Scampering around a cage with trembling whiskers and small, beady eyes, the particular mouse in question looks no different from any other mouse to the naked eye, but deep within the molecular makeup of this mouse there lies a key difference. This mouse comprises not only natural mouse genetics and an array of proteins but also human proteins, human DNA sequences, and the potential for human furtherance. Early in January of 2006, Nils Lonberg of Medarex Inc. and Harry Meade of GTC Biotherapeutics created a startling new transgenic mouse [7]. While transgenic mice are not newcomers to the biotechnology and medical research fields, this particular mouse houses a complete human immune system. In the realm of medical sciences, the search for new therapies and miracle cures takes on a life of its own, and medical applications abound with this new possibility on the frontier of genetic engineering. Limitless possibilities for applying transgenic animals to medicine currently exist. These include harvesting human antibodies from chimera milk and the subsequent creation of novel drugs. Fifty biotech and pharmaceutical companies are already pursuing the utilization of these chimeric products.

Chimeras are by no means a new idea in the world of bioengineering. The process of inserting human DNA into mice and goats was already in practice prior to 1984. Even then mice were the transgenic animal of choice for researchers, and Sherie Morrison and her associates [4] led the way with mice that produced chimeric human antibodies. These transgenic mice maintained mice-antigen-binding domains on their cells, but the constant region domains remained human. Specifically, the mice were engineered by transfecting mouse myeloma cell lines with human genes that produced an IgA anti-phosphocholine antibody. The transformed cells synthesized chimeric mouse-human IgG anti-phosphocholine antibodies despite being oncogenic to the mice upon maturation. As expected by those involved in this early research [4], their work paved the initial steps in constructing and efficiently expressing human immunoglobulin molecules, and the techniques facilitated the development of antibodies for immunodiagnosis and immunotherapy.

Lonberg, Sherie, and Meade’s approach to generating human antibody production in chimeras parallels another technique involving a microscopic approach. In 1990, John McCafferty published an article in Nature on certain bacteriophages that could be manipulated to express human antibody domains on the surface of their cell membranes [3]. Their work demonstrated that functional antigen-binding sites could be expressed on the surface of fd phages (M13 filamentous phages). Amplified genes from hybridoma B cells were cloned into expression vectors; these vectors allowed for the construction of antibodies using the chimeric human antigen-binding sites introduced to these phages. But the impact of this parallel tract pushed the field of transgenic animal research forward. McCafferty’s research also demonstrated that fd phage vectors could select and clone genes using the binding properties of their expressed surface proteins, a monumental development to later aid Lonberg and Meade’s project.

A leading expert of the time, in 1994, Lonberg’s work towards a viable means of manufacturing specific human antibodies yielded a vital step in his journey [2]. While at GenPharm International, Lonberg engineered mice that produced antigen-specific human antibodies. The mice underwent four distinct genetic modifications in order to accomplish the expression of human IgM, IgG, and Ig? as well as to remove mouse IgM and Ig?. The genetic modifications were human sequence transgenes that underwent mutations to generate human specific immunoglobulins. The final immunization of the mice with human proteins proved that it was not only feasible but possible to isolate hybridoma (mouse-human) cells that secrete IgG? antigen-specific antibodies. This discovery held vast significance for Lonberg and Meade’s later work, demonstrating the viability of hybridoma technology to obtain human antibodies from mice. It brought to life the possibility of chimeras as a source of human antibodies that target corresponding antigens as a feasible means for future therapeutic design.

The future of human protein production using transgenic animals, specifically antibodies, foreshadows an economically advantaged world of biotherapeutics from these aforementioned processes. In addition, it offers a favorable alternative to using bacteria or other microbial systems which often improperly fold human proteins due to their lack of post-transcription modification of mRNA. Currently, cell cultures for bioreactors represent the current substitute to microbial or animal cell cultures, but these bioreactors often include an exorbitant startup cost with comparatively low yields. To replace these methods, the most promising possibility of transgenic animals for use in biotherapeutics comes from the expression of human proteins in animals’ milk. This so called transgenic milk could easily yield higher amounts of desired components for drug design/manufacture without the initial costs or difficulties of protein mis-folding. A stock of transgenic dairy animals could be easily established at minimal cost, and the property would pass through to the offspring of the current animals, propagating and ensuring the availability of human proteins through the generations. According to Pollock et al. the ideal dairy animals for transgenic milk production are goats and cows [6]. Dairy goats’ milk production ranges between 600 to 800 liters per 300 day lactation and yields of greater than 1000 liters have been recorded. Additional research finds that recombinant antibody concentration in transgenic goat milk ranges from 1 to 5 grams per liter with easily reproducible results. Considering dairy goats only achieve a theoretical yield of transgenic milk production of 1 to 300 kilograms of proteins per year, these numbers fall into the low or middle range of high-volume protein required for most therapeutic antibodies currently in development. The establishment of transgenic milk production for goats would require only 16 to 18 months [6]; this is quick enough to support expansion of production herds within the time frame needed to establish a stock of transgenic bovines.

The amount of transgenic milk available from mice is impractically small such that their popularity among researchers is for a solely research focused reason; however, because mice are easily bred and genetically engineered at low cost, they are the optimal species for research with transgenic species. Meade’s work in 1999 [5] with Newton and Pollock, the main author of “Transgenic Milk as Method for the Production of Recombinant Antibodies” [6], demonstrates for the first time the capability of antibodies to be expressed in large quantities in mammary glands of mice. Their work specifically targets the creation of a mouse model for breast cancer treatment research. The mice produced, in their milk, a mouse-human chimeric antibody—fused to DNA for human angiogenic RNase angiogenin (Ang)—that targeted the human transferrin receptor. These chimeric antibodies are very similar in structure and expression to human breast cancer cells. Antibody-Ang fusions are milk specific promoters that can induce the expression of active immunotoxins during lactation. Implications include the possibility to engineer a targeted expression of immunotoxins to prevent or alter the progression of a disease. While this research works toward a specific goal of producing chimeric or strictly human antibody-protein fusions for therapeutic use, Meade’s work in this project was crucial to his later work with Lonberg in the creation of a transgenic mouse with a complete human immune system.

The research undertaken by Newton et al. [5] predicts the future use of such transgenically manufactured human or mouse-human chimeric antibodies and proteins for therapeutic advances in disease treatment and prevention. The final goal was made manifest with the compilation of products from these past discoveries: the methods of engineering transgenic mice, including pronuclear microinjection and fusion of yeast protoplasts to deliver yeast artificial chromosome (YAC)-based minilocus transgenes, and the complete expression of these transgenes in model transgenic organisms such as mice or goats. In the present, these technologies have finally realized their potential in their therapeutic uses. Currently, several therapeutic drugs are in testing from a multitude of biotherapeutics companies and research laboratories. Mederax, the company Nils Lonberg is associated with, has numerous therapies in clinical trials. Their targeted diseases include melanoma, rheumatoid arthritis, lymphoma, psoriasis, multiple sclerosis, and autoimmune diseases such as HIV/AIDS. The future for biotherapies abounds with possibilities and promise for disease treatment and prevention as the field of transgenic technology continues proliferating.

Lonberg and Meade, the engineers of our transgenic, human-immune-system mouse, derived their discovery not only from these past works and research experiments but also from the desire for more effective novel therapies for such lethal diseases as cancers and autoimmune diseases. The introduction of human DNA sequences into mice and bacteriophages, the effective production of human-mouse chimeric DNA, and the expression of human proteins in the transgenic animals have all led to a cumulative discovery and success in the form of our not-so-everyday, everyday mouse. Those beady eyes and trembling whiskers hold the future for many possible applications. Drugs for combating disease can be tested in a transgenic mouse to determine their efficacy in treatment and how they affect and interact with the human immune system’s natural response. The future of farming for pharmaceutical ingredient through Lonberg and Meade’s mouse also highly outweigh the current means of production in biotherapeutics. The use of mice instead of cell cultures or bioreactors not only proves economically advantageous but also excels in efficiency and quality. The future of advances in preventative and therapeutic medicine could progress by enormous proportions with the advent of this single, spectacular mouse.

References: