Rapid development of human antibodies to stem cell proteins using scFv yeast & phage display
The purpose of the program set forth in these pages is to describe an innovative technology we will perfect that will eliminate a current troubling limitation on the wide-spread use of human embryonic stem cells. A bit of explanation is in order. Antibodies are highly valuable research tools in biological research including stem cell biology. Without having specific antibodies available a majority of biological experiments could not be accomplished successfully.
For example, novel antibodies to unique stem cell markers are required to determine the functional characteristics of the unique proteins and for isolating individual stem cell populations. The general structure of all antibodies is similar with the exception of a small region at the tip of the antibody, which is extremely variable, allowing billions of antibodies with slightly different tip structures to exist. Specific antibodies are normally produced by injecting a protein antigen into a mammal, such as a mouse, rat, rabbit, goat, sheep or horse. Blood isolated from these animals contains polyclonal antibodies in the serum. To obtain an antibody that is specific for a single site of an antigen (that may exhibit multiple potential sites for antibodies), antibody-secreting lymphocytes are isolated from mice and fused with a cancer cell line. The fused cells, which are called hybridomas, will grow and secrete antibodies in the culture. These methods take many months to obtain a single antibody.
The limitation on the use of human embryonic stem cells mentioned earlier is a lack of methods to characterize, isolate and enrich the individual lineage cells from the heterogeneous progenitor populations and from undifferentiated cells which potentially can cause cancer if injected into a patient. The availability of antibodies against unique stem cell proteins is essential in order to overcome this limitation. Recent advances in recombinant technology now allow us to produce antibodies to any antigen. Engineered antibody fragments can be manipulated and then produced in phages and yeast.
Our research program will bring the antibody selection process up to date and avoid using animals as a source of antibodies. Instead, we will use the human antibody libraries (approximately one billion individual antibodies) expressed on the surface of yeast (AIM 1) and phages (AIM 2). The value of these libraries especially multiplied by our experience and by advanced enrichment and selection processes will allow us to rapidly select the individual high potency and selectivity antibodies to multiple unique stem cell markers. We envision highly rewarding experimental results because of our efforts and because multiple novel antibodies to stem cell proteins will be available to the CIRM community. The availability of novel antibodies will be publicized through our interactive web site to promote their use and to allow scientists to make requests to the statewide Antibody Core we will have established.
The term “pluripotent stem cells” identifies a population of cells, which is the source of other, more specialized cells. We have only recently begun to gain the necessary understanding of the specific individual steps, among the many molecular events involved, in embryonic stem cell differentiation. To date, our means to control and to modulate these differentiation processes have been limited, primarily, because of the restricted number of tools, which are available for the analyses of stem cells.
High affinity and selectivity antibodies are essential tools in biology including stem cell research. Traditional immunization methods using animals take months to create an antibody. Recent advances in recombinant technology, however, now allow us to produce high quality antibodies to any antigen and to avoid using animals as a source of antibodies. Engineered antibody fragments can be manipulated and then produced in phages and yeast. The short doubling time of phages and yeast allows the rapid isolation of antigen-specific scFv (Single Chain Fragment Variable) antibodies.
Our research program is focused on the development and the use of the scFv yeast and phage libraries (over one billion individual clones) as a source of antibodies to stem cell proteins. Our proposed research will lead us to a far better and very necessary understanding of the role of multiple stem cell proteins in human embryonic stem cell and induced pluripotent somatic cell differentiation. We also believe our results will lead us to develop novel and effective means to control and to specifically modulate the differentiation processes of ES cells. The means to control stem cell differentiation will ultimately lead to novel, stem cell-based, therapies the development of which will benefit the citizens of the state of California.
We suggest that a broadly understandable way to describe the benefits to the State of California that will flow from the stem cell research we propose to conduct is to couch it in the familiar business concept of “Return on Investment”. The technology, assays, tests and experimental findings that will be developed and accomplished as a result of our research program and the many related programs that will follow will provide direct benefits to the health of California citizens through the many therapies that will use human embryonic stem cells. This program and its many complementary programs will also generate potentially large monetary benefits. These financial benefits will derive directly from two sources. The first source will be the sale and licensing of the intellectual property rights that will accrue to the state and its citizens from this and the many other stem cell research programs that will be financed by the CIRM. The second source will be the many different kinds of tax revenues that will be generated from the increased bio-science and bio-manufacturing businesses that will be attracted to California by the success of the CIRM.