Tuesday, February 24, 2015
Broad 100

New Frontiers in Biological Engineering

Genetically Engineered Antibodies Show Enhanced HIV-Fighting Abilities

Capitalizing on a new insight into HIV's strategy for evading antibodies—proteins produced by the immune system to identify and wipe out invading objects such as viruses—Caltech researchers have developed antibody-based molecules that are more than 100 times better than our bodies' own defenses at binding to and neutralizing HIV, when tested in vitro. The work suggests a novel approach that could be used to engineer more effective HIV-fighting drugs.

"Based on the work that we have done, we now think we know how to make a really potent therapeutic that would not only work at relatively low concentrations but would also force the virus to mutate along pathways that would make it less fit and therefore more susceptible to elimination," says Pamela Bjorkman, the Max Delbrück Professor of Biology and an investigator with the Howard Hughes Medical Institute. "If you were able to give this to someone who already had HIV, you might even be able to clear the infection."

The researchers describe the work in the January 29 issue of Cell. Rachel Galimidi, a graduate student in Bjorkman's lab at Caltech, is lead author on the paper.

The researchers hypothesized that one of the reasons the immune system is less effective against HIV than other viruses involves the small number and low density of spikes on HIV's surface. These spikes, each one a cluster of three protein subunits, stick up from the surface of the virus and are the targets of antibodies that neutralize HIV. While most viruses are covered with hundreds of these spikes, HIV has only 10 to 20, making the average distance between the spikes quite long.

That distance is important with respect to the mechanism that naturally occurring antibodies use to capture their viral targets. Antibodies are Y-shaped proteins that evolved to grab onto their targets with both "arms." However, if the spikes are few and far between—as is the case with HIV—it is likely that an antibody will bind with only one arm, making its connection to the virus weaker (and easier for a mutation of the spike to render the antibody ineffective).

To test their hypothesis, Bjorkman's group genetically engineered antibody-based molecules that can bind with both arms to a single spike. They started with the virus-binding parts, or Fabs, of broadly neutralizing antibodies—proteins produced naturally by a small percentage of HIV-positive individuals that are able to fight multiple strains of HIV until the virus mutates. When given in combination, these antibodies are quite effective. Rather than making Y-shaped antibodies, the Caltech group simply connected two Fabs—often from different antibodies, to mimic combination therapies—with different lengths of spacers composed of DNA.

Why DNA? In order to engineer antibodies that could latch onto a spike twice, they needed to know which Fabs to use and how long to make the connection between them so that both could readily bind to a single spike. Previously, various members of Bjorkman's group had tried to make educated guesses based on what is known of the viral spike structure, but the large number of possible variations in terms of which Fabs to use and how far apart they should be, made the problem intractable.

In the new work, Bjorkman and Galimidi struck upon the idea of using DNA as a "molecular ruler." It is well known that each base pair in double-stranded DNA is separated by 3.4 angstroms. Therefore, by incorporating varying lengths of DNA between two Fabs, they could systematically test for the best neutralizer and then derive the distance between the Fabs from the length of the DNA. They also tested different combinations of Fabs from various antibodies—sometimes incorporating two different Fabs, sometimes using two of the same.

"Most of these didn't work at all," says Bjorkman, which was reassuring because it suggested that any improvements the researchers saw were not just created by an artifact, such as the addition of DNA.

But some of the fabricated molecules worked very well. The researchers found that the molecules that combined Fabs from two different antibodies performed the best, showing an improvement of 10 to 1,000 times in their ability to neutralize HIV, as compared to naturally occurring antibodies. Depending on the Fabs used, the optimal length for the DNA linker was between 40 and 62 base pairs (corresponding to 13 and 21 nanometers, respectively).

Taking this finding to the next level in the most successful of these new molecules, the researchers replaced the piece of DNA with a protein linker of roughly the same length composed of 12 copies of a protein called tetratricopeptide repeat. The end product was an all-protein antibody-based reagent designed to bind with both Fabs to a single HIV spike.

"That one also worked, showing more than 30-fold average increased potency compared with the parental antibodies," says Bjorkman. "That is proof of principle that this can be done using protein-based reagents."

The greater potency suggests that a reagent made of these antibody-based molecules could work at lower concentrations, making a potential therapeutic less expensive and decreasing the risk of adverse reactions in patients.

"I think that our work sheds light on the potential therapeutic strategies that biotech companies should be using—and that we will be using—in order to make a better antibody reagent to combat HIV," says Galimidi. "A lot of companies discount antibody reagents because of the virus's ability to evade antibody pressure, focusing instead on small molecules as drug therapies. Our new reagents illustrate a way to get around that."

The Caltech team is currently working to produce larger quantities of the new reagents so that they can test them in humanized mice—specialized mice carrying human immune cells that, unlike most mice, are sensitive to HIV.

Along with Galimidi and Bjorkman, additional Caltech authors on the paper, "Intra-Spike Crosslinking Overcomes Antibody Evasion by HIV-1," include Maria Politzer, a lab assistant; and Anthony West, a senior research specialist. Joshua Klein, a former Caltech graduate student (PhD '09), and Shiyu Bai, a former technician in the Bjorkman lab, also contributed to the work; they are currently at Google and Case Western Reserve University School of Medicine, respectively. Michael Seaman of Beth Israel Deaconess Medical Center and Michel Nussenzweig of the Rockefeller University in New York are also coauthors. The work was supported by the National Institutes of Health through a Director's Pioneer Award and a grant from the HIV Vaccine Research and Design Program, as well as grants from the Collaboration for AIDS Vaccine Discovery and the Bill and Melinda Gates Foundation. Nussenzweig is also an investigator with the Howard Hughes Medical Institute.

Kimm Fesenmaier
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Getting a Better Grip on HIV
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Friday, February 13, 2015
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Backpocket Barnburner: A Lightning Quick Overview of Educational Theory

Why Do We Feel Thirst? An Interview with Yuki Oka

To fight dehydration on a hot summer day, you instinctively crave the relief provided by a tall glass of water. But how does your brain sense the need for water, generate the sensation of thirst, and then ultimately turn that signal into a behavioral trigger that leads you to drink water? That's what Yuki Oka, a new assistant professor of biology at Caltech, wants to find out.

Oka's research focuses on the study of how the brain and body work together to maintain a healthy ratio of salt to water as part of a delicate form of biological balance called homeostasis.

Recently, Oka came to Caltech from Columbia University. We spoke with him about his work, his interests outside of the lab, and why he's excited to be joining the faculty at Caltech.


Can you tell us a bit more about your research?

The goal of my research is to understand the mechanisms by which the brain and body cooperate to maintain our internal environment's stability, which is called homeostasis. I'm especially focusing on fluid homeostasis, the fundamental mechanism that regulates the balance of water and salt. When water or salt are depleted in the body, the brain generates a signal that causes either a thirst or a salt craving. And that craving then drives animals to either drink water or eat something salty.

I'd like to know how our brain generates such a specific motivation simply by sensing internal state, and then how that motivation—which is really just neural activity in the brain—goes on to control the behavior.


Why did you choose to study thirst?

After finishing my Ph.D. in Japan, I came to Columbia University where I worked on salt sensing mechanisms in the mammalian taste system. We found that the peripheral taste system has a key function for salt homeostasis in the body by regulating our salt intake behavior. But of course, the peripheral sensor does not work by itself.  It requires a controller, the brain, which uses information from the sensor. So I decided to move on to explore the function of the brain; the real driver of our behaviors.

I was fascinated by thirst because the behavior it generates is very robust and stereotyped across various species. If an animal feels thirst, the behavioral output is simply to drink water. On the other hand, if the brain triggers salt appetite, then the animal specifically looks for salt—nothing else. These direct causal relations make it an ideal system to study the link between the neural circuit and the behavior.


You recently published a paper on this work in the journal Nature. Could you tell us about those findings?

In the paper, we linked specific neural populations in the brain to water drinking behavior. Previous work from other labs suggested that thirst may stem from a part of the brain called the hypothalamus, so we wanted to identify which groups of neurons in the hypothalamus control thirst. Using a technique called optogenetics that can manipulate neural activities with light, we found two distinct populations of neurons that control thirst in two opposite directions. When we activated one of those two populations, it evoked an intense drinking behavior even in fully water-satiated animals. In contrast, activation of a second population drastically suppressed drinking, even in highly water-deprived thirsty animals.  In other words, we could artificially create or erase the desire for drinking water.

Our findings suggest that there is an innate brain circuit that can turn an animal's water-drinking behavior on and off, and that this circuit likely functions as a center for thirst control in the mammalian brain. This work was performed with support from Howard Hughes Medical Institute and National Institutes of Health [for Charles S. Zuker at Columbia University, Oka's former advisor].


You use a mouse model to study thirst, but does this work have applications for humans?

There are many fluid homeostasis-associated conditions; one example is dehydration. We cannot specifically say a direct application for humans since our studies are focused on basic research. But if the same mechanisms and circuits exist in mice and humans, our studies will provide important insights into human physiologies and conditions.


Where did you grow up—and what started your initial interest in science?

I grew up in Japan, close to Tokyo, but not really in the center of the city. It was a nice combination between the big city and nature. There was a big park close to my house and when I was a child, I went there every day and observed plants and animals. That's pretty much how I spent my childhood. My parents are not scientists—neither of them, actually. It was just my innate interest in nature that made me want to be a scientist.


What drew you to Caltech?

I'm really excited about the environment here and the great climate. That's actually not trivial; I think the climate really does affect the people. For example, if you compare Southern California to New York, it's just a totally different character. I came here for a visit last January, and although it was my first time at Caltech I kind of felt a bond. I hadn't even received an offer yet, but I just intuitively thought, "This is probably the place for me."

I'm also looking forward to talking to my colleagues here who use fMRI for human behavioral research. One great advantage about using human subjects in behavioral studies is that they can report back to you about how they feel. There are certainly advantages of using an animal model, like mice. But they cannot report back. We just observe their behavior and say, "They are drinking water, so they must be thirsty." But that is totally different than someone telling you, "I feel thirsty." I believe that combining advantages of animal and human studies should allow us to address important questions about brain functions.


Do you have any hobbies?

I play basketball in my spare time, but my major hobby is collecting fossils. I have some trilobites and, actually, I have a complete set of bones from a type of herbivorous dinosaur. It is being shipped from New York right now and I may put it in my new office.

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Saturday, January 24, 2015
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The personal side of science

Wednesday, February 4, 2015
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Meet the Outreach Guys: James & Julius

Wednesday, February 18, 2015
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HALF TIME: A Mid-Quarter Meetup for TAs

Four Caltech Professors Elected to National Academy of Inventors

Caltech professors Frances Arnold, David Baltimore, Carver Mead, and Axel Scherer have been named fellows of the National Academy of Inventors (NAI).

Election as an NAI fellow is an honor bestowed upon academic innovators and inventors who have "demonstrated a prolific spirit of innovation in creating or facilitating outstanding inventions and innovations that have made a tangible impact on quality of life, economic development, and the welfare of society." Fellows are named inventors on U.S. patents and were each nominated by their peers for their contributions to innovation.

According to its website, the NAI "was founded in 2010 to recognize and encourage inventors with patents issued from the U.S. Patent and Trademark Office, enhance the visibility of academic technology and innovation, encourage the disclosure of intellectual property, educate and mentor innovative students, and translate the inventions of its members to benefit society."

Frances Arnold is the Dick and Barbara Dickinson Professor of Chemical Engineering, Bioengineering and Biochemistry, and director of the Donna and Benjamin M. Rosen Bioengineering Center at Caltech. Her group has pioneered methods of "directed evolution" to engineer new proteins in the lab. The method is now widely used to create catalysts for use in industrial processes, including the production of fuels and chemicals from renewable resources. The Arnold group also uses the results of laboratory evolution experiments to elucidate principles of biological design. Arnold is a coinventor on more than 40 U.S. patents. She was inducted into the National Inventors Hall of Fame in 2014 and received the National Medal of Technology and Innovation in 2013.

David Baltimore is the Robert Andrews Millikan Professor of Biology, and President Emeritus of Caltech. His research group focuses on two major areas: investigation of the development and functioning of the mammalian immune system, and translational studies of the viral transfer of genes into immune cells to increase their ability to fight disease and resist cancer. He was awarded the Nobel Prize in Physiology or Medicine in 1975.

Carver Mead (BS '56, MS '57, PhD '60) is the Gordon and Betty Moore Professor of Engineering and Applied Science, Emeritus, in computation and neural systems. Mead has significantly advanced the technology of integrated circuits by developing a method called very-large-scale integration (VSLI) that allows engineers to combine thousands of transistors onto a single microchip, thus exponentially expanding computer processing power. He received the National Medal of Technology and Innovation in 2002.

Axel Scherer is the Bernard Neches Professor of Electrical Engineering, Medical Engineering, Applied Physics and Physics, and the director of the Caltech Global Health Initiative. Holding more than 100 patents in the area of nanofabrication and device design, Scherer has most recently developed ways to integrate optics, electronics, and fluidics into sensor systems. Much of his work is currently focused on systems for medical diagnosis and health monitoring through molecular pathology and wireless implants. Over the past decades, technology from his group has been commercialized through several start-up ventures in telecommunications and health care devices.

Arnold, Baltimore, Mead, and Scherer will join Vice Provost Morteza Gharib, the Hans W. Liepmann Professor of Aeronautics and Bioinspired Engineering, and Robert H. Grubbs, the Victor and Elizabeth Atkins Professor of Chemistry, as Caltech's Fellows of the NAI. They will be inducted during the fourth annual conference of the National Academy of Inventors, which will be held at Caltech on March 20, 2015.

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SKIES App Aids Learning in Caltech Classrooms

When you first walk into Bruce Hay's genetics class, it looks like any other 21st-century college lecture hall: the professor, backed by his PowerPoint slides, faces a room full of students with iPads. However, as Hay delivers a lecture about the mechanisms that inhibit gene expression, a rectangular yellow bubble suddenly pops up on the screen below his lecture slide. It's from a student, asking a question about RNA interference. Soon another bubble pops up, this one with a link to a video that explains how microRNAs can affect the color pattern of flower petals. Other bubbles branch off from each other, and hands rise into the air.

Suddenly, you realize you're in the classroom of the future.

Those pop-up bubbles are a key component of a new interactive lecture format made possible by an app developed by two Caltech alums and brought to campus by Caltech's Center for Teaching, Learning, and Outreach (CTLO). The iPad app—produced by Su-Kam Intelligent Education Systems, or SKIES, named after cofounders Julius Su (BS '98, BS '99, PhD '07) and Victor Kam (PhD '08)—is now being used in several classes on campus.

"It's been known for a long time that lecturing—just a professor teaching and the students passively listening—is not an optimal way for students to learn. You always want to have students doing some thinking, some processing, and some recall during the lecture," says Su, who is a CTLO program manager in addition to being one of the cofounders of SKIES.

With this in mind, Su and his colleagues at CTLO have been experimenting with ways to change the traditional Caltech classroom. The SKIES app is based on one such approach—active learning, which gets students participating in a variety of ways, and is supported by a great deal of evidence indicating its effectiveness.

The wiki-like app facilitates this kind of active learning by allowing students to directly interact with lecture materials both inside and outside the classroom. The app compiles and connects notes, links, videos, and other materials—contributed by students, teaching assistants, and the professor—into a branching tree of collective knowledge stemming from an initial seed of slides or other multimedia material.

This concept of students and teachers interacting to create knowledge together is what drew Professor of Biology Bruce Hay to become one of the first users of the SKIES app in 2012.

Students can ask questions during Hay's lecture in the SKIES app. The questions or comments pop up below the lecture slide in real time.

"The big struggle that I have—and that lots of people have—is just getting students to ask questions; getting people to turn the class into more of a discussion rather than just the lecturer speaking," Hay says. "I knew that Julius and Victor were developing this prototype, and I thought, I've been teaching this same course for 15 years now and maybe this would be a good idea, to just try something new that might make it a little more interactive."

In the three years Hay has used the app in his genetics class, he says the app has done just what he'd hoped it would: provided an alternative channel by which students can participate. During class, Hay says students often add cards to his lecture slides as a form of public note-taking; for example, in one lecture, a student added a card to a particular slide, saying, "Professor Hay says this would be an excellent exam question." After class is over, he says, students often post cards with questions about the day's materials; these can then be addressed by Hay or the class's teaching assistants, either directly in class or through another branch of cards in the app.

Hay says that the app lets him monitor what students are contributing—and it allows him to promote and highlight information he considers particularly helpful and relevant to understanding the lecture material. Conversely, the app also allows students to rate how well they understand his lecture slides, as well as the cards added by TAs and other students.

"After class, I take a look at the reviews to see how well the students understood what we talked about in class that day," Hay says. "If I see that a slide is rated green [the app's version of a thumb's up], I can assume that it was pretty straightforward and understandable. But if the students have rated it red, I can add extra material to the slide, like additional text or figures. Then, in the next class, I can go back and say, 'It looks like this part was a little bit difficult. Let's just go back and review it again before we go on to the new stuff.'"

Although these continuous double checks require a bit more effort on the part of the professor and the students, they seem to be paying off: Hay says the average grades in his class went up by almost a full grade point after his first year using the app. The improvement was so noticeable, he says, "it was actually almost frightening. That was probably the biggest indication this was making a difference in terms of learning, instead of just making it fun for me and them."

In the SKIES app, students create dialogue by adding 'cards' that branch off of the professor's lecture slides.

Hay says the added content from students in the SKIES app over the past three years has enabled his course "tree" to grow and improve each term. "I'm not just repeating all the same slides every year. Many of the slides stay the same, but now I add new things, based on what the students found helpful in terms of explanations, quiz questions, and examples. So everyone is involved in making the course better," he notes.

After hearing about some of the app's early successes, other instructors across campus began using SKIES in their courses. This includes Bill Goddard, Charles and Mary Ferkel Professor of Chemistry, Materials Science, and Applied Physics, who has used SKIES to teach lectures and manage group projects in his computational and theoretical chemistry classes; Jeff Mendez, lecturer in chemistry, who has used SKIES to teach the lecture portion of his freshman solar chemistry lab; and Yaser Abu-Mostafa, professor of electrical engineering and computer science, who has used SKIES to promote and manage discussions in the in-class portion of his well-regarded Learning From Data MOOC, which has attracted over 200,000 students within and outside Caltech since its inception.

The app is also being used by many Caltech outreach programs, such as the Summer Research Connection, the Community Science Academy, and Harry Gray's Solar Army, to broaden the impact of university initiatives at the K–12 level; and is being used to teach several classes at Pasadena City College, as well as in several local middle schools and high schools.

SKIES is currently only available on iPad and iPhone, and as the app's popularity has grown, support from the Provost's Innovation in Education Fund and the Bechtel Foundation fund has allowed CTLO to expand the use of SKIES on campus through the purchase of more iPads. In the future, Su hopes that the app's reach will grow even further, both on iPads and eventually by expanding the app to work on other operating systems.

Aside from those few comments on the hardware limitations, Su says the feedback he and his colleagues at the CTLO have received from professors and students who have been using SKIES has been overwhelmingly positive.

"CTLO is continually providing Caltech faculty and TAs with evidence about what helps students learn more effectively. Active and collaborative approaches tend to work well," he says. "This app is just one way to foster more active and collaborative learning, but I think we can already see that it's providing new ways for professors to make classes even more lively and engaging for students at Caltech."

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An App for the Teacher
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Tuesday, December 2, 2014
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