title: Addition of an extra immunoglobulin domain to two anti-rodent TNF monoclonal antibodies substantially increased their potency.
abstract: The functional valency of a monoclonal antibody (mAb) has important influences on such things as antigen avidity, Fc-mediated immune effector functions, and clearance of immune complexes. cV1q, a neutralizing rat/mouse chimeric anti-mouse tumor necrosis factor (TNF) monoclonal antibody (mAb), and Rt108, a neutralizing mouse anti-rat TNF (anti-raTNF) mAb, appear to be functionally monovalent for TNF-binding despite containing two antigen binding sites. The functional monovalency of these two independent anti-rodent TNF mAbs is presumably a result of steric hindrance from one TNF molecule binding to one Fab arm that prevents binding of a second TNF molecule to the other Fab arm. To test whether this steric hindrance could be overcome by introducing extra space and flexibility between the Fab arms, these mAbs were engineered to contain an extra CH1 immunoglobulin domain between the CH1 and hinge domains of their heavy chains. In vitro binding data showed that, compared to the original mAbs, the modified mAbs (S-mAbs) had greater capability of binding two TNF molecules simultaneously. In vitro activity assays showed that, compared to the original mAbs, the S-mAbs had significantly greater TNF-neutralization potency, with the S-mAb version of cV1q (S-cV1q) being 200-fold more effective at blocking mouse TNF (muTNF) and the S-mAb version of Rt108 (S-Rt108) being 20-fold more effective at blocking raTNF. Similar results were observed in vivo, where S-cV1q was between 100- and 500-fold more protective than cV1q in mice challenged with endotoxin. These data reveal that introduction of another constant region immunoglobulin domain into two unrelated mAbs dramatically enhanced their neutralization potency. Other mAbs may also show more potent activity using this engineering approach, particularly mAbs that recognize homopolymeric antigens.
authors.affiliation: Discovery Research, Centocor Inc, 200 Great Valley Parkway, Malvern, PA 19355, USA. bscallon@cntus.jnj.com
authors.forename: Bernard
authors.initials: B
authors.lastname: Scallon
authors.forename: Ann
authors.initials: A
authors.lastname: Cai
authors.forename: Jennifer
authors.initials: J
authors.lastname: Radewonuk
authors.forename: Michael
authors.initials: M
authors.lastname: Naso
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000493:pharmacokinetics / Q000494:pharmacology*; D001666:Binding Sites, Antibody; D002460:Cell Line; D007136:Immunoglobulins / Q000737:chemistry*; D051379:Mice; D009500:Neutralization Tests; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000633:toxicity
chemical_list: D000911:Antibodies, Monoclonal; D007136:Immunoglobulins; D014409:Tumor Necrosis Factor-alpha