Adapted from [published 2008, Vol. a practical approach for making less immunogenic protein therapeutics from non-human functional proteins. This approach requires grouping of the epitopes to identify targets for de-immunization followed by quantitative analysis of the decrease in affinity produced by the mutations in B cell epitopes. Keywords: Deimmunization, mutagenesis, antigen, antigenicity, antibody, biopharmaceutical 1. Introduction Biologically useful proteins originating from nonhuman species are an attractive source of biopharmaceuticals, due to their high selectivity and potency. However, because they are recognized as foreign by the human immune system, the number of treatment cycles that can be administered to patients is limited by the formation of antibodies [1C5]. GADD45B Nevertheless a few foreign proteins have proven to be clinically useful. Streptokinase is usually a bacterial protein secreted by hemolytic that is an effective clot-dissolving medication for myocardial infarction and pulmonary embolism [6]. Although streptokinase is usually highly immunogenic and inactivating antibodies can be present from prior streptococcal infections, the levels of antibodies are generally of little clinical significance, when streptokinase is used in the large doses recommended. Streptokinase has relatively less bleeding risk for patients than newer brokers and is still utilized for the first collection treatment of acute myocardial infarction. Another example of a foreign protein in clinical use is usually Botulinum toxin, a neurotoxic protein produced by the bacterium [7]. Botulinum toxin is usually a very potent toxin and minute doses are used to treat muscle mass spasms. The very small protein load (usually less than 100 ng) needed for its medical effect does not usually induce significant antibody responses; only 5C15% of patients injected serially with Botulinum toxin became unresponsive due to the production of neutralizing antibodies [8]. These examples show that highly immunogenic foreign proteins can be utilized for medical purposes. Another important factor is that non-human proteins are unlikely to produce auto-immunity that could neutralize endogenous protein function. This suggests that it is not necessary to aim for the complete removal of the immunogenicity for medical benefit [9]. We have successfully used a 38 kDa portion of exotoxin A (PE38) as a cytotoxic moiety in recombinant immunotoxins for the therapy of malignancy [10C12]. In these immunotoxins, PE38 is usually genetically linked to the Fv portion of a monoclonal antibody, Lycopodine guiding the PE38 toxin to malignancy cells that express the antigen on their cell surface. We have been actively pursuing the reduction of immunogenicity of recombinant immunotoxins to expand their usefulness in malignancy treatment. Clinical trials revealed that over half of the patients with life threatening drug resistant Hairy Cell Leukemia achieved a complete remission after 3 to 10 cycles of treatment with BL22, a recombinant immunotoxin made up of PE38 [13, 14]. However, such multiple cycles of treatment are not possible, in patients with normal immune systems, because neutralizing antibodies usually develop within three weeks. These antibodies almost always react with the bacterial toxin and very infrequently with the Fv, and limit the number of cycles of therapy that can be given. Fortunately, patients with leukemias and lymphomas make antibodies to the immunotoxin relatively infrequently, because the chemotherapy used to treat this disease is usually toxic to the immune system and because leukemias and lymphomas infiltrate and damage the immune system. The success in treating drug resistant leukemia suggests that immunotoxin therapy can be useful in the treatment of other types of cancer, if we can reduce immunogenicity to a level, which permits multiple cycles of treatment to be given. One approach to de-immunize a protein is to identify B-cell epitopes around the protein and eliminate them by mutagenesis Lycopodine [15, 16]. PE38 is usually a highly immunogenic protein and de-immunizing appeared to present a formidable task. Our success over the last 5 years in substantially reducing the immunogenicity of PE38 made up of immunotoxins [17, 18] suggests that B cell epitope removal can also be accomplished for other foreign proteins. In this review, both theoretical aspects and experimental evidence around the reduction of immunogenicity by B cell epitope removal will be discussed. 2. Theoretical basis of B cell epitope removal for reducing immunogenicity There are several essential prerequisites for deimmunization by B cell epitope removal. They are: (1) the presence of antigenic warm Lycopodine spots on a protein surface that more frequently serve as epitopes to the antibodies than other surface regions of the protein; (2) the antigenic structural signatures of the warm spots can be altered so that they are less immunogenic by point mutations in amino acids located at these sites; and (3).
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