Cancer Vaccines from a Blue-Blooded Mollusk Posted on June 2, 2013 by Andrew Porterfield Megathura crenulata, or the giant keyhole limpet, runs up to 10 inches in size, and uses an unusual molecule for breathing: hemocyanin. Instead of its red-blooded terrestrial oxygen carrier hemoglobin, hemocyanin is blue in color, carries a copper molecule instead of iron, and is used by marine snails and mollusks for gill-based respiration. The molecule is fortuitous not only for the mollusk. Hemocyanin, especially keyhole limpet hemocyanin (KLH), also has long shown potential as a delivery vehicle for vaccines—especially cancer vaccines. Vaccines work by mimicking the actions of a disease-causing cell or other foreign body. As they enter the blood, they'll trigger a response by the body's white blood cells (called T or B cells), which multiply and start to attack the foreign body. Once the vaccine is killed, the immune response will remain strong, often permanent. But anti-cancer antigens are often too small to elicit an immune response. Without such a response, the body won't be able to kill abnormal cells before they become full-fledged tumors. But KLH, which is a very, very large molecule, does elicit a slight immune reaction, resulting in T cells (and sometimes B cells) in the host that are specific to the cancer vaccine molecule. These KLH-vaccine combinations are undergoing clinical trials in a number of research institutions, for follicular lymphoma, non-Hodgkin lymphoma, glioblastoma, and melanoma, and bladder, prostate and ovarian cancer. A biotechnology company in California, Stellar Biotechnologies, is harvesting KLH by farming keyhole limpets, and hopes that KLH will be approved as a drug (or in this case, a vaccine enhancer). While many natural substances are chemically tweaked for better performance, enhanced absorption by the human body, or other factors, KLH seems to work on its own. In addition, Stellar Biotech says, the KLH molecule is so large it defies artificial synthesis. While the cancer-vaccine possibilities of KLH have been studied for years, its molecular structure was discovered only two years ago, which has created more potential for targeted vaccines. KLH is one of the largest carrier molecules in existence: resembling a hollow tube, it is assembled from two 500 kDa polypeptide subunits. By comparison, most proteins top out at about 200 kDa. KLH has eight functional units, each of which carries a single copper active site to reversibly bind to oxygen. This structure, as well as recent studies showing specific B cell homing activity for various KLH-antigen combinations, may finally provide a clinically available vaccine from the giant limpet. The KLH story is a relatively new one in drug discovery. Many possible therapies may arise from organisms living in the ocean, but the discovery of these therapies has only seriously started over the past two decades or so. So, when in California or Mexico and you stumble upon a nearly foot-long flattish shell sticking to a rock, you might consider thanking it.
Keyhole limpet hemocyanin KLH is purified from the hemolymph of Megathura crenulata by a series of steps that typically includes ammonium sulfate precipitation and dialysis, and may involve chromatographic purification to obtain the highest purity. KLH purification may also include endotoxin removal, but this step is often unnecessary because the endotoxin serves as an adjuvant when injected for antibody production. If the protein becomes denatured or if the copper ions are lost in the purification process, the opalescent blue color disappears and the solution becomes a dull grayish color. Denaturation of KLH also results in a tendency of the protein to aggregate and precipitate from solution.
Use in biotechnology Keyhole limpet hemocyanin (KLH) is used extensively as a carrier protein in the production of antibodies for research, biotechnology and therapeutic applications. Haptens are substances with a low molecular weight such as peptides, small proteins and drug molecules that are generally not immunogenic and require the aid of a carrier protein to stimulate a response from the immune system in the form of antibody production.[3] KLH is the most widely employed carrier protein for this purpose. KLH is an effective carrier protein for several reasons. Its large size and numerous epitopes generate a substantial immune response, and abundance of lysine residues for coupling haptens allows a high hapten:carrier protein ratio, increasing the likelihood of generating hapten-specific antibodies. In addition, because KLH is derived from the limpet, a gastropod, it is phylogenetically distant from mammalian proteins, thus reducing false positives in immunologically-based research techniques in mammalian model organisms. KLH can also be a challenging molecule to work with because of its propensity to aggregate and precipitate. Aggregates remain immunogenic, but limit the ability to conjugate haptens, and are difficult to manipulate in the laboratory. A high-quality KLH preparation with clear opalescent blue color is the best indicator of KLH solubility.
Hapten coupling Haptens can be coupled to KLH using several methods. A simple one-step coupling can be performed using the carbodiimide crosslinker EDC to covalently attach carboxyls to primary amines. This method is the simplest to perform and the "random" orientation allows for antibody generation against all possible epitopes, but it generally results in some degree of polymerization, which decreases solubility making the conjugate more difficult to handle. KLH can be activated with the crosslinker Sulfo-SMCC, which converts lysine residues to sulfhydryl-reactive maleimide groups. A sulfhydryl-containing hapten can then be reacted with the KLH to complete the immunogen without causing polymerization. The specificity of this reaction is ideal for situations where the cysteine is located away from the desired epitope (e.g. in peptides where a terminal cysteine can be added to either end of the peptide). Maleimide activated KLH, where the first part of this two step procedure has been completed, is commercially available.
Other carrier proteins Concholepas concholepas hemocyanin (marketed as Blue Carrier) Bovine serum albumin (BSA) Cationized BSA (cBSA) Ovalbumin
Use in cancer therapy KLH is being tested in a variety of cancer vaccines, including non-Hodgkins lymphoma, cutaneous melanoma, breast and bladder cancer. These vaccines contain specific tumor-associated antigens conjugated to KLH to stimulate anti-tumor immune responses which can destroy tumor cells. The rapidly growing interest in therapeutic vaccines (i.e. active immunotherapies) for cancer and the documented efficacy of KLH as a superior carrier protein for cancer vaccines are creating a significant biopharmaceutical market for KLH formulations. Assays to monitor humoral immune responses against KLH in human serum have been developed to facilitate optimal use of biomedical KLH applications.
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