Variants of the Antibody Herceptin That Interact with HER2 and VEGF at the Antigen Binding Site

Abstract

The interface between antibody and antigen is often depicted as a lock and key, suggesting that an antibody surface can accommodate only one antigen. Here, we describe an antibody with an antigen binding site that binds two distinct proteins with high affinity. We isolated a variant of Herceptin, a therapeutic monoclonal antibody that binds the human epidermal growth factor receptor 2 (HER2), on the basis of its ability to simultaneously interact with vascular endothelial growth factor (VEGF). Crystallographic and mutagenesis studies revealed that distinct amino acids of this antibody, called bH1, engage HER2 and VEGF energetically, but there is extensive overlap between the antibody surface areas contacting the two antigens. An affinity-improved version of bH1 inhibits both HER2- and VEGF-mediated cell proliferation in vitro and tumor progression in mouse models. Such "two-in-one" antibodies challenge the monoclonal antibody paradigm of one binding site, one antigen. They could also provide new opportunities for antibody-based therapy.

Cross-domain inhibition of TACE ectodomain

“…However, given that V L exchange was recently shown to modify a V Hbiased Her2 antibody to also bind VEGF (21), this reselection technique clearly has considerable potential beyond monoantigen affinity maturation. Our work further demonstrates how manipulating initial selection conditions (e.g., by blocking undesired epitopes with SMIs or cofactors) can produce primary antibodies : AREG) and HeLa cells stably over expressing HB-EGF-Alkaline Phosphatase were used to assay cell-surface TACE activity.…”

“…As solution-phase phage display typically produces antibodies with conformational (i.e., nonlinear) epitopes, this technology is also theoretically capable of providing the intricate macromolecular cross-domain binding desirable in an ADAM inhibitor. Recent technical advances in phage display have produced antibodies recognizing multiple disparate antigens (21) and different conformations of the same antigen (22,23). These approaches use a combination of calculated selection conditions and/or antibody variable-domain chain-shuffling to produce antibodies against previously unique epitopes.…”

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

“…[1][2][3][4][5][6][7][8][9][10][11][12] Bispecific molecules described thus far can be divided into five classes based on their molecular format: 1) BsAb with IgG1-like structure, which are monovalent for each antigen; 2) BsAb prepared by appending binding domains to the IgG, which are bivalent for each antigen; 3) BsAb prepared using antibody fragments, which are monovalent for each antigen and often lack the Fc region; 4) bispecific fusion proteins, which are formed of an antibody fragment genetically linked to a protein to add additional functionality or specificity; and 5) bispecific conjugates, which are prepared by chemical conjugation of antibody fragments. While some such bispecific molecules have demonstrated manufacturing robustness, in vivo drug-like properties, suitable pharmacokinetics (PK) and efficacy have been achieved, platforms that yield multifunctional bispecific antibodies with different spatial configurations and flexibility suitable for a variety of target and disease applications are still needed.…”

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

“…Other designs of IgG-Bs have been described, but with the exception of IgG-Bs that are restricted to the same heavy chain on both binding arms 24 and the 'two-in-one' IgG, 25 these other IgG-Bs are either variations or optimizations of the methods described thus far. [26][27][28] Although these approaches generate IgG-Bs, the deviation from the IgG chain sequence and structure remains a substantial issue, [6][7][8][9][10][11][12] and novel antibody engineering solutions are thus still needed.…”

“…The iMab design offers a unique engineering solution to overcome some of the limitations of the described previously IgG-Bs, [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] such as the potential formation of half-antibodies and homodimers, which can be difficult to eliminate and require complex multi-step purification methods.…”