later used by others in work that went on to win a Nobel Prize. He introduced methods that have since been applied to create several useful drugs, including human growth hormone. And with his longtime colleague Ajay Garg, who is speaking here today, he worked to develop drought and salt tolerant transgenic rice, which of course has the potential to greatly increase rice yields.
The Chinese Academy of Engineering elected him a CAE Foreign Academician in 2001. And he earned the Annunzio Award from the Christopher Columbus Fellowship Foundation in 2002 for his pioneering work in genetic engineering.
He was elected a Fellow of the American Association for the Advancement of Science in 2003, and then was named the Liberty Hyde Bailey Professor, as you heard, from Dean Henry. He was honored in 2005 with the Outstanding Faculty Award. And that's a very major achievement. Very few faculty members at Cornell win this award. And Dr. Wu was recognized for all facets of his life as a Professor with this award.
He continued until a few weeks before his death to work full time at Cornell, running his laboratory, presenting papers, attending conferences, and serving on advisory committees throughout the world. One of the key qualities of his scientific leadership was his ability to encourage those in his laboratory to look forward, to take risks, not simply to do incremental science, but to look for ways to contribute that would be 5 to 10 years ahead of what others were doing.
His favorite quotation was, today's impossibilities are tomorrow's miracles. He worked at the forefront of his discipline, and inspired others to take a long term view.
He was also committed, as you heard from Dean Henry, to the land-grant ideal. He took that seriously, and it's important to underline this facet of Ray Wu's career. He did not see the land-grant ideal as simply something to pay lip service to on occasion. He believed in it and he practiced it.
He took ideas from the laboratory into human society, whether it was business or social improvement. He made certain that the ideas he discovered and that his students discovered could find application in the world. He believed that was the most important part of the work that he was doing, to enable it to help other people.
This ideal I think is what characterizes Cornell. It gives Cornell a special mission which is different from the mission of many other very fine universities, because it ensures that Cornell is always interested in how to apply the work that goes on here to the world in ways that help other human beings.
This dimension then is not simply an intellectual dimension, it is a moral dimension. This means that Cornell faculty members, when they're practicing the land-grant mission, are carrying out not simply an academic or intellectual enterprise, but an ethical one which is designed to help other people.
If you just look at, Ray Wu's face you see clearly that this was someone who took that obligation seriously. He meant it. He wanted you to benefit from his work. He wanted you to learn more. He wanted you to achieve. And he wanted you to have a better life.
That's what Ray Wu's face always said to me. And it said so very directly. That's the face of a humanitarian. And I often think that if all of us went around during the day every day the way Ray Wu did, with a smile on our face, it would make a positive contribution in and of itself to humanity, because that's what Ray always meant to those who knew him.
His research was cutting edge. As his colleague Bill Lucas, Professor of Plant Biology at the University of California Davis, noted, "He moved around the world with a mission to help all people." And he was particularly focused on world hunger. That is what he considered to be the most important scientific enterprise.
And so he stressed, in an email to Dr. Garg a few years ago, "both of us," he said, "have agreed to give this new technology in transgenic rice seeds free of charge to developing countries, including India." Free of charge to developing countries, including India-- that's a humanitarian message. With that philosophy, he and Dr Garg continued to make major contributions to the world.
He was also committed to graduate education. Beginning in 1981, he initiated and coordinated the China-US Biochemistry Examination and Admission Program with the Ministry of Education in China-- 1981. This was way ahead of its time. No one else was doing anything remotely like this in 1981. Ray Wu was an innovator decades ahead of his time.
As our Cornell Colleague and Dr. Wu's longtime friend, Professor Emeritus Tsu-Lin Mei, has noted, in the early '80s, students from China began to apply to American graduate schools in large numbers, but American graduate schools had no way to evaluate their credentials.
Ray Wu and Edmund Lin of Harvard Medical School got together to create the Examination in Biochemistry, assembled from exams they had given at Cornell and Harvard. And so they set up a mechanism to enable Chinese students to apply, to be evaluated, and to gain admission to major graduate programs in the United States.
This was an enormous service, an enormous service to a country that, at that time, needed opportunities in excess of this kind. And Ray provided that through farsightedness, through commitment, and through his wonderful vision.
He helped to place more than 400 top Chinese PhD students in nearly 90 different universities in the United States. Think of that, 400 students in 90 universities in the United States, at a time when no one else was working on this issue.
Kelly Liu, Associate Professor of Molecular Biology and Genetics at Cornell, and one of the organizers of this symposium whom you've just met, was among those who came to the US through this program. And although she was never a student in Ray's lab, she gratefully acknowledges that she is at Cornell today because of Ray Wu's program.
Another fellow wrote in a blog post after Ray Wu passed away, quote, if one's legacy can be measured by what becomes of his influence, then all the Fellows in this program stand to testify to the great vision and insight of Dr. ray Wu.
He personally trained more than 30 PhD students or visiting scholars in his laboratory at Cornell. And his students tell many wonderful stories about what it was like to study under Ray. One of his very first students, in fact his very first student, Xiao-Hong Sun, who was at Cornell from 1982 to 1987, is now Eli Lilly distinguished Chair and full member of the Oklahoma Medical Research Foundation.
As she told a reporter for the Science Times shortly after Dr. Wu passed away, scientifically, she said, he encouraged my independence. The scientific skills I learned from him have propelled me throughout my scientific career. In terms of career development, he made me take scientific writing classes from which I am still benefiting.
He and his wife, Christina, carefully corrected every piece of writing I did. In terms of helping me adjust to the new environment in America, I, as many other Chinese students and scholars, spent every holiday in his house. He even paid for my driving lessons.
[LAUGHTER]
This is something that is above and beyond. This is more than having a great laboratory, teaching a great class. This is having a great friend, a friend when you need a friend, when you're in a strange country, and during holidays.
All of the other students are going home. You have nowhere to go. Ray and Christina always provided a place for students from abroad to go where they could feel welcome and confident during a period that otherwise might have been quite awkward.
As Dr. Sun recalled, many of us can confirm from personal experience, he lived a very simple life himself, but he gave generously to others. In the five years I was in his lab, I saw him bringing the same lunch every day. And he wore the same sweater with a hole at the elbow--
[LAUGHTER]
--every day. He had a modest house, but he provided financial support, large or small, to so many people that we will probably never know the precise number. Now, that's the kind of generosity that typified Ray and continues to typify Christina.
It is unusual to find anywhere, but especially in the Academy, where people do not make large sums of money. But Ray and Christina were always generous with what they had. And generations of students have benefited from their generosity.
He would always also, another student said, find time to give timely advice to his students, not only on specific research projects, but more importantly on the long-term development of their scientific careers.
In addition to weekly lab meetings for us to present and discuss individual research projects, we were fortunate to have ready access to Dr. Wu for his advice on a regular basis. He would always schedule a time to spend with you to help you with your career planning. He also gave written advice as handouts at weekly lab meetings.
And this could be from his own personal experience or from professional journal articles, on how to identify a worthwhile and exciting research project, how to make specific plans to carry it out, how to get connected with people in our research field, and how to prepare for a job interview. He helped many of us practice our English.
All of this, I think, gives you a picture of an unusual Professor, a Professor who stands out among other professors in his generosity. Another student said every year Dr. and Mrs. Woo would host a Thanksgiving dinner for the lab members and their families. For us, this would be the best dinner of the entire year.
In addition to the big turkey, there was always a whole lot of food. Dr. Wu himself would serve everyone to make sure every one of us got what we liked. One year, I don't remember which, Dr. Wu, with a broad smile as usual, showed us his new sunroom that could hold 10 to 15 people.
To our surprise, Dr. Wu himself actually constructed this new addition to the side of the existing house. He was very proud of it, because it meant that more of us students could come for thanksgiving dinner. All of that, it seems to me, gives us an idea of this remarkable human being.
In 1999 I had the honor of recognizing Dr. Wu for endowing a Graduate Student Fellowship in the Department of Molecular Biology and Genetics. Now, that's a rare thing for a Professor, to endow a position at his University. That is an extremely rare thing. And Dr. Wu did this out of sheer generosity.
He understood how important it is to be able to attract excellent graduate students, students who will extend the work of their faculty mentors, contribute new insights, and enliven the graduate student experience, yet another case in which Dr. Wu stood out beyond his peers.
At the reception to celebrate the creation of the Ray Wu Fellowship, Dr. Wu noted that he had been planning to establish such a fellowship for some time, originally thinking that he would include such a provision in his will. Then he said he realized that by making the commitment earlier while he was still alive, he would be able to enjoy the results. That was a much better plan, he said.
Through his generosity, he helped make Cornell an especially attractive place for gifted young molecular biologists and geneticists to come for graduate work. And the fellowship, which came on top of everything else he had done, has helped to attract outstanding graduate students by providing support in their critical first year at Cornell.
I knew Ray Wu best for his fourth contribution to Cornell, extending knowledge internationally across political divides. He was the only American scientist to serve on Biotechnology Advisory Committee for both mainland China and Taiwan, putting him in an ideal position to promote cooperation across the straits.
And as a relatively new Cornell president, I was grateful to have the benefit of Dr. Wu's knowledge and his stature within Chinese scientific circles as Cornell explored the extension of its ties in Asia. We went together to China and Taiwan in January of 1996.
My colleagues Tsu-Lin Mei, Catheryn Obern, Norm Scott, and I visited Academia Sinica in Taiwan, where Ray Wu was working to establish a new Institute of Agricultural Sciences. He was himself a member of Academia Sinica, and he had earlier played a leading role in establishing the Institute of Molecular Biology, and served as its first director there. Later, we traveled together to Beijing on Cornell's behalf.
He was a wonderful traveling companion. Everyone gets very tired on these trips. Some people lose their patience on these trips, especially when airplanes are late. Dr. Wu, no matter what the situation, had the same smile on his. Face and that was a comfort, I must say, and a very good example to set for the rest of us when we got tired and testy, which I'm afraid we did.
Dr. Wu never got testy. He may have gotten tired, but he didn't show it. He came from a scholarly family. His father was a respected biochemist who had prepared at MIT and Harvard. And his father had also served as the first Professor of Biochemistry at Peking Union Medical College in Beijing.
His mother earned a graduate degree from Columbia University in Food chemistry and Nutrition, and joined her future husband's laboratory at Peking Union Medical College in 1923. They emigrated to the United States in 1949, where Ray and his siblings received their college educations.
So you can see that this was an academic family, an intellectual family, a family that put learning, and particularly scientific learning, at the top of its agenda. Ray became a US citizen in 1961. And like his parents, he was at home in both his native and his adopted lands.
In addition to his leadership roles as a member of Academia Sinica he served as an Honorary Professor at more than 12 universities in mainland China, including Peking University and Peking Union Medical College where his father had spent his most important years.
He helped establish the National Institute of Biological Sciences in Beijing. And in 2007, the Chinese Academy of Sciences invited him to Beijing to Tsinghua University to thank him for his contributions, especially through the program that he had developed to bring Chinese graduate students to the United States.
His passing was noted with sadness by colleagues throughout the world. In addition to today's gathering, there will be, I'm happy to say, Memorial Symposia in his honor this fall in both Taiwan and Beijing. He is being honored appropriately in this country, in China, and in Taiwan. He should be for the contributions that he made to all three places.
And the Ray Wu Memorial Fund, an independent non-profit organization, is working with the Chinese Biological Investigators Society, the Society of Chinese Biologists in America, and other professional organizations to fund the Ray Wu Prize of excellence, the Ray Wu Prize of Excellence to be awarded to graduate students in the Life Sciences in mainland China, Hong Kong, Taiwan, and Singapore. Over $100,000 has already been raised for this distinguished new fellowship.
Now, I'm a student at the classics. I study Ancient Greece and Rome. Socrates, the Athenian philosopher of the fifth century BC, used to be thought of as a thinker who went well beyond his particular small city, the city of Athens. He called himself a citizen of the world.
Ray Wu was a citizen of the world, a scholar of the world, not simply a Chinese scientist or an American scientist, although he was both. He was a citizen of the world whose concern for humanity stands as an inspiration to all of us.
Like so many of you who have come to Cornell for this Memorial Symposium, I feel honored and privileged to have known this great man. He stands above his peers. He was ahead of his time. He was a great humanitarian. Congratulations Christina, on a wonderful, wonderful life and partnership. Thank you all.
[APPLAUSE]
KELLY LIU: Thank you President Rawlings. So now that President Rawlings mentioned about the different funds, so in the abstract book, on the first few pages-- I can't remember the number of the pages-- there are foundations that you can donate your money to, either to the Cornell Graduate Fund Fellowship that Dr. Ray will set up, or to Ray Wu Memorial Fund. Actually, the Ray Wu Memorial Fund is going to have a poster, number 17, that's going to be in the record room this afternoon during the reception period, all right.
So our next speaker is Ajay Garg. He's a Senior Research Associate in the in the Wu lab. His talk is Feeding The Hungry, Developing Transgenic Rice Plants Tolerant to Drought and Salt Stress.
AJAY GARG: Good morning, everybody. It's a privilege and honor for me to join all of you in remembrance and celebrating Dr. Wu's lifetime contribution to science and humanity. My presentation, I want to humbly dedicate it to Christina Wu.
So here is Dr. Wu and Christina Wu, Dr. Wu's parents and Christina's parents, and Wu's parents, brother and sisters here. And this is Dr. Wu's family, Dr. Wu's grandchildren, Alex, [? Adriana ?] Sam, and [INAUDIBLE].
So the philosophy of Dr. Wu was be kind and helpful to others, and be honest, try to contribute something to mankind, and trust in God. So as you see from his philosophy, he was always want to help something-- want to do something for the mankind. And it's very, very inspiring for all of us.
For the mission in the lab, Dr. Wu's lab, was feeding the hungry. And developing transgenic rice plants tolerant to drought and salt stress was the goal of the lab. As you can see, the major rice growing areas in the world are in Asia. And 90% of the rice is grown and consumed in Asia.
So he really wanted to do something for the people in the developing countries, because he realized that the population growth in the developing countries is much higher than the developed countries. And the per capita income is much less in the developing countries. And he also realized that the agricultural yield increases are declining, especially in developing countries, mainly due to drought and salt distress.
So the importance of rice is it feeds more than 2.7 billion people. It is a half of humanity. And rice is grown in about 130 million hectares, which is 10% of arable land. And annual rice production is about 600 million tons.
So if you have a 10% of loss due to salinity, which amounts to about 60 million tons. And in general, the 30% of the crop land contains high salt. So our lab was mainly focusing on developing transgenic plants which are tolerant to drought and salt stress, which is controlled by multigenes.
So in the lab, we are using transgenic approach to produce more stress-tolerant rice, which could eventually benefit farmers. So it can also serve as a tool to test whether a candidate gene is beneficial or not.
So why do we want to work on rice? If you see the consumption of rice, it is increasing from 1997 to 2002, and more recently it's much higher. But the buffer stocks are declining. And there is a stagnation in the production.
So he wanted to see whether transgenic approach can increase the yield by using different genes, because he realized the drought risk in Asia is much higher, especially in India, China, and other Asian countries.
So here is an example, estimated yield loss of rice due to two abiotic stress factors, drought and cold. So if you see the overall abiotic stress factor, the yield loss is significant in both India and China. So by using the tools of genetics and plant breeding during the first Green Revolution, many Asian countries were benefited, especially India, China, Bangladesh, Vietnam, Philippines.
So these are the rice-- if you see during the first Green Revolution, these are the chief beneficiaries. So he thought whether we can use something of this kind by using the transgenic approach. So the current rice production is about 600 million tons, which in terms of economic value is about $120 billion.
So by 2020, we need to increase the production to 950 million tons, which is roughly about the value of $250 billion. So his strategy was, if we could decrease the yield loss is due to drought and salinity, and increase the production by 130 million tons, and decrease the loss due to insect and diseases about 105 million tons, and then increase the grain yield by using other candidate genes. So this is the strategy or potential of agricultural biotechnology to increase the rice production.
And our lab is mainly focused on drought, salinity, and low temperatures. So these are the three major abiotic stresses due to which we'll lose significant grain yield. So the current actual in the field conditions, but there is a potential we can increase by using other approaches.
So the advantages of using transgenic technology is to allow us to experimentally test whether a potentially beneficial gene is controlling to an increased stress tolerance, and then to be able to introduce genes from any source, including bacteria, into rice plants to greatly extend the capability of plant breeding.
So we can increase and enhance the genetic variability by introducing the different candidate genes from bacteria, and then help in integration by plant breeding, and then eventually produce superior transgenic plants to benefit farmers and consumers.
So as an example, we were choosing a potentially beneficial gene of interest. And we were using different constructs, plasmid constructs. As an example, here we are using an ABF stress-inducible promoter, and trehalose biosynthetic gene, and a selection [? cassette. ?]
We transform rice by agrobacteria method. And we regenerate at least 50 independent transgenic lines, and confirm the integration of the gene of interest, and determine copy number by Southern blot, and choose the plants with a single copy of intact transgene, and determine the expression levels, and then test for stress tolerance, both under greenhouse conditions as well as in field conditions.
So over the years, in the lab, we have tested more than 10 candidate genes, which are involved in, for example, LEA3 gene, proline biosynthetic gene, polyamide gene, trehalose biosynthetic gene, and transcription factor, or glycine betaine.
So we were using either a constitutive promoter like actin 1 gene, or we are using an ABRC promoter, which is stress inducible.
So from this study, we found that so far, trehalose biosynthetic gene gives high level of tolerance to salt as well as drought. We also found that medium to high level of tolerance we can achieve by using the proline biosynthetic gene.
So what is the role of Trehalose? For example, in resurrection plants there is about 10% of dry weight, Trehalose accumulates. So this, for example, Selaginella or [INAUDIBLE], they were kept dry for five years. And when they were kept in water, they get back to life. So this is a lesson from nature.
Trehalose is a non-reducing disaccharide of glucose. And it is present in bacteria, yeast, fungi, insect invertebrates, and probably in trace amounts in all the plants. And it is known to play a major role in stress protection, particularly desiccation, salt, and freezing.
So Trehalose biosynthetic and degradation pathway-- So UDP-glucose and glucose-6-phosphate are the substrate. And Trehalose-6-phosphate synthetase is an enzyme which converts into trehalose-6-phosphate. And trehalose-6-phosphate is de-phosphorylated into trehalose. So it's a 2-step reaction to synthesize trehalose.
So there are the two genes, otsA and otsB gene in E. coli. So what we did is we used the fusion gene. We fused the otsA and otsB to make the trehalose. So these are the two plasmid constructs. So by using a linker, we fused this gene.
The advantage of having a fusion gene is it has a higher catalytic efficiency compared to having the otsA or otsB gene alone. And then we use an [? ABS ?] stress-inducible promoter for cytosolic expression, or we use the rbcS promoter-- that is the light-inducible promoter with the transit peptide-- for targeting the gene product to the chloroplast.
And we have a selection [? cassette ?] here. So by using agrobacteria media transformation, we regenerated large number of independent transgenic lines. And I'm very fortunate to work in Dr. Wu's lab, because his lab was pioneering in genetic engineering of rice. So I am very lucky. I very much benefited in learning the techniques of agrobacteria media transformation in his lab.
So after the efficiency of the transformation is very high, in indica rice, even though it is very difficult to transform indica rice compared to japonica rice. So he was able to succeed regenerating large number of independent transgenic lines in indica rice.
So by Southern blot analysis, we found that at least 40% of the plants contain a single copy of the transgene, because it is ideal to have the plants with a single copy of the transgene, because multiple gene leads to gene silencing and pleiotropic effects.
So in the T1 generation we choose the lines which segregate in a Mendelian fashion for 3 is to 1. So we choose the lines which are homozygous and are segregating for 3 is to 1.
So the next step is, after making the transgenic plants, we did the HPLC to see the trehalose content in the transgenic lines. So the non-transgenic plants have low amount of endogenous trehalose. But in the transgenic lines, we see 3- to 10-fold higher trehalose under abiotic stresses.
So this is the trehalose peak. And we confirm that trehalose peak by digestion with a trehalose enzyme to make sure that the peak is trehalose peak. So here is the data for the trehalose accumulation in rice lines. This is the non-transgenic line. This is the R line with rbcS promoter, and A line with the [? AB ?] inducible promoter.
So if you see, under drought stress, the transgenic line has tenfold higher trehalose than the non-transgenic line under drought stress. So we make these transgenic lines homozygous. And in the T5 generation, we evaluate the lines which has normal plant growth and higher grain yield.
So we pick up those lines which are more drought tolerant based on the physiological experiments. And then we evaluate further in the greenhouse and under contained conditions. So here is the screening procedure for salt tolerance.
So this is the-- under non-stress sodium chloride, and with 100 millimolar of sodium chloride. So indica rice is highly sensitive at 3 decisiemens per meter sodium chloride. But we are screening around 10 decisiemens per meter. So these transgenic lines are highly tolerant to salt stress.
And if you see the root growth of the transgenic lines, is similar to non-transgenic, non-stress plants, whereas the non-transgenic stressed plant, the root growth is stunted. So the transgenic lines have better root growth.
Then, in collaboration with Dr. Leon Kochian, we do the mineral nutrient analysis. And what we found is the transgenic lines have more potassium in the root compared to the non-transgenic stressed plants. And the transgenic lines have low sodium content in the shoot compared to the non-transgenic stressed plants.
So essentially, because of the change in the sodium by potassium ratio, the transgenic lines are more tolerant to salt stress. And the dry weight of the transgenic lines is much higher compared to non-transgenic plants.
Both root and shoot dry weight is lower in the non-transgenic stressed plants compared to their transgenic lines. So if you see the sodium by potassium ratio, the transgenic plants have much better sodium by potassium ratio compared to the non-transgenic plants.
We do both methods of screening, hydroponic screening, and soil culture experiments for soil tolerance. So this is a pot culture experiment where we are using 100 millimolar of sodium chloride for 4 weeks of salt stress. Again, we see the non-transgenic plants are almost dead, whereas the transgenic plants are having continuous growth under salt stress.
And in this experiment we are screening the transgenic plants throughout the life cycle under 5 decisiemens per meter electrical conductivity. So you can see the transgenic plant still can grow at that salinity level.
For drought resistance experiment, we use two cycles of 100 hours of drought stress and then recovery experiment. So the non-transgenic plants, and after drought stress, they are almost dead, whereas the transgenic plant, they continue to grow after re-watering.
And in collaboration with Dr. Tom Owens, we did the-- for [? PSII, ?] or photosynthetic efficiency of the transgenic plants, is much higher than the non-transgenic control plants. So this is the preliminary grain yield data for the TPSP transgenic lines after drought stress at flowering stage.
So A line means, A means [? AB ?] inducible line, and R [? means ?] rbcS promoter, and this is the non-transgenic line. You can see the transgenic lines have significantly higher grain yield compared to the non-transgenic plants under drought stress. So these are the T6 generation plants.
The next step is we want to see whether, is there any genetic variability in indica or japonica rice for endogenous trehalose accumulation without any transgene? So without any transgene, we see that there is a low amount of endogenous trehalose indica rice, or slightly higher in japonica rice. So there is a slight variability. But the amount of trehalose is very low in non-transgenic plants. So that's why we are interested to overexpress that trehalose biosynthetic gene.
So more recently, what we did is we did a phylogenetic analysis from the four different genomes, rice, Arabidopsis grape, and poplar to see how many trehalose biosynthetic genes are there in plants. And to our surprise, what we found is there are more than 21 trehalose biosynthetic genes are there in Arabidopsis. And there are about 22 genes in rice. And in grape, there are about 14 genes. And in poplar, there are similarly 21 or 22 genes.
So we were very surprised, why there are so many genes, and still these plants doesn't accumulate trehalose? So that means there are other important role which we don't know yet of the trehalose biosynthetic pathway in plants.
And this classification is based on their TPS domain and TPP domain. And class 1 looks like are more important in terms of the trehalose biosynthesis, whereas class 2 has both TPS and TPP domain. And class 3 has only TPP domain, and class 4 are the trehalose one.
So earlier when people overexpress the trehalose biosynthetic gene in dicot plants using either otsA or otsB gene with constitutive promoter, they found a low amount of trehalose in the transgenic plants. But they also found that when they express their individually, this gene, that leads to stunted plant growth or pleiotropic effects.
So that's why we chose to use the stress-inducible promoter, or light-inducible promoter, and see whether we could overcome the pleiotropic effects problem, and still increase more trehalose content. What we found is that transgenic plants have a higher trehalose levels, sustained plant growth under drought, salt, and cold stress, less photo oxidative damage, and more favorable mineral balance under abiotic stresses.
And most important thing, what we found was increased photosynthetic capacity under both stress and non-stress conditions. And more recently we did experiment in maize and wheat with a stress-inducible promoter. What we found is similar to rice, the transgenic plants have higher trehalose content, and changing the soluble carbohydrate pool.
So what we found is that transgenic plants have more soluble carbohydrate. And we also saw increased capacity in photosynthesis, differences in plant growth, development, and grain yield per plant, and multiple stress tolerance. So this is the last manuscript which Dr. Wu was editing just before he passed away.
So this is the unpublished data which I'm showing you. So this is the maize work in collaboration with Tim Setter. So these are the maize transgenic lines. So after 12 days of drought stress, the non-transgenic plants are almost dead, whereas the transgenic plants, they recover well after rewatering.
And then we also measured the [? PSII ?] activity in the transgenic and non-transgenic lines after 12 days of drought stress, or after 10 days of recovery. So the transgenic plants recover well after 10 days of rewatering, whereas the non-transgenic plant, the recovery is much less.
Another interesting observation was the transgenic plants have better root growth in maize, and especially the phosphorus content is much higher in the transgenic lines compared to the non-transgenic lines. And we also see change in the magnesium content in the transgenic lines compared to the non-transgenic line, whereas the potassium and zinc are more or less same in transgenic and non-transgenic lines.
The conclusion of our experiments is, our data indicate that the stress-inducible or tissue-specific overexpression of trehalose biosynthetic genes in rice and maize plants confers high levels of abiotic stress tolerance and improves grain yield.
The modest increase in trehalose levels in transgenic plants resulted in concomitant decrease in the extent of photo-oxidative damage during drought stress. In addition, trehalose must be acting with other physiological processes to account for changes in soluble sugars and mineral nutrient content.
So this effect supports the role of trehalose as a key regulatory molecule in a number of physiological process, rather than as a simple osmoprotectant as seen in resurrection plants. So in resurrection plant, the amount of trehalose is 10% dry weight basis, whereas in our transgenic plants we see only 500 micrograms per gram fresh weight, so which is too low to act as an osmoprotectant.
So what we believe is-- so trehalose may be interacting with other physiological process to account for these changes and to confer drought and salt tolerance. So there is emerging evidence that trehalose and trehalose-6-phosphate are the regulators of carbon metabolism in plants. So this is a recent article in current opinion plant biology.
So trehalose or trehalose-6-phosphate has been shown to improve photosynthesis, leaf development, embryo development, cell wall deposition, cell division, starch synthesis, drought tolerance, and inflorescence architecture. So it looks like this particular pathway is involved in multiple physiological and biochemical aspects of the plant.
So the future opportunities for transgenic lines is we can use that-- by 2010, we can see the transgenic improved rice with C4 plants. And by 2015, we can have transgenic plants for drought and salt tolerance.
Well, Dr. Wu said anything we can do to help crop plants cope with environmental stressors will also raise the quality and quantity of food for those who need it most, so thank you.
[APPLAUSE]
KELLY LIU: Any questions?
AUDIENCE: What was the yield of the transgenic versus non-transgenic under non-stress conditions?
AJAY GARG: The transgenic plants with rbcS promoter had a higher yield, about 8% to 16%, than the non-transgenic plants. Of course, this is under greenhouse conditions.
AUDIENCE: What about the taste?
AJAY GARG: Pardon?
AUDIENCE: The taste of rice.
KELLY LIU: Can you speak louder?
AUDIENCE: The taste of the rice.
AJAY GARG: We haven't tested the taste or quality of the rice, yeah, yeah.
AUDIENCE: [INAUDIBLE].
KELLY LIU: Any other questions? [INAUDIBLE].
AUDIENCE: Yes. Do you know if the trehalose is accumulating in the cytoplasm, in the vacuole, in both?
AJAY GARG: Probably in the cytoplasm, but we haven't done any experiment to show that it is [INAUDIBLE], because I don't know whether the substrates are there in the vacuole for trehalose synthesis.
KELLY LIU: No more questions. Thank you.
AJAY GARG: Thank you.
[APPLAUSE]
KELLY LIU: So as Dean Henry and President Rawlings mentioned, one of the major legacies of Ray Wu is the establishment of the CUSBEA program, which brought over 400 talented Chinese students to US for graduate studies. Many of these former CUSBEAns are now leaders in academia or industry around the world.
So the organizers wanted to showcase the success of the CUSBEA program by picking one member from each of the eight years of the CUSBEA classes. So our next eight speakers, five speakers today and then three speakers on Saturday morning, represent the former CUSBEA students.
So the first speaker representing the CUSBEA students is Dr. Junying Yuan, who's a Professor of Cell Biology at Harvard Medical School. She is a class 1 CUSBEAn.
[APPLAUSE]
[SIDE CONVERSATION]
JUNYING YUAN: Thank you. I'm honored to speak to you as a representative for the first class of CUSBEA program at this special occasion honoring Professor Ray Wu. Let me first introduce you to class.
[LAUGHTER]
For those of you yet to learn how to read Chinese, the top says, the first class of CUSBEA students for study in America. This picture was taken in 1982.
AUDIENCE: Which one is you?
[LAUGHTER]
JUNYING YUAN: This one. Do I look the same? Let me first introduce our class. This was a very special class, I must say. We were also the first class to enter college after 10 years of cultural revolution. During that time, all the colleges and university were shut down all over China.
So as a result, this is a very diverse class. By the time we entered the College, the youngest one were still teenagers, while the oldest one already had a children of their own. But we also have a highly motivated class, because we can all cherish the opportunity that many of us might otherwise never had a chance to go to college.
We were very, indeed, very fortunate, because by the time we graduated, China is beginning to open up. But the communication between East and West was still difficult. So it was Professor Ray Wu and others effort provided this opportunity for go to America to go to another world to study.
Although I must say that at a time when we're taking this picture, we were all very excited to go to America, but we had very little understanding what was ahead of us and what was a great opportunity that Professor Ray Wu has given us.
26 years has passed since this picture was taken. And many of us went on to do extremely well in different areas of biology, whether it's academic science, biotech, or investment, and various other areas of biology.
And if we say Professor Ray Wu has made a transgenic plant to help conquer the hunger in the world, he had planted us in the rich soil of American science so we can grow, prosper, contributed to science in the world in general, and become the link between East and West.
So here I want to share with you a little my personal journey for the last 26 years. I was into the Harvard Medical School in this program Neuroscience in 1982. I must say, at that time, my English was not nearly as good as some of the Chinese student today.
And I remember, although I could have a personal conversation one on one, but it was really difficult for me to have a discussion in the seminar course or discussion group. And by the time I understood what people were talking about, they already went on to talk about something else. But nevertheless, I did catch something very interesting in my class.
So it was in a class that called neurodegenerative diseases. In that class I learned a variety of diseases cause neurodegeneration, which have remained largely incurable even today. But what strikes me is the specificity of a neuronal loss, whether it's the Huntington's disease, or Parkinson's disease, or Alzheimer's disease, there was no wholesale loss of the neurons, rather only specific populations of neurons would die.
And one of the most typical example is the Huntington's disease here. And this is a coronal section of a normal brain. And this is a coronal section of an HD brain. As you can see, the area of the caudate and putamen are completely gone in this Huntington's disease patient.
So it was the specificity of a neurodegenerative disease in causing neuronal cell death made me thought about another class I was taking at the time, namely it's called neural development. At that time, we already appreciate that there also are significant neuronal loss in the early embryonic development, except that that is a genetically programmed the process so to ensure precise pre and postsynaptic connections.
So I thought it was a very strange that you can have a selective neurodegeneration later on in life. But then, yes, neuronal cell death it can be a perfectly normal process in the beautiful process of embryonic development.
So I made a, you can say, naive connection of a first year graduate student. I said, there may be some connections between the mechanism-wise between the cell death in early embryonic development and the later on in the neurodegenerative diseases.
So I went to discuss my ideas with some professors in the neurobiology department. And one professor answered in the precise word. What he said was, cells that die cannot be important. And, of course, I wasn't very persuaded by the argument. And I decided I want to study cell death.
But at that time, indeed, the prevailing notion at the time, what everybody was excited about, were growth factors. And everybody knows a growth factor like a neurotrophins, when you add it to the neurons, the developing neurons, you can prevent cell death. So the neurons that die during development most people believe were because they are starved to death.
But nevertheless, I decided I want to work on this mechanism. And I look around and, however, nobody was studying cell death. So I went to a program director Ed Kravitz and I said that I cannot find the lab I want to work with.
And thinking back, it was very bold statement I was making, and although I was perfectly sincere and honest, because I had a choice of over 200 or 300 labs that I can work with at Harvard.
[LAUGHTER]
And but most people, you probably say that you just came from China. You can hardly speak English. And are you sure you're saying the right thing? But nevertheless, Ed was very kind to me. And he said, oh, if you cannot find the lab here, you can go to main campus.
For those of you familiar with Harvard, if you cross the river, go to the main campus, it might as well be a different institution. And then he said, you can even go to MIT. And when I hear that, I was very happy, because at that time I just heard a talk given by Bob Horvitz.
For those of you who know Bob, he went on to earn a Nobel Prize in 2002. But at that time, he was still a junior Professor at MIT and not yet tenured. In Bob's talk, he talked about the post-embryonic lineage study of C. elegans.
And I was very impressed that the C. elegans development is so well mapped out. And most interesting, what he talked about is that there's a set of cells, which is marked by a cross here. Those cells undergo programmed cell death at a precise time during the C. elegans development. And those are truly programmed cell death, because in every individual worms, their same set of cells would die at precisely the same time.
And furthermore, they just found two mutations that can block all of those cells that die, although they didn't know what those genes were doing. So for me, that was most exciting genes that I learned at the time I saw that, because I cannot study mammalian cell death, and nobody was studying that, that's probably my best opportunity to study cell death. And [INAUDIBLE] allowed me to work at MIT. And so that's why I went to Bob's lab and did my PhD.
And my thesis work involved a discovery that, interestingly, ced-3 ced-4 will act cell autonomously within dying cells. And in contrast, some of the prevailing proposal was that these genes may be encoding hormonal factors, to act non-cell autonomously, like [? insect, ?] to control cell death. So that was actually the first discovery that cells may have a suicide machine acting within the dying cell to control the cell fate.
And my other part of the thesis consists of identifying the gene product of a ced-3, ced-4. But at that time, we still found it to be a novel gene product. So I published it as a novel gene, but highly expressed during the period of programmed cell death.
And after I graduated, that was the time that I decided that I want to go back to my mamallian system, because I started to work on C. elegans because we cannot work on mammalian cells. And the time that I graduated, I decided that probably I can come back to the mammalian system.
And with Bob's help, and I was very fortunate to be able to have my own laboratory right after I graduated without any training in mammalian biology, but decided to work on the mechanism of mammalian programmed cell death by finding the homolog, if it exists at all, look like ced-3 Indeed, two years down the road we discovered that the ced-3 is actually homologous to mammalian interleukin 1 beta converting enzyme, [? that ?] published a study by [? Merck, ?] independently, at the time.
And that lead to the discovery that ced-3 is probably-- or very likely to be-- assisting a protease that controls the programmed cell death. And so that suggests that-- it was unbelievable to me at the time that the C. elegans programmed cell death mechanism actually is also conserved in mammals to regulating a phenomena called apoptosis, that was named by Andrew Wyllie independently.
And in my own laboratory at MGH, we discovered that indeed if we express interleukin 1 beta converting enzyme, later called caspase 1, we can see induction of apoptosis. And furthermore, if we inhibit this class of enzyme using a cowpox virus encoded caspase inhibitor named CRMA or CrmA, we can inhibit neuronal cell death that in culture induced by trophic factor deprivation, a model mimicking developmental neuronal cell death induced by trophic factor deprivation.
And furthermore, in collaboration with Mike Moskowitz' lab at MGH-- we have ongoing collaboration for more than a decade now-- we show that the inhibition of a caspase can reduce the ischemia injury, brain injury mimicking stroke. And that lead to the proposal that inhibiting apoptosis or caspase is a therapeutic possibility for stroke therapy.
And by that time, it was already discovered that a disease like a Huntington's disease is caused by what are called a polyglutamine expansion disease, because in this class of disease, what's wrong is that the polyglutamine stretch in the wild-type gene product get expanded to much longer. And this is somehow the expanded gene product can cause cell death.
And interestingly, what we found is that if we express polyglutamine, we can indeed induce cell death, and that cell death can be inhibited by expression of CrmA, the caspase inhibitor, or by Bcl-2 or by peptide inhibitor with caspase.
So you can say that it's really a remarkable, almost a graduate student fantasy that the development, maybe there's some connection between the embryonic and neuronal cell death and end stage neurodegenerative cell deaths, actually the same mechanistic link mediated at least in part by the caspases.
Then you might say, OK, I can spend the rest of my life to studying caspase, because mammals have 11 caspase, much more complicated than our C elegans. Indeed, we spent many years trying to make all the knockout and study the mechanism of caspase.
However, for my own satisfaction, there still have many problems. And first of all, nobody can develop a small molecule inhibitors of caspase to go into the CNS. For whatever the reason, cytoprotease-- which is a caspase-- inhibitors all go to the liver. You cannot even detect it in the blood.
So for the therapeutic purposes, it is really no good. And this is really not because a lack of effort. Almost all the major pharmaceutical companies has a caspase program, including Merck. And they have to dump it after many years of effort.
And secondly, what we discovered, that for certain cells, cells may still die in the presence of a caspase inhibitor in the presence of a proapoptotic stimuli. So I was very interested in this phenomenon. In particular, death receptor mediated signaling, for example, if you treated cells with a TNF, is known to induce a very canonical path with apoptosis.
For example, stimulation of a TNF, interaction of TNF with type 1 TNF receptor results in recruitment of intracellular signaling complex, including RIP, TRADD, FADD, and TRAF that lead to NF-kappa-B activation, which can inhibit cycloheximide protein synthesis inhibitor.
But the FADD is a key adaptive molecule that recruits caspase-A, and leads to activation with caspase and downstream apoptosis. So if we inhibit caspase activation by a peptide inhibitor such as zVAD.fmk, can lead to inhibition of apoptosis.
On the other hand, what we found, in a subset of the cell types that these cells also will be-- apoptosis will be inhibited, but the cells will die through a necrotic morphology. Which was very interesting to us, because apoptosis and necrosis are traditionally-- a very distinct type of cell death.
Apoptosis is characterized by marginalized chromatins and cytosol condensation. But most importantly, the plasma membranes remain intact until very late stage in apoptosis. On the other hand, necrosis is characterized by early plasma membrane permeabilization and swollen mitochondria.
So this is the appearance of necrotic-like cell death in the very defined condition such as stimulating cell with a TNF in the presence of caspase inhibitor, suggesting to us there actually be a regulated necrotic cell death mechanism.
And traditionally, cell death has been classified to at least two different classes, while apoptosis is known as energy-dependent cell death mechanism regulated by genetic encoded programs, necrosis is supposed to be caused by overwhelming stress. And that cannot be regulated.
Although necrosis is very common in human pathology, in particular acute pathological cell death, but because this notion that necrosis is a type of a passive cell death, nobody's actually paying attention, trying to inhibit necrotic cell death.
So the proposal that the possibility that stimulating cells with the pro-apoptosis stimuli, but the cells for whatever reason, cannot undergoing cell apoptosis, they end up going in necrotic cell death, suggesting the cells may have a backup cell death mechanism that characterized by necrotic cell death.
So we decided to study this mechanism by a chemical approach. The notion is that because when we discover the caspase, we found that use a gene approach first. And we end up discovering caspase. Although it was a very exciting discovery by itself, but it ended up to be no good for the patient, because we cannot develop inhibitor.
We decided to use a chemical approach to ask whether in this necrotic cell death mechanism we turn to necroptosis, whether any of these steps may be chemically specifically inhibited. So in other words, we turned the discovery process upside down using the chemical approach to ask whether we can inhibit this necrotic cell death.
Indeed, the first screening that we came up with is very specific inhibitor that we called a necrostatin-1 or nec-1. It's a derivative of a thyohydantoin tryptophan. And it's very specific, because we know that if we remove this methyl group, it's no longer active.
And since then, we have screened a half million compounds that lead to identify 20 highly potent micromolar chemically distinct small molecule that inhibit this type of cell death. Now, we want to define that the apoptosis process is a caspase-dependent cell death with the apoptotic features, while necroptosis is a caspase-independent cell death induced by the activation of death receptor pathway, and probably others as well, with the necrotic cell death and necrotic features.
In collaboration with medicinal chemist Greg Cuny, we have searched for and improved the derivative of nec-1 that lead to the improvement of a specificity and chemical stability. Now we enter the fourth generation of a necrostatin nec-1 with EC50 of a 50 nanomole [INAUDIBLE] culture.
And what's most remarkable is the specificity of nec-1. And these are necroptosis of a FADD-defficient [? gerbil ?] [? cell ?] that cannot undergoing apoptosis. You can see the cell burst in to pieces. In the presence of TNF a nec-1, the cells are completely protected. They look completely normal, as if TNF is not present.
On the other hand, the wild-type [? gerbil ?] [? cells ?] stimulated with cycloheximide and TNF, the cells undergoing apoptosis, and nec-1 doesn't touch anything at all. And these cells-- I don't have data here, but that they actually proliferate just as normally as if TNF is not present. So it's a completely normal, complete 100% inhibition.
And what we [? learned ?] about necroptosis pathway is this RIP molecule. And RIP has come to be a very important molecule. Now it contains a death domain responsible for the recruitment to this death receptor complex, as well as a kinase domain that's required specifically for necroptosis.
So we suspected that at least some of the necrostatins are inhibiting RIP1 kinase. , Indeed what we discovered that the nec-1 can specifically inhibit RIP1 kinase. Now, the new study shows that the IC50 is actually much better than the numbers shown here, as through a better assay is actually a 3 nanomolar inhibitors of RIP1, and remarkably it doesn't touch the closest analog of RIP1, RIP 2.
So we want to define that-- better define the apoptosis is the biochemical apoptosis requires the caspase and shares the morphology apoptotic morphology, and inhibit by caspase inhibitor, whereas the necroptosis, biochemically, it's RIP1 kinase-dependent, and morphology is a necrotic inhibited by necrostatins.
And now availability of necrostatin actually allows us to directly answer whether this pathway is relevant pathologically. And I just want to show you a few examples that have been published. And our study shows that the nec-1 can inhibit ischemia brain injury. With expended time window, we can deliver as late as six hours after the onset of injury, and shows a significant inhibition.
And this is done by another group in England independently without our knowledge. And they showed that the nec-1 has a cognitive protective effect. This actually requires a cyclophilin-D, which is actually known to be a necrotic component in the mitochondrial permeability transition pool.
And thirdly, in collaboration with Mike Whalen and Mike Moskowitz, we studied a traumatic brain injury model, and shows that the nec-1 can reduce the histopathology and the functional outcomes after traumatic brain injury.
Now, you may ask, OK, so if cells has this backup mechanism, why, what is the significance? Because this mechanism cannot be evolved as a mechanism to mediate injury-induced cell death. And that's really a good question.
And that's-- actually the answer I think we come to appreciate from a recent genome-wide sRNA screen, this lead to the identification of 400-something-plus genes that, when inhibited, will inhibit necroptosis pathway.
Interestingly, through bioinformatics analysis, more than 70 of these genes can be connected into a large network of innate cell host defense response involving TNF, toll pathway, RIP kinase.
So suggesting that this necroptosis is also a host defense response, in particular because we know that many viruses has caspase inhibitor. So they encode caspase inhibitors. When cell-- in particular, like host defense response cells like macrophages, monocytes, when they're infected by virus, the caspase activation is inhibited by the viral encoded caspase. But the cells also receive apoptotic stimuli because the virus themselves, and that constitute a death signal to activate the necrotic cell death pathway.
So we hypothesized that necroptosis maybe evolved as a host defense mechanism activated by virus infection, as virus often includes inhibitor caspase. Examples includes CrmA, the cowpox virus, or p35, the baculovirus.
So now I want to switch gears a little bit further now to go to the chronic neurodegenerative disease. Although I believe that the inhibiting cell death constitute a great therapeutic opportunity for acute neurologic injury, for the chronic neurodegenerative diseases, such as Alzheimer's, and Huntington's, and Parkinson's, inhibiting cell death is not going to do the same, because oftentimes these chronic neurodegenerative diseases started by neural dysfunction.
So if you just inhibit cell death per se, which actually cell death is often the last thing that happens in late-stage disease. So we need to, for treatment of chronic neurodegenerative disease, we need to go to the source of the neural dysfunction. So how we solve that?
For the chronic neurodegeneration, so we often know that a hallmark of this disease is accumulation of a misfolded proteins. And these misfolded proteins are often marketed by ubiquitin. And now in the recent years, we come to appreciate that autophagy is very important cellular mechanism to remove not only expelled organelles, a large protein complex, but more importantly are misfolded proteins. My time is running out, so I'm not going to go to details.
And autophagy is really efficient in removing these misfolded protein, because conditional knockout of autophagy genes mostly created by Japanese schools, and shows selective accumulation of a misfolded proteins in neurons that's marked by polyubiquitin.
And one of the most interesting phenomena for these chronic neurodegenerative diseases is the late onset. In particular, Huntington's disease patient, although if you inherited a mutant allele of Huntington's, there's a 100% probability you're going to develop disease. But you can live up to 40, 50 years completely normally, disease free.
It's not because this gene product, the mutant gene product is not expressed. Actually, it's expressed from the very beginning of embryonic development. But for some reason, the young neurons can handle these mutant proteins.
But when people turn 40 or 50, we still think we're completely normal. But there's something biological change in ourselves that may reduce our capability to dealing with these mutant proteins. The question is, what is the mechanism?
Now, [? I want ?] [? to ?] hypothesize that-- because autophagy is so important in removing these misfolded proteins, I hypothesized that there may be a reduction of autophagy function during aging that contributing to the onset of chronic neurodegenerative disease.
And that-- I apologize for the lousy quality of the slide, because this is a very new slide. In the recent human genome-wide RNAi screen for genes that are regulating autophagy, we identified something like over 200 genes [? where ?] [? knockout ?] [INAUDIBLE] is up- or downregulating autophagy.
When we analyze these hits against a set of normal human brain that collected by [? Bruce ?] [INAUDIBLE] group, what we discovered, interestingly, in the aging brain, the genes that are downregulating autophagy or up, the genes that are important for autophagy are down.
So this is consistent our hypothesis that, in the aging plan, it's not that old. In the mid age, this expressed-- the reduction autophagy function can be reflected in gene expression pattern. So what can we do? I don't think we have lost all the hope. We still have some autophagy gene expression.
So the question is whether we can find a pharmacological agent that can induce autophagy without inducing injury. So to cut a long story short, in the paper that we published end of last year, we discovered that we did an image-based screen in a [? known ?] bioactive compound library. We discovered there's seven FDA-approved drugs that actually can induce autophagy without causing cell injury.
And interestingly, three of the drugs already, CNS drug and-- [? actually, two of ?] [? the ?] drugs already have one paper each for use in the Huntington's disease patient for improved movement disorders. And the three other drugs are cardiovascular drug used for anti-arrhythmia. And the other one is a very common drug probably many of you have used, loperamide for diarrhea.
So we're not starting now to just see whether any of these drugs can reduce misfolded protein injuries and the accumulations in the Huntington disease model. My dream is that for-- because there's a large lag between in the onset age of Huntington's disease patient, maybe for this patient we can somehow develop a drug that can give them three days a month so we can reduce the accumulation of misfolded protein to the extent that they will delay the onset of the disease. And that will be as good as a cure.
And, finally, I want to thank all the collaborators that had a fortune to work with over many years. I don't have to name one by one specifically. And most importantly, I want to thank Professor Ray Wu for giving me this opportunity of lifetime to have a profession that I can truly enjoy. Thank you very much.
[APPLAUSE]
KELLY LIU: Questions for Dr. Yuan?
AUDIENCE: [INAUDIBLE] that the hypoxia is one of the most severe conditions, induce necrosis. And so you guys have [? looked at ?] [? the ?] [? link ?] how hypoxia can actually-- [INAUDIBLE] induce, actually, the necrosis [INAUDIBLE]?
JUNYING YUAN: We haven't looked at that extensively. But there's a paper published. I think it's relating to that fact, on the necrostatins, the effect.
AUDIENCE: Do you think that any of the drugs that promote autophagy that are used in Huntington's would also be useful in later-onset neurodegenerative diseases like Alzheimer's?
JUNYING YUAN: The reason that I want to use a Huntington's disease as the first example is because it's very clean mechanisms. We know very clearly what's the cause, what's-- with Alzheimer's, I think it's more complicated. The vesicular trafficking is involved.
If you induce autophagy, what is the effect on vesicular trafficking? Process [INAUDIBLE]. So that need to be much more extensive study before we can say anything that one way or the other. But I think in general, reducing the misfolded protein would be a good strategy.
AUDIENCE: So if I remember right, the Huntington degrees and a couple other neural disease have this aggregated accumulation of ubiquitin. So is that ubiquitin activated by itself, or is the ubiquitin-- how do you get it to a misfolded protein?
JUNYING YUAN: The ubiquitin conjugated in misfolded protein.
AUDIENCE: Do you know why it cannot be [INAUDIBLE]?
JUNYING YUAN: Yeah, so originally the accumulation of ubiquitin positive misfolded protein we're taking as evidence that-- because the proteasome degradation requires ubiquitin. And you have accumulation your good and positive misfolded proteins.
So in the aging, there must be a reduction of function or dysfunction of a ubiquitin system that lead to accumulation of a ubiquitin positive misfolded proteins. But actually, I searched the literature extensively.
Nobody actually bothered to make a conditional knockout of a proteasome, key proteasome subunit, and show that lead to accumulation of a ubiquitin positive misfolded proteins. I'm not saying that cannot happen, but just nobody have done that.
On the other hand, there's unequivocal evidence that if you knock down autophagy, you can lead to ubiquitin positive misfolded proteins. And that included neural dysfunction and everything we would expect that what is the deleterious function. So I think that for whatever reason, ubiquitin may also be involved in autophagy degradation.
AUDIENCE: So autophagy [INAUDIBLE] synchronization in activating the immune cells. Can you comment on the side effect of those drugs, the possible side effects, for example, the [INAUDIBLE] or--
JUNYING YUAN: Yeah, see, the reason I'm proposing that you're going to take three days of autophagy inducer per month is I want-- precisely that I don't want to propose that you're going to take autophagy inducer every single day. If you're taking that, I think it's going to have problems no matter what. And I think it's probably true for all the drugs. And it's also probably true for apoptosis inducers.
The beauty, I think, of the autophagy inducers is such an effective process. If you just turn it on, you can reduce the accumulation misfolded protein for a while. Then you don't have to take it for rest of month. And then you don't worry about the immune system.
AUDIENCE: You also published a paper on the cell [INAUDIBLE] that would activate the [INAUDIBLE] maybe a year ago.
[INTERPOSING VOICES]
AUDIENCE: Several years ago.
JUNYING YUAN: [LAUGHS]
AUDIENCE: [? One of the ?] [? things about ?] that [INAUDIBLE], that future, that activating part of [INAUDIBLE].
JUNYING YUAN: That compound is a tool. It's not like a necrostatin actually have pharmacokinetic properties that are compatible to be drug. Actually, we show that it can IV into-- if you inject IV, you can go to the brain.
So it's much better than the caspase inhibitor, which doesn't go to the brain. For [INAUDIBLE], no, I think it has same problems. I think that in vivo [? property ?] is questionable.
KELLY LIU: All right, we can continue the discussion during the break, and also during the reception this afternoon. So we'll take 20 minutes break. We'll come back at 11:10.
[APPLAUSE]
Our next speaker, Dr. Guo-Liang Yu is a class 3 CUSBEAn. He's the Chairman, President, and CEO, of Epitomics, of his own company. He will talk about discovery and development of monoclonal antibody drugs using RadMAb technology.
GUO-LIANG YU: OK, good morning. It's an honor for me to representing the class 3. Can you hear me?
SPEAKER 2: Is it turned on?
GUO-LIANG YU: Yes, it's on. OK, now it works. All right, OK, it's my honor to representing the class 3 CUSBEAns.
Dr. Ray Wu certainly made a profound impact in many of our lives. And I just want to, before I start my talk, I want to give you a short story that certainly made, in my life, a very memorable and big impact.
So it's about time in 1992, in one of the conference, I met Dr. Ray Wu. And I try to seek his advice what I should do after my postdoctoral training. So I said, after publishing a few nice papers, I'm ready to look for a job.
And he actually said to me, in Chinese, he said, well, don't rush it. And try to build a solid ground. And there's always opportunity. So of course, the take-home message is that you need to be patient. And that really registered in my heart. And actually, what I'm about to tell you is something that I decided to embark on which require lots of patience and lots of persistence, which is to develop a therapeutic drug using monoclonal antibody.
OK, so let me start my talk to honor Dr. Ray Wu. My story, it's actually a rather short story, because we haven't really done much. Unlike what Junying has talked about, 25-year history in her scientific career, this whole activity, initiative, was only probably two years old.
So I really don't have a whole lot of scientific data to show you, but kind of give you an overview what we like to do, and what technology can change the way that the scientific discovery and translate into medicine that, of course, would benefit patient and human life.
So what I'm going to talk about is therapeutic antibodies. And you probably know this is the emerging field of drug discovery, development. About 50% of the new drugs are antibody drug now. And pretty much all the pharmaceutical company, most biotech company, working on antibody drugs.
It's the fastest growing market, as well as activity R&D expenditure on this class of antibodies-- I mean drugs. So on the left side I showed a bar graph where the cells of antibody drug, starting from 1997 to 2007, you see the growth. So last year sale was $26 billion already.
Yet developing antibody drugs is very difficult and take a long time. And typically, actually still today, average time to develop an antibody drug is about 16 years, and it costs more than a billion dollars to develop one drug. So it cost a lot of money.
On the other side, to the patient, cost of antibody drug is still very high. Typically each treatment could easily above $10,000 per treatment. Much of the reason is that we need a lot of antibody. We need about several hundred milligram to 1 gram, couple of gram of proteins, antibody drugs, to treat each of the patients.
So the cost itself is quite an issue. Then, of course, when you inject such large quantities of protein into human body, the side effect start to show.
So one of the passion that we had at Epitonics is to develop platform technology so that we can develop a new class of antibody therapeutics for the treatment or better treatment of disease. So, in other words, we try to make [INAUDIBLE] drugs, or drugs that already had scientific validation, but we try to make a better class of drug.
To do that, we need two basic technologies. One is ability to make really high quality, high affinity, and high specific antibodies. In this case, we use a technology platform we call RabMAb, which I'll tell you in a minute. And another technology we need is to convert an antibody that derive from animal, in this case is from rabbit, into a human form, because we can't simply inject rabbit antibody into humans. That it will cause immunogenicity. So humanization technology is another important platform we need. And I listed some of the benefits and the features down there.
OK, so just very simply describe the RabMAb technology, well, it is the rabbit monoclonal antibody using cell-cell fusion. It's a hybridoma technology which originally was applied in mouse using a fusion partner cell fused with B cells isolated from immunized rabbit. So [? just ?] [? schematically ?] shows how hard it works.
Now, why rabbit? Rabbit actually has very different immune system. I think I have the slides.
[LAUGHTER]
These just talk about the technology doesn't invent overnight. This actually has a history of 16 years, when, in 1995, Dr. Kathryn Knight at Loyola University in Chicago developed a cell line called the 240E, which is a myeloma-like cell line, which is able to fuse with B cell.
Just like any technology, when it first showed up, doesn't really work very well. So it took many years of hard work by the founders of Epitomics at UCSF, and then, subsequently, in the company, to develop a technology that is very robust today. So we're able to develop thousands of monoclonal antibodies a year right now.
I just mentioned, rabbit has a very unique immune system in that they develop antibodies rather different from those come from mouse and from human. They have a very diverse structure in terms of IgG gene structure.
And they go through extensive gene conversion and point mutation to create diversity, therefore have a larger repertoire. And also, they employ a rather different way, how the diversity is created. That is, instead of using a lot of recombination that happen in mouse and human, VDJ recombination. They use a gene conversion a lot more.
Now, the net result of that is that if you isolate a RabMAb from a rabbit, the average affinity of rabbit monoclonal antibody is about 100-fold higher than average mouse monoclonal antibody. You all know, high affinity potentially leads to high potency in drug. So that's what. We like to do
A lot of people ask me, OK, what are different ways to generate antibodies? Typically in the industry there are three ways to generate antibodies. The classical mouse monoclonal antibody technique, that's where most of the drugs on the market come from. And phage display has been emerging, a new technique. There is one drug that derived from phase display technology.
And we try to use the rabbit system. So, again, I'm not going to go through line by one just to say rabbit has a number of advantages, one is a very high affinity, two is it's recognize a very diverse epitope.
In other words, you may find the epitope that mouse immune system may not be able to recognize. And you may find antibodies that phage display or mouse won't be able to find. And the other thing I want to mention is molecular cloning of IgG genes from rabbit monoclonal antibody or humanization of it is relatively easy because of a unique structure of rabbit IgGs.
And so just one slide to-- a cartoon to illustrate how humanization work in our system. So we actually have our own intellectual property and our own way of humanized monoclonal antibody, so just kind of a schematic. So if you have a rabbit antibody sequence and your goal is to moving up the ladder, so make it closer to human, as human as possible, and so the molecule look very much like a human.
And there are really two ways. The classical humanization technique involving CDR grafting. You know, CDR is the regions that bind to the antigen. So the classical way of humanizing it is to try to convert any other sequence to human as much as possible, mainly in the framework, not in the CDRs. Typically, CDR is not touched.
The way we use, we call MLG humanization, basically utilize phylogenetic information. OK, so if you think about how a B cell evolved, B cells evolve very, very much like a mini-evolution within the system. So if you sequence many bioactive antibody and put all the sequences together into a lineage, you actually can trace back, say what is the progenitor B cell that result into a population of IgGs that have similar functions.
So the sequence function relationship will lead us to know which residue is important, which residue is not important. Therefore we can use that knowledge plus the 3D structure modeling to humanize it. So don't have the time to go into much of the detail. Hopefully I give you some idea how the humanization step. But it's a completely new IP to us.
So over the last couple of years, this is really only a two-year-old story. Many of you have not heard. In my previous talk, always talk about something else.
So we have built a capability from antigen preparation. And we try to make antigen as native as possible in a cell. And then we develop a whole set of assays. This is just a small list of assays where we would screen for biological activities of those antibodies. Then, of course, we check, across species, reactivities, with mouse, with monkey, because ultimately, [INAUDIBLE] test those drugs in animals.
Then we also try to profile the antibodies in different pathways, different tissue, and so forth, then, of course, try to analyze the sequence, map the epitope, and humanize it.
What I'd like to do quickly is to give you two examples of two drug candidates that we've been working on. First is antibody against TNF-alpha for treatment of rheumatoid arthritis. Actually, Junying already give you a very good introduction. TNF alpha is a pro-inflammatory cytokine, actually has-- it's quite a [INAUDIBLE] involving quite a lot of functions.
But mainly it's involving inflammation and immune system. And it's also involved in cell death, and cell proliferation, and so forth. And also overexpression of TNF alpha has been implicated in many diseases, especially in the inflammatory diseases.
Those are the different types of diseases that people try to develop a drug against those diseases. There are three major TNF alpha inhibitors. Enbrel is sold by Amgen. It's actually a soluble receptor. And Remicade and Humira are two monoclonal antibodies. One is derived from mouse. It's a chimeric molecule. Humira is an antibody come from phage display.
OK, and I just want to mention, collectively, these three drugs sold $10 billion last year, so it's a quite, quite large market. What do we try to do? Rheumatoid arthritis is quite a devastating disease, actually affects a large population. About 1% to 2% of world population eventually develop rheumatoid arthritis.
And it's a prevalent in women. Onset could vary from young age, 25 years old, all the way to 55 years old. Females tend to get more. And there's also other effects. So this is just a picture of that. It's actually quite a disease [? that suffers. ?]
As Junying mentioned earlier on, TNF alpha signaling, it's a trimer ligand bind to the receptor, ultimately activate downstream signal transduction pathways. So there is activation of NF-kappa-B. Eventually leads to inflammation, and also involving apoptosis and activate some of the MAP kinase pathways.
So what we decide to do is that we would like to develop degrade monoclonal antibody against TNF alpha. Basically, this is just a sequence of the event. We start looking for binders, monoclonal rabbit monoclonal antibody that binds, eventually screens through different affinity specificity, and humanized and tested in vitro. This is just a number of the clones that we handle. This is the assays that we go through.
So at the end, we have decided that clone 858 is the one that we will move forward. Then that molecule eventually becomes engineered into a human version. Just want to show you that the humanized version look like a human and behave like human, although we haven't tested in humans. But that's probably the best we can do.
This is the parental molecule. If you take the sequence, you do a blot analysis of heavy chain, light chain. And you see the target is a rabbit. And it actually cross-reacts-- I mean, it has homology to human, to rabbit, to dog.
But most other animals, after humanizing it, you take the molecule and blast the sequence, you see pretty much they look like human. There is no trace of rabbit at all.
It has significant higher affinity compared to the existing drug Remicade and similarly Humira as well. It has already a nanomolar affinity, but we're pushing down to a picomolar affinity. So after humanization, we're looking at almost 100-fold improvement in affinity. So the question is, does that translate into in vivo efficacy?
OK, this is still in vitro, so this is to test as the cytotoxicity activity of a TNF. As you can see, the parental molecule as well as a humanized molecule shows significant more potency than the leading drug on the market.
So then we decided to test this in mice model. This is actually a model that widely used for anti-TNF therapy. It's a human transgenic mice overexpressing human TNF alpha, also known as Taconic mice. So basically, this mice will develop spontaneously rheumatoid arthritis at a certain age. So we decided to treat them at 1 mg per kg, using our compound as well as the leading drug, Humira, through IP injection.
And normal mice, if you just look at [INAUDIBLE], normal mice has a paw open up. Arthritic mouse look like this. The paw is closed. If you're treat it with a drug, you see it look very much like normal.
And there's a whole series of clinical scores. You can look at it. OK, so what's really encouraging to us is you have three bars here. If you already have a set of mice that has disease onset, say let's say clinical score 3, 0 is normal, then the disease will progress over time if you just treat with saline, the control.
If you treat it with the leading drug Humira, disease stabilizes. But if you treat it with our drug, disease improves the symptoms. And that is also very visible in the histology analysis.
The joint of normal mice look like this. A rheumatoid arthritic mice joint look like this. If you're treat it with Humira, there's a big improvement here. But if you trade with our TNF drug, anti-TNF drug, it almost reverted back to normal mice.
So this is actually a very new test that was done by our collaborator [INAUDIBLE]. It's a very interesting observation. So let's say if you put the mice on a bar, grab on the bar, a normal mouse would grab on the bar for 60 seconds in the minute-- in other words, all the time.
And if you take the rheumatoid arthritis mice, they can only grab 10 seconds in a minute. And if you treat it with Humira, it will double the time. But if you treat it with our antibody, it goes back to almost normal in all three experiments. So quantitatively, this is significantly better.
OK, just very quickly, to summarize this, we have a novel humanized anti-TNF antibody. It comes from a rabbit. And it's picomolar affinity. It's got a better in vitro potency compared to the leading drugs, and also has better clinical scores in animal models as well.
And now we're actually looking into the downstream of the development process [INAUDIBLE], and try to figure out how to manufacture the compound. We hope we're able to push this into human in probably two to three years.
Give you an example number two, there's another drug that we attempt to develop. This is a humanized anti-VEGF antibody for treatment of a cancer. So as you know, VEGF is a growth factor that involved in the proliferation and growth of vessels involving both vasculargenesis and angiogenesis.
There are actually-- it's quite complex interacting. There's a number of VEGFs. You have A, B, C, D. And there are three different receptors. VEGF receptor 1, 2, and 3. Receptor 2 is really the one that involving angiogenesis.
So as the angiogenesis and the vasculargenesis involving cancer and the macular degeneration, age-related macular degeneration, and rheumatoid arthritis and diabetic retinopathy. So currently, there are two drugs, both sold by Genentech. One is called Avastin.
It's a humanized monoclonal antibodies for treatment of three types of cancer-- colon cancer, breast cancer, and lung cancer. And they also have a fragment of the same antibody, called Lucentis, for the treatment of age-related macular degeneration. And Avastin I think sold more than $2 billion last year.
So we decided to develop a better version of anti-VEGF antibody. And there's a similar scheme, as I showed you, for the anti-TNF antibody. So we basically identified more than 200 binders, and ultimately we identified the clone we call clone 21 being the best candidate.
So the process is very similar. Basically, we identify the binders. And then we do receptor-ligand binding [INAUDIBLE] assay, and identify the IC50. Then we also make sure it binds to the receptor KDR. And then we identify the ones that have a very high affinity.
Then we also study the stimulation of a phosphorylation and signal transduction pathway specificity-- then, of course, the molecule analysis of the sequence, and eventually humanize it. Then we also test them in the animal model. This is a mouse xenographic tumor models and also angiogenesis model. And then, of course, right now, we're starting [INAUDIBLE] and figure out how to deal with CMC and enabling the R&D filing.
OK, so this is some data on specificity. It's actually very interesting on the way we started. So the immunogen was VEGFa, human VEGFa. In this experiment, we tried to test specificity. There's two things here. One asks the question whether the antibody cross-react with mouse. Remember, mouse VEGF is different from human VEGF.
And in order to study the animal model, you actually would have preferred to have an antibody across species. Just remind you, most of the monoclonal antibody drugs are derived from mice. And therefore they do not cross react with mouse. Avastin does not across react with mouse.
OK so we actually were purposely looking for an antibody that recognizes both mouse and human. As you can see in this population, some only recognize human, doesn't recognize mouse. Some recognize mouse as well. So they probably represent different epitopes
The second thing we are looking for is, does the antibody-- is antibody specific to VEGFa, but does not recognize the VEGF b, c, and the d. And that is indeed the case. So it is a highly specific to VEGFa. All right, again, just like a anti-TNF antibody, with humanized it.
And this is, again, a schematic figure to show that the parental molecule looks like a rabbit. So it's a rabbit anybody. And after humanize it, again, there's no trace of rabbit anymore. It looks just like humans, probably more human than some of the other human you can find. Is my time up?
KELLY LIU: You have five minutes. But leave some time for questions.
GUO-LIANG YU: OK, almost there. So, again, when we measure the Kd affinity of our antibody bench mark with Avastin, this is a difference. So, again, we have also made antibody that is several hundred-fold higher in affinity.
The binding to the receptor actually shows very similar potency when you compare it to the Avastin. But then when we look at the phosphorylation, VEGF stimulated receptor phosphorylation, we actually see marked difference. The unhumanized as well as the humanized version look more potent than Avastin.
This is the data on the animal model. This is a xenographic model of lung cancer. So basically what we're looking at is over the time we measure the tumor size. And this is untreated control. You can see tumor growth, continue to grow.
And Avastin, you treat it with 5 mg per kg. That's Avastin. And these two are the unhumanized and the humanized version, basically completely inhibits the tumor growth. And we also have a colon cancer model. Quantitatively, it shows much better inhibition in terms of tumor growth.
We also look at the vascularization in those tumors. So this is just a picture. If you don't treat the mice with any drug, you see a lot of vascularization. Treated with Avastin, it's-- treated with Avastin, you see a lot of inhibition of vascularization, and same as using our antibodies.
But if you try to quantify it, that's what the result is. [? Contrary, ?] you see a lot of vascularization. Using Avastin, you see inhibition in both unhumanized and the human. This is actually just the rabbit version. You see significantly inhibition of angiogenesis.
So, again, to summarize it, a novel humanized anti-VEGFa antibody RabMAb with picomolar affinity, it actually recognizes different epitope from what Avastin recognize. It block the receptor binding, and blocks the phosphorylation, and block microvascular formation. So we show a better in vivo efficacy compared to Avastin in the animal model. And, again, this would be a very interesting candidate to move forward.
So just to conclude, RabMAb technology enabled novel drug discovery. We're talking about a completely new chemical entity-- well, a new biological markers. Even with old target, what I showed you are probably, by far, the two most validated targets, TNF alpha and VEGF. And we were able to demonstrate, in both cases, that RabMAb technology is able to make better version of it. And, of course, demonstration of in vivo efficacy of RabMAb is the first step.
I was commenting on what Dr. Ray Wu told me-- be patient. And developing a drug certainly requires a lot of patience. And I certainly take that word very much into my heart. And I'll probably devote my next 10 years, 15 years, into developing a new class of antibody drugs.
And then I'd like to acknowledge my colleagues and collaborators, both in the US, in China, the scientists at Epitomics, and our team in-- [? Econ's ?] our wholly-owned subsidiary in Hangzhou. We have about 120 people there.
And [INAUDIBLE] [? Bao ?] is our collaborator working on the anti-TNF drug. Actually, most of the data I presented here generated by their team. So I appreciate the collaboration and hard work together. Tychan is a company in [INAUDIBLE]. They're actually our partner for manufacturing antibodies.
And we also have [? Zhejiang ?] University, Professor [? Xiaoming, ?] and the Zhejiang Chinese Medical University Animal Center help us to build animal models and do animal studies. So with that, I'll take questions.
[APPLAUSE]
KELLY LIU: Questions?
AUDIENCE: You mentioned several times that these are new chemical entities. So when you compare this with a mouse sequence, particularly in the CDR, [INAUDIBLE] any of the long sequences, [INAUDIBLE] bind to the same antigen [INAUDIBLE]?
GUO-LIANG YU: Yeah, well as I mentioned in terms of VEGF anti-VEGF, we know it's different epitope from that bind by Avastin.
AUDIENCE: So it binds the same epitope [INAUDIBLE] different?
GUO-LIANG YU: Different, different epitope. And sequence, protein sequence is completely different.
AUDIENCE: [INAUDIBLE] so it is actually really interesting to hear that, actually, the rabbit immune system is just better [INAUDIBLE] antibodies. And [INAUDIBLE] comparing to llamas and a camel, with those single-peptide antibodies, which systems that actually have the most advantage if you say, OK, want to choose one system to generate antibodies?
GUO-LIANG YU: Yeah, it's actually a very interesting question. I always, after I did my work at a human genome sciences where we studied a large number of human genes, I said we need a technique that can make antibody in very large scale.
So when I look at the animals, OK, typically we use mice in little cage. And I said, the rabbit probably is the ideal size. You can handle them in a relatively contained space. If you talk about going llama or sheep or camel, it's going to be very difficult, and especially if you're talking about larger scale.
So in our company we have a facility that house about 4,000, 5,000 rabbit any given time. I just can't imagine, how can I have 4,000 camels.
[LAUGHTER]
It's just difficult. And the other thing, other important thing is, in terms of fusion partner, as far as [? now, ?] there's only mouse fusion partner and a rabbit fusion partner. There's no other type of fusion partner. So if you need to make monoclonal antibodies, you're going to have to use, say, phage display, or use other molecular means to get-- well, you won't get hybridoma-- get a cell that express IgG. So in terms of hybridoma, there's only rabbit and the mouse. Any other questions?
KELLY LIU: All right, thank you.
GUO-LIANG YU: OK, thank you.
[APPLAUSE]
[SIDE CONVERSATION]
KELLY LIU: All right, our next speaker is Ning Wei from Yale University Department of Molecular Cellular and Developmental Biology. She's a class 5 CUSBEAn.
NING WEI: I'd like to thank meeting organizers for this fantastic [INAUDIBLE]. And it's truly an honor to come here to talk in Dr. Ray Wu Memorial Symposium representing my class.
And Dr. Ray Wu really is a icon and role model for us. I don't know him personally very well, but I think I properly represent many of us, hundreds, one of the hundreds who Dr. Ray Wu don't know very well personally, but who benefited tremendously from his work, and from his work on CUSBEA, from his work on science.
Now, today I'll talk about something that I have been working on for many years. And I started by trying to understand-- what happened to this-- by trying to understand a very classic question in plant biology, photomorphogenesis.
We can go on with this slide. That's not a problem.
[SIDE CONVERSATION]
NING WEI: OK, I'm sorry for the break. OK, where-- yes, we all know that plants use sunlight as an energy source for photosynthesis, but light actually means a lot to a plant than photosynthesis. It's an important signal that guide development of plant.
So when plant germinate in the dark, it elongate and try to break the soil, break the surface and see the sunlight. And once they do exposed to sunlight, they open up and start their life. And this is called photomorphogenesis.
So to understand this process, genetic screening has been carried out-- OK.
SPEAKER 3: Let me see if we can get this working out for you.
NING WEI: Take another break.
SPEAKER 3: Sorry to interrupt you all. [INAUDIBLE] escape out of this for a moment. Is that OK?
NING WEI: OK, yes, that's fine.
SPEAKER 3: And full screen. I'm not very adept with PowerPoint at this--
NING WEI: All right, thank you.
[APPLAUSE]
[INAUDIBLE] I was talking about this genetic screen. And in this particular screen, mutants were isolated that, when they grow in the dark, it looks like it has been exposed to light. So it's called constitutive photomorphogenic, or it's also called de-etiolated, FUSCA.
This screen was carried out predominantly in [INAUDIBLE] lab and [INAUDIBLE] lab. And this is a saturation screen. And a few loci has been recovered. And the majority of those loci encode subunits of a [INAUDIBLE] protein complex. Now we call the COP9 signalosome. So this is the template [INAUDIBLE].
And this mutant, I want to mention, those mutant, all of those mutants, no mutant are lethal. They are essential genes in plants too. CSN or COP9 signalosome contains eight subunits. At the time when we had just identified this complex, I was thinking that I was working on a plant-specific mechanism because of the morphogenesis.
As it turned out, I was wrong. CSN it's found in fungi and animal cells. And I wondered at the time what CSN do in animal cells where there's no photomorphogenesis. So I'll come back to this point. But first of all, I want to talk about the biochemistry of CSN a little bit.
While all the CSN subunits were identified is clear that they are similar to a subset of genes from the 26S proteasome. And the proteasome can be developed to 20S catalytic particle and 19S regulatory particles. And all the subunits similar to CSN were from 19S.
And so at the time, we hypothesized that there must be a [? SOC ?] complex within 19S [INAUDIBLE], that is sort of like a cousin to CSN. And sure enough, [INAUDIBLE] lab, at the time, worked on the budding yeast proteasome. They identified a complete set of 19S subunits, and then separate-- they can break them apart into base and lid.
And lid has eight subunits. And each subunits pairs can pair with CSN. So this-- the fact that CSN is paralog of lid, which lid different from core and the base, which has-- that they are more ancient. They have primitive types in [INAUDIBLE]. And lid evolves with ubiquitin system. So this really suggests that CSN might be related to ubiquitin proteasome system.
And a few years later, the work from [? Rita ?] [? Shea's ?] lab found that CSN is in fact a protease, [INAUDIBLE] peptidase. And the substrate of CSN is cullin-RING ubiquitin E3 ligase. And this is a huge family of ubiquitin E3 ligase. It consists of a cullin, a RING finger protein, it has an adaptor, and substrate receptor.
And there are seven cullin members in human. And one of well known, the best known, this family of E3 ligases, this ICF complex. And all of the family of CRL ligase are modified by NEDD8 ubiquitin-like protein. This is more like similar to mono-ubiquitination. But this is not a ubiquitin. It's a NEDD8. It's a ubiquitin-like protein.
What CSN do is CSN cleaves NEDD8 from cullins, [? so ?] [? eight. ?] It carries out this reaction. So in a cell extract, if you add CSN, increase the amount of CSN, you can see the modified form of Cul1. Or all the cullins, in fact, get reduced and accumulate the unmodified form.
And similarly, and if you knock out CSN subunits, this is in fact a Arabidopsis protein extract. All the cullins, the majority of cullins, hyperneddylate. They accumulate in neddylated form, in modified form. And then, in vitro, they can be cleaved again by adding CSN back to the system. So that's a protease.
Going back to the function of CSN in different organisms, CSN can be found in yeast. But it's not that critical in yeast. They don't really care too much about it. But once you go to a more complex organism such as a Drosophila, now CSN mutants die at larvae stage. And in plant, as I mentioned, the CSN mutant die at seedling stage.
And we and others have done CSN knockout in mouse, and they die very early, and during embryo development at 7 and 1/2 days. So those, because they die so early, we cannot really learn too much about what CSN do in animals.
And we then developed conditional knockout system, and in collaboration with Richard Flavell's lab at Yale Medical School, we carried out-- there's a T-cell specific knockout of CSN8 subunits. And this is using CD4-cre deleter string.
So this is our knockout animal. And in this animal, CSN8 is deleted only in T-cells when CD4-cre is expressed. And this is the sibling control. So T-cells are generated in bone marrow. And they go to thymus to mature.
And eventually, the mature T-cells migrate to peripheral lymph organs where they rest there. And they rest until they are activated. They are stimulated, for example, by antigen stimulation. And then they undergo T-cells activation, which has a few components, T-cell expansion and differentiating into a effector T-cells.
CD4-cre express a late thymus stage. And we found in thymus, CSN still expressed and there's not much changed. But in peripheral system, CSN8 is [? completely ?] knocked out, is depleted. And in our system, once we knock out one CSN subunit, the complex falls apart and they all get disrupted. And then we can see the inactivation of the complex. And you see the cullins are hyperneddylated.
So this tells us it's a good system to look at T-cell activation during-- in peripheral organs. So here I will only concentrate, focus on one story. I don't have a lot of time here. And that is this the work is done by [? Sue ?] [INAUDIBLE] and [INAUDIBLE].
And they found that cell proliferation of knockout cells is severely, profoundly affected by knockout of CSN8. And those cells, the normal cells, when stimulated by antigen, they can undergo robust proliferation. But in knockout cells, this doesn't occur.
So let's dissect this, the process. The T-cell, when at the resting stage, they are quiescent. They're at G0 state. They must enter the cell cycle to become an effector cell prior to rapid proliferation phase.
And so in the previous slides, I showed you when we knockout CNS8, at this stage, it cannot proliferate. So we like to know whether it has a problem here, or here, or both. So we wanted to knock out CSN at this stage. So [? Sue ?] isolated CSN containing naive T-cells. She then turned into effector cells. And then she delete CSN8 in culture. This is in vitro.
And the [? fun-- ?] [? in ?] [? fact, ?] [INAUDIBLE] proliferation as [? the ?] CSN is [? deleted, ?] the knockout cells don't differ too much from other control cells, so they can proliferate once they turn into the effector T-cells. And this tells us that CSN8 or CSN complex is not too much involved in the housekeeping activity of cell cycle progression, but it is awfully important for cell cycle entry.
Now, this is-- the function of CSN in cell cycle entry not specific just to T-cells. We also see [? MEF ?] cells, and you see CSN deletion, the knockout, don't affect continuously cycling cells too much.
They can also exit cell cycle when [INAUDIBLE] were withdrawn. But the problem is cell cycle entry. They just simply cannot enter, re-enter the cell cycle, cannot initiate proliferation. So, yeah, so we like to understand what happened, and why it cannot initiate proliferation.
We look at the signaling process. Obviously, if you block the signaling, the cells won't know that they're supposed to replicate. But the signaling, when you look at pathways, they're all fun. [INAUDIBLE] was phosphorylated. And they can also degrade upon activation.
And [? MEF ?] [INAUDIBLE] look OK. And in short, all the signaling pathway we look at, they are normal in knockout cells. We also look at some substrate of SCF. Some of the substrates are cell cycle inhibitors such as Pdcd4 and p27.
And those proteins can be degraded upon stimulation by antigen in knockout cells, as well as just in normal cells. So all these substrates of SCF are not affected by CSN deletion. So we look at-- then we look at Rb phosphorylation, because Rb is an important molecule during cell cycle entry.
And this Rb has to be phosphorylated at specific sites. And this phosphorylation is absent in knockout cells. And phosphorylation of Rb [INAUDIBLE] are mediated by G1 cyclin-CDK complexes. And they are also inhibited and controlled by p21. So we look at the protein level of those genes in knockout cells.
And as it turned out, this is where the problem lies. And if we just look at the cyclin E expression, normally, before activation, cyclin E is expressed at very low level. And they are tremendously induced once you stimulate by antigen.
And if you look at the knockout cells, the cyclin E expression is already at a pretty high level-- not very high, slightly higher level. And they stay flat. Their expression is constitutive. And this happens to most of the G1 cyclin-CDK molecules.
And also P27 is strongly induced in the knockout cells compared to the normal cells. And this is why that Rb cannot be properly phosphorylated during the cell cycle entry. And that cause the phenotype of cell cycle problem.
And all of this effect in the expression level of the T1 cyclins and the CDKs, they are all at mRNA level. And you see here, in a knockout, the expression is pretty much constitutive. And then they are higher at 0 hours before stimulation. And they don't get much induced after stimulation. And [INAUDIBLE] protein level.
Not all genes are affected. For example, there's A20. That's a deubiquitinase. These genes can be induced perfectly fine in the knockout cells. And so this indicates that CSN has a function in gene expression. And the [INAUDIBLE] IP experiment shows that indeed the CSN subunit can be recruited to the promoters of the cyclin D2 gene.
And that indicates the CSN actually have the capacity to directly regulate gene expression. In the past, we thought the function of CSN with gene expression has been indirect. But the fact that they are being recruited to the promoter, to a chromatid, indicate they probably can directly regulate it in the vicinity of transcription complexes and the transcription factors.
All this is, I just trying to make a point. And the point is here that the function of CSN in signal-dependent gene expression is in fact conserved from Arabidopsis to mouse. And this function is consistent with two-- it has a dual role. And then the function in silencing gene expression in the absence of-- in the signal. And it also facilitate gene induction in response to signal activation.
And this is not new. in fact, when we look at a very old Northern blot that is published in 1992. And this, we're working on looking at light-induced genes in Arabidopsis COP9 mutant. That is the CSN8 mutant.
And if you look at these two genes, the [? RBS ?] gene, which is highly induced in the light compared to the dark, but this gene, the mutant, in the dark, is already highly induced. So expression of this gene is pretty much constitutive. And it is the lack of repression of those light-induced genes that probably contribute to the constitutive photomorphogenic phenotype in plants.
And in the meantime, we also see the lack of induction as you look at the cab gene expression. And cab gene is acutely induced in the light. And in the mutant, it can never express at this high level. So it also has the problem in gene induction.
And this I think is probably contribute to the lethality of the mutants. And at the time, we didn't really focus on this aspect of the phenotype. And in the mouse, we see both the lack of repression before stimulation and the lack of induction after stimulation.
And in this particular case, in T-cell activation, there's a [INAUDIBLE] activation that leads to failed cell cycle initiation. We haven't found a [INAUDIBLE], the cellular phenotype, due to this problem, [? as yet. ?]
So, finally, I just want to make the point that what CSN do in plant and animals are really not much different, really. And because it's conservation, the [? method ?] of CSN is probably general and it's probably fundamental.
And it is probably involved in developmental transitions, and involved in the transition of different physiological state. In plant, it could be photomorphogenesis. And in animal cells, it can be T-cell activation.
And, finally, I'd like to thank the people who did this work. The knockout work is mostly done by [? Sue ?] [INAUDIBLE], a very talented graduate student, in collaboration with [INAUDIBLE] from [? Richard ?] [INAUDIBLE] lab. And he now has his own lab at St. Jude's. And [INAUDIBLE] did the [INAUDIBLE] IP experiment. And thank you.
[APPLAUSE]
AUDIENCE: I assume a process this complicated must be regulated pretty closely. Is that why there's so many [INAUDIBLE] that are involved in forming this complex structure?
NING WEI: If forming the complex structure? Yeah, this complex is-- it's a isopeptidase. It's a protease, but it's actually a lot more than that.
And as you said, it's a very potent protease. If you put it in vitro, it carries out this activity in minutes. It's very fast. So it has to be regulated in a certain way.
And so the fact that it's a complex, it's a part of the complex, means it's tightly regulated. It's regulated by several ways. One is the interaction with the substrate, which is cullin ligases. And there are some studies shows that some signal, for example UV, can triggers the association of CSN for one type of cullin ligases and [INAUDIBLE] another type.
So that's one type of regulation. And there are also other ways to regulate. And in addition to being a protease, it's also an important one-- we have done this experiment. We preserve the deneddylation activity and knock out the other domains of CSN-- one of the CSN subunits. This also causes lethality. So it does [INAUDIBLE] them being a protease.
AUDIENCE: [INAUDIBLE] So one of the mechanisms proposed [INAUDIBLE] for the function of a signalosome is to deneddylate. And the neddylation preventing the binding of CAND1.
So I wonder, in your knockout cells, can you still detect any cullin and CAND1 binding? So the idea is, if every cullin is deneddylated and the CAND1 is supposed to be unable to bind anymore-- have you checked--
NING WEI: We didn't do this experiment. But this has being done in other models. Like in plant, they have done that. And yeah, once it's neddylated, CAND1 cannot bind to cullins because is only binds to un-neddylated form of cullins. We didn't test this, the interaction. But I thought this is-- it's already pretty much established that [INAUDIBLE].
AUDIENCE: No one has [INAUDIBLE] method you have. [INAUDIBLE] are neddylated so that you can really confirm by showing there's no detectable binding of CAND1 and cullin. [INAUDIBLE].
NING WEI: Yeah, yes, yeah.
AUDIENCE: Is the transcriptional activity involved in neddylation?
NING WEI: That's a very good question. And I think this is a question that the field should actively address. And there ubiquitin has been shown to tightly-- to a link to the transcription. But the mechanism, I don't think it's established.
A lot of hypotheses on how ubiquitination may regulate transcription elongation process. And there are several models being proposed on how the ubiquitination of transcription factors that in fact activate the ubiquitin factor mediate the transcription.
And I think-- which is [INAUDIBLE] at the moment-- that CSN may be involved in this process, because it regulates turnover of transcription factor, many of which are substrates of cullins. But NEDD8 has been found to target other proteins other than cullins.
And some of those studies indicated that NEDD8 may have a role in transcription. But, again, this is not very established. It's not completely accepted in the field at the moment. It's still quite vague. And I think there's a lot of room to study.
We ourselves, we have found that CSN bind to transcription complex, in histone [INAUDIBLE], transcription effect complex. And not only us, and also people from Drosophila field also found association of CSN with histone acetylation complexes.
And so there may be something there that may be interesting. And then I also want to mention that, you just mentioned CAND1. And the CAND1 bind to cullins in a neddylation-specific manner, and only to unneddylated cullins.
But then CAND1 was previously also isolated as a TPP-- in [INAUDIBLE] protein. It was isolated in connection with transcription, and which may suggest there's something there. But right now, it's really an open field really. It's not understood.
AUDIENCE: Have you looked at a stem cell [INAUDIBLE] and see whether it regulated G0 to G1, cell cycle [INAUDIBLE]?
NING WEI: I'm sorry, I didn't--
AUDIENCE: Have you looked a stem cell activity in terms of G0 to G1 cell cycle [INAUDIBLE]?
NING WEI: Yeah, this is a very attractive model. And I think there are people working on this. Yeah, it is. It's immediately suggested there could be many functions that CSN is involved, many processes, [INAUDIBLE].
KELLY LIU: Any other questions? All right, let's thank all the speakers [INAUDIBLE].
Ray J. Wu, late Cornell University professor of molecular biology and genetics, is widely recognized as one of the fathers of genetic engineering. He developed and sought to feed the world with a higher yielding rice that resists insects and drought. Colleagues, friends and former students of Ray Wu came together at a symposium, October 3, 2008, to celebrate his lifetime contributions to science and humanity.
Speakers:
Opening remarks by Susan Henry, Ronald P. Lynch Dean of the College of Agriculture and Life Sciences, Cornell University Hunter Rawlings, President Emeritus, Cornell University: "Remembering Ray Wu" Dr. Ajay Garg, Cornell University: "Feeding the hungry -- Transgenic rice plants tolerant of drought and salt" Dr. Junying Yuan, professor of cell biology, Harvard Medical School: "A quest to understand the mechanisms of neurodegeneration" Dr. Guoliang Yu, chairman, president and CEO, Epitomics, Inc.: "Discovery and development of monoclonal antibody drugs using RabMAb technology" Dr. Ning Wei, principle investigator, Department of Molecular, Cellular and Developmental Biology, Yale University: "The COP9 Signalosome: A regulator of protein degradation and gene expression in plants and animals"
