ErbB3 inhibitory surrobodies inhibit tumor cell proliferation in vitro and in vivo.

ErbB3 is an important regulator of tumorigenesis and is implicated in development of resistance to several currently used oncology drugs. We have identified ErbB3 inhibitors based on a novel biologic scaffold termed a surrobody. Two of these inhibitors appear to work by a previously unrecognized mechanism of action. As a consequence, they not only inhibited cell proliferation and intracellular signaling driven by stimulation with the ErbB3 ligand neuregulin (NRG), but also inhibited signaling and proliferation that was driven by overexpression of ErbB2 in the absence of ligand stimulation. In addition, the surrobodies inhibited tumor growth in vivo in both ErbB2-overexpressing and nonoverexpressing cells. In ErbB2-overexpressing cells, both of the anti-ErbB3 surrobodies significantly augmented the activities of trastuzumab, lapatinib, and GDC-0941, agents that inhibit cell proliferation by different mechanisms. Moreover, although NRG diminished the efficacy of these agents, when they were combined with anti-ErbB3 surrobodies the affect of NRG was abrogated. In this capacity, the anti-ErbB3 surrobodies were more effective than the ErbB2/ErbB3 dimerization inhibitory antibody pertuzumab. Despite the fact that these surrobodies appear to engage ErbB3 differently than previously described anti-ErbB3 antibodies, they retain all of the beneficial characteristics of this class of agents, including the ability to augment drugs that inhibit EGF receptor. These anti-ErbB3 agents, therefore, show substantial promise for development as single agents or in combination with other ErbB-directed antibodies or small molecules and may provide for a broader range of therapeutic indications than previously described anti-ErbB3 antibodies.

Combinatorial surrobody libraries.

A unique type of combinatorial protein libraries has been constructed. These libraries are based on the pre-B cell receptor (pre-BCR). The pre-BCR is a protein that is produced during normal development of the antibody repertoire. Unlike that of canonical antibodies, the pre-BCR subunit is a trimer that is composed of an antibody heavy chain paired with two surrogate light chain (SLC) components. Combinatorial libraries based on these pre-BCR proteins in which diverse heavy chains are paired with a fixed SLC were expressed in mammalian, Escherichia coli, and phagemid systems. These libraries contain members that have nanomolar affinity for antigen. We term this type of antigen-binding protein a "surrobody" to distinguish it from the canonical antibody molecule.

A general method for greatly improving the affinity of antibodies by using combinatorial libraries.

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-alpha antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (K(d) = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each V(H) and V(L) domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-alpha. These beneficial mutations in both V(H) and V(L) were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-alpha neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.