title: Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.
abstract: The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv). Firstly, the relationship between affinity and potency was examined in a panel of four scFv immunotoxins generated from different anti-CD3 monoclonal antibodies fused to the 38 kDa fragment of Pseudomonas aeruginosa exotoxin A (PE38). Of these four scFv-PE38 immunotoxins, the one derived from the anti-CD3 monoclonal antibody UCHT1 has highest cell killing potency. Analysis of these four scFv-PE38 immunotoxins indicated a correlation between antigen binding affinity and immunotoxin potency in the cell killing assay with the exception of the scFvPE38 immunotoxin derived from the antibody BC3. However this scFv appeared to suffer a greater drop in affinity ( approximately 100x), relative to the parent Mab than did the other three scFvs used in this study (2-10x). Secondly, the scFv(UCHT1)-PE38 immunotoxin was then compared with a further panel of scFv(UCHT1)-derived immunotoxins including a divalent PE38 version and both monovalent and divalent Corynebacterium diphtheriae toxin (DT389) fusion proteins. When the scFv-UCHT1 domain was amino-terminally positioned relative to the toxin, as in the scFv(UCHT1)-PE38, an approximately 10-fold higher antigen-binding affinity was observed than with the C-terminal fusion, used in the DT389-scFv(UCHT1) molecule. Despite this lower antigen-binding activity, the DT389-scFv immunotoxin had a 60-fold higher potency in the T-cell-killing assay. Thirdly, a divalent form of the DT389-scFv construct, containing tandem scFv domains, had a 10-fold higher binding activity, which was exactly reflected in a 10-fold increase in potency. Therefore, when comparing immunotoxins in which scFvs from different antibodies are fused to the same toxin domain (DT or PE) a broad correlation appears to exist between binding affinity and immunotoxin potency. However, no correlation between affinity and potency appears to exist when different toxin domains are combined with the same scFv antibody domain.
authors.affiliation: Transplantation Research, Novartis Pharmaceuticals, 556 Morris Avenue, Summit, NJ 07901, USA. mark.hexham@pharma.novartis.com
authors.forename: J M
authors.initials: JM
authors.lastname: Hexham
authors.forename: D
authors.initials: D
authors.lastname: Dudas
authors.forename: R
authors.initials: R
authors.lastname: Hugo
authors.forename: J
authors.initials: J
authors.lastname: Thompson
authors.forename: V
authors.initials: V
authors.lastname: King
authors.forename: C
authors.initials: C
authors.lastname: Dowling
authors.forename: D M
authors.initials: DM
authors.lastname: Neville
authors.forename: M E
authors.initials: ME
authors.lastname: Digan
authors.forename: P
authors.initials: P
authors.lastname: Lake
mesh_terms: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology*; D001427:Bacterial Toxins; D017252:CD3 Complex / Q000276:immunology*; D005098:Exotoxins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology*; D019169:Jurkat Cells; D020298:U937 Cells; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D017252:CD3 Complex; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C055966:immunoglobulin Fv