CAR-T Pioneer and Noted Mentor Art Weiss, MD, PhD

A. I was very surprised to receive the Coley Award since my work has not focused directly on translational cancer research. Moreover, since it largely recognizes work done in my lab almost 30 years ago, the Coley Award was particularly unexpected. However, it was gratifying to receive the award because it is recognizing the value and importance of very fundamental research, in this case focused on the question of how T cell antigen receptor signals. Bryan Irving, a BMS student in my lab, using a chimeric receptor, showed that the zeta chain cytoplasmic domain was sufficient to induce TCR like signaling via a much simpler chimeric receptor construct. Many people contributed to the concept that chimeric receptors containing the zeta chain cytoplasmic domain could be harnessed to allow T cells to be redirected to respond to antigens in a non-MHC dependent way. I am particularly pleased to share the Coley Award with key contributors from that era, Zelig Eschar, Larry Samelson and Brian Seed.

A. I was the only child of Holocaust survivors. Born in Landskrona, Sweden, I immigrated with my parents as Romanian refugees to Chicago when I was two years old. I grew up in Chicago where I had a wonderful biology teacher (Harold Kiehm) in a Chicago public high school who recognized my strong interest in biology and convinced me to participate in NSF summer research programs. I worked in a public health lab in Grand Forks, North Dakota the summer after my sophomore year and the next summer at the Jackson Lab in Bar Harbor Maine, the home of many genetically inbred mice that have been so important for many scientists over decades. These experiences had a big influence on hooking me on research.

While an undergrad at Johns Hopkins, my interest in research was reinforced when I worked in Professor Michael Edidin’s cell biology lab studying membrane fluidity using fluorescent antibodies. I had a blast doing research and even published two papers with Edidin. However, I wanted more relevance to my research and thought medicine might be the best pathway forward for me.

I was fortunate to be admitted to the University of Chicago medical school but by the winter quarter, I was missing working in the lab. My exposure to working with antibodies in the Edidin lab kindled an interest in immunology and there seemed to be a lot to learn about this very important system. I explored several labs and eventually settled on studying transplantation tolerance in the lab of Professor Frank Fitch. I relished my elective time working in his lab. In the spring of that year, funding that had been withheld for MSTP (MD/PhD) program was released and Fitch encouraged me to apply. After a lot of soul searching, I applied and was admitted to this program which funded the remainder of my education at Chicago.

I had a wonderful time under Fitch’s mentorship, studying the immunologic mechanisms responsible for a form of antigen specific tolerance in a rat kidney transplant model. Because of funding shortfalls for the seventh year of my MSTP training, Fitch convinced me to spend six months in the Swiss Institute for Experimental Cancer Research in Lausanne, Switzerland, where he had previously spent two sabbaticals. It was a great scientific and cultural experience for me and my wife, Shirley.

A. I chose to rank the UCSF internal medicine residency program as my first choice for two reasons: UCSF had a very large kidney transplantation program; and the residents seemed happier than the other places I interviewed. I was fortunate to match at UCSF. However, my second month here, I was an intern on the kidney transplant unit, but was disappointed in the lack of complexity of the patients. I was largely interested in solving unusual medical puzzles, patients that were difficult to diagnose, and with unusual disease pathogenesis. My third month as an intern I rotated on the internal medicine service at the VAMC and met Professor Bill Seaman, a rheumatologist consulting on one of our patients who posed a diagnostic dilemma. Seaman made astute observations on the patient and discussed the unusual differential diagnoses that might be involved. He also took an interest in me and my background in immunology. He and others sold me on rheumatology in which many patients present with autoimmune manifestations of disease. There seemed to be a lot to learn about disease pathogenesis which was centered around abnormalities of the immune system.

UCSF was an exciting place at the time because the revolution in molecular biology was taking place here based on the work of Herb Boyer (recombinant DNA technology) and Bishop and Varmus (oncogenes). We didn’t have much space on the Parnassus campus, so junior and senior scientists were constantly running into each other. The basic science seminars and journal clubs were all on this campus and the collaborative spirit was very strong. The immunology community, however, was tiny, as were many other programs. With the establishment of the Mission Bay campus, there came growth and new opportunities but also a dispersal and separation of many scientists and programs. I think we’re still trying to figure out how to optimize our lost interactions. I see less of old colleagues and friends. It takes more effort to collaborate across campuses.

However, one consequence was the immunology graduate program could grow from about six original faculty to more than 50 today, with most of them based at Parnassus. Immunology, which was regarded weak in the ‘80s and early ‘90s, is one of the strongest research programs at UCSF and is among the top immunology graduate programs nationally. The establishment and generous funding of ImmunoX has extended the influence of immunology within the research community and offers great opportunities for the future.

A. I wound up in Professor Jack Stobo’s lab as a rheumatology fellow in 1983.  At the time, so little was known about the normal immune system- few relevant genes had been cloned - that it seemed hopeless to try to understand how the system was abnormal in rheumatic diseases.  So, I decided to characterize the normal immune system first.   I set out to identify the T cell antigen receptor (TCR), a task that had eluded many.  Within two months in the lab, it was identified - but not by me, nor at UCSF.   Others identified clone-specific disulfide linked ab hetero dimers on the surface of clonal T cell populations.  One group headed by Ellis Reinherz at Harvard suggested the clone specific ab heterodimer was associated with a complex of other proteins now called CD3 chains.  Monoclonal antibodies to CD3 could induce T cells to proliferate.  So, it was speculated that CD3 might be involved in signal transduction by the TCR.  I tried testing this hypothesis using somatic cell genetics by isolating mutant cell lines that lacked either the ab chains or CD3 from the Jurkat T cell leukemia line.  It wasn’t possible.  The mutant lines lost both ab and CD3.  It turned out that all the lines lacked a or b chains.   Ultimately, we had to reconstitute these lines.  We did with the help of Pam Ohashi and Tak Mak.  We had to use protoplast fusion (fusing e-coli protoplasts to a b-chain deficient mutant of the Jurkat line using polyethylene glycol) to reintroduce a missing b chain cDNA.  Thus, I showed that all the chains were required for expression of the oligomeric receptor on the cell surface.  It was later shown that a newly discovered chain, the z-chain was also a component of the TCR.  During my fellowship, together with John Imboden and Dolores Shoback, we showed that stimulation of the TCR increased intracellular calcium.  However, how this occurred was still not clear.  Nonetheless, these accomplishments led Stobo to offer me a faculty position in the rheumatology division and with that position came the advantage of an appointment to the Howard Hughes Medical Institute.  Stobo left to become Chair of Medicine at Johns Hopkins on the day that I became a faculty member in July of 1985.  So, I was on my own.

When I started my own lab, we focused on the oligomeric nature of the TCR.  What was the basis for its assembly?  What were the functions of the CD3- and z-chains?  The a and b chains had short cytoplasmic domains of only ~ 5 residues – they were unlikely to mediate signaling.  But the CD3- and z-chains had much longer cytoplasmic domains of unknown function.  While the CD3 chains had been speculated to be signaling molecules, papers at the time had suggested that z-chain modulated the pathways by which the TCR signaled.   We noted that there were unusually placed basic and acidic residues in the typically hydrophobic transmembrane domains of ab, CD3- and z-chains.  One of my postdocs, Lee Tan, had just joined me from Sherri Morrison’s lab (Columbia) where she had learned how to use recombinant DNA technology to modify immunoglobulin genes.

Lee showed that she could create chimeric molecules that would associate with CD3- and z-chains if she linked extracellular domains (in this case CD8) to the transmembrane domains of the a- and b-chains.  This was difficult technology because PCR techniques were not yet available.  She used restriction enzymes, partial DNAase digestion and blunt-end ligation.  It was a heroic effort.  She used CD8 since it was a dimer and antibodies to it were readily available.  Stimulating these CD8 chimeric molecules linked to the CD3- and z-chains recapitulated the signaling effects seen with antibodies against ab- or CD3-chains. This provided evidence that the transmembrane domains were sufficient to couple to the CD3 and z-chains and by doing so she linked CD8 to the signaling apparatus of the TCR.     This was a critical observation. Bryan Irving, a BMS grad student in my lab, hypothesized that if we separated the relatively long cytoplasmic domains

of CD3- or z-chains from their transmembrane domains, we could separate them from their obligatory interaction with the oligomeric TCR complex.  Bryan decided to test the function of the z-chain cytoplasmic domain by linking it to the extracellular and transmembrane domain of CD8.  Like Lee, Bryan also used CD8 because it was a disulfide-linked dimer and so was the z-chain.  His task was easier because PCR technology had just been widely introduced by then.   Indeed, Bryan was able to express CD8-z-chain chimeras independently of the TCR.  To our great surprise and delight stimulating these chimeras with anti-CD8 monoclonal antibodies induced signaling and responses that mimicked those induced by simulating the oligomeric TCR. 

A. We realized from Bryan’s chimera experiment that it would theoretically be possible to make such chimeric receptors that had any extracellular domain and thus free T cells from their requirement for MHC restriction and more directly to target T cells to pathogens and tumors. The TCR recognizes short peptides derived from pathogens, tumors or self-proteins that become associated with MHC molecules. Each individual expresses their own unique constellation of allelic variants of MHC molecules (there are hundreds to thousands of alleles for each type of MHC molecule).

T cells that mature and leave the thymus only express TCRs that can recognize their own allelic variants – hence MHC restriction. We realized that chimeric receptors could be produced with other receptors or antibodies that would free them from recognizing peptides only associated with MHC molecules; and the same chimeric receptor could be used in any patient’s T cells for tumor immunity or infectious diseases. In 1991, when we published this work, we thought such approaches were beyond available technologies accessible to our lab to grow T cells and transduce genes into them to any useful extent. However, during the course of our work, scientists from a small biotechnology company called Cell Genesys were interested in developing this technology for cell-based therapy.

They licensed the technology from UCSF and two patents were issued which included Bryan Irving and me as inventors. The Cell Genesys scientists learned how to expand human T cells (with the advice of Carl June, a consultant) and to transduce chimeric genes into T cells with viral vectors. They called their chimeric receptors “Universal T cell Receptors.” Cell Genesys started clinical trials for Kaposi’s sarcoma patients in HIV infected individuals with CD4- chimeras but during the course of their clinical trials anti-retroviral therapy rendered the program obsolete.

They tried using an anti-MUC1 single chain antibody linked to the CD8 transmembrane domain and the -chain cytoplasmic domain for colon cancer, but that trial failed. Cell Genesys discontinued these efforts. However, through the efforts of academic and industry investigators such as Carl June (U. Penn.), Michele Sadelin (MDKCC), Margot Roberts (Kite Pharma) and many others, CD19 CAR-T therapy for childhood and adult B cell malignancies became an incredible success story. They are now the basis for FDA-approved therapies available from Novartis and Gilead. This success required evolution of the chimeric receptors, to include costimulatory receptor signaling domains, as well as many other advances.

A. All you require is a vivid imagination. Much effort is directed towards other malignancies, but ongoing early studies are starting to direct T cells in autoimmune diseases.

This area of cell-based therapy research has spawned incredible efforts in large number of academic labs and of small and large biotech companies. New training programs in bioengineering are actively developing research programs in cell-based therapy. Most efforts are still aimed at treating malignancies, particularly aiming at other hematologic malignancies. However, many scientists are trying to extend CAR-T therapy towards solid tumors, which to some extent have been intractable.

The specificities and nature of the receptors are evolving rapidly. Very creative approaches are being used. An example of such advancement is the development of Notch-receptor based chimeras developed in Wendell Lim’s lab at UCSF. Promise of being able to rewire cells to express different functions has been shown to be possible in early experimental settings.

Different cell types are being exploited as new vehicles. For instance, T regulatory cells that suppress responses are being developed to target autoimmune diseases instead of malignancies. Jeff Bluestone at UCSF and others are developing such therapies. Chimeric receptors consisting of autoantigens linked to chimeric receptors are targeting autoantibody producing B cells. Much effort is being directed towards generating a universal T cell donor. That would be a truly exciting development!

We are just at the very early stages of effort cell-based therapies and predictions are likely to be very inaccurate. A major hurdle in cell-based therapies is and will continue to be cost.

A. I had generous and unselfish mentors in my undergraduate, graduate and postdoctoral studies who gave their trainees a lot of independence. I strongly believe they had an enormous impact on the way I practice science and upon how I treat my trainees. My mentors have enabled and supported my advancement and research accomplishments. I am forever grateful to them.

While I recognize that I have had success in my research career, it would not have been possible without a remarkable cadre of trainees who have worked hard and made sacrifices. It has been important to recognize that we all think differently and solve problems in creative and different ways. I learn a great deal from my trainees. While I learned giving trainees some degree of independence is important, collaboration and exchange of ideas is critical for success. While I consider the contributions that my lab has had to be significant, the collective impact that the research efforts of my former trainees will vastly overshadow those that I have made. I take great pride and satisfaction in their accomplishments and contributions.

A. Think creatively about big questions and problems. I would focus on fundamental questions that are likely to be relevant to disease pathogenesis or therapy. Let the question drive your work, not the technology. You can acquire technology in your own lab or through collaborators to tackle problems.