88


I. Pastan et al.


The known steps required for PE to kill cells include (1) binding to a cell-surface antigen, (2) internaliza- tion into an endocytic vesicle where the toxin unfolds at low pH, (3) proteolytic cleavage by furin between amino acids 279 and 280, leaving a disulfide bridge between Cys265 and Cys287, (4) reduction of the disulfide bond, (5) transport of the carboxyl terminal portion of the toxin from the transreticular Golgi to the endoplasmic reticulumby theKDELreceptor, (6) translocation to the cytosol, and finally (7) catalytic ADP-ribosylation of elongation factor II, leading to apoptotic cell death. Thus, recombinant immunotox- ins, unlike vaccines, kill cells directly without help from the immune system. On the contrary, a humoral immune response against recombinant immunotox- ins will block their function, and preventing immu- nogenicity is an important goal for the development of recombinant immunotoxins.


Identification of immunogenic epitopes in PE38


To identify immunogenic epitopes, a panel of 60 anti-PE38 MAbs were isolated and tested pairwise (3600 combinations) in an immune complex capture enzyme-linked immunosorbent assay (ELI- SA). Each experiment utilized one MAb as an indicator and another as a competitor [6]. As shown in Figure 1(A), these experiments identified seven major epitopes. The next step was to determine where on the PE38 molecule these epitopes were located. To accomplish this, point mutations to alanine or glycine were performed in PE38, targeting amino acids predicted to be highly surface-exposed. As shown in Figure 1(B), several of the point mutants reacted with anti-PE38 MAbs with510% of the affinity of native PE38, disclos- ing the locations of the immunogenic epitopes.


Figure 1. Summary of steps to identify and remove B cell epitopes in PE38. (A) Mutual competitive binding assay of 60 mouse monoclonal antibodies against PE38 produced for B cell epitope analysis leading to deimmunization of PE38. Binding of indicator antibodies (in rows) in the presence of competitor antibodies (in columns) was measured by immunocytochemistry-ELISA. Color code (red to blue) shows the degree of competition from 100 to 0%. (B) Binding of each antibody (in rows) to each point mutant of PE38 (in columns). Mutations were made in highly exposed amino acids in a crystal structure. The binding assay measured the affinity difference between mutant and wild-type PE38; mutations that reduced affinity to the antibody under 10% compared to wild-type are shown in red. (C) Locations of mutated amino acids that decrease binding of PE38 to antibodies in an epitope-specific manner (from data shown in B) are displayed in different colors on a structural model of PE38. Clustered residues shown in different colors indicate the location of each epitope. The two different approaches (topographical epitope mapping and location of epitopes on the crystal structure determined by mutagenesis) are in close agreement. This concordance verifies the epitope locations without analyzing crystal structures of the antigen–antibody complexes. Adapted with permission from The Journal of Immunology [6].


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68  |  Page 69  |  Page 70  |  Page 71  |  Page 72  |  Page 73  |  Page 74  |  Page 75  |  Page 76  |  Page 77  |  Page 78  |  Page 79  |  Page 80  |  Page 81  |  Page 82  |  Page 83  |  Page 84  |  Page 85  |  Page 86  |  Page 87  |  Page 88  |  Page 89  |  Page 90  |  Page 91  |  Page 92  |  Page 93  |  Page 94  |  Page 95  |  Page 96  |  Page 97  |  Page 98  |  Page 99  |  Page 100  |  Page 101  |  Page 102  |  Page 103  |  Page 104  |  Page 105  |  Page 106  |  Page 107  |  Page 108  |  Page 109  |  Page 110  |  Page 111  |  Page 112  |  Page 113  |  Page 114  |  Page 115  |  Page 116  |  Page 117  |  Page 118  |  Page 119  |  Page 120  |  Page 121  |  Page 122