Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity.

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

Galiximab (anti-CD80)-induced growth inhibition and prolongation of survival in vivo of B-NHL tumor xenografts and potentiation by the combination with fludarabine.

Galiximab is a primatized monoclonal antibody that targets CD80 expressed on malignant B cells and is being studied in the clinic as a potential treatment for follicular NHL. We have recently reported that galiximab signals B-NHL cells in vitro and inhibits cell growth and sensitizes resistant tumor cells to apoptosis by chemotherapeutic drugs. This study was designed to validate the in vitro findings in in vivo in mice. Thus, we examined in vivo the antitumor activity of galiximab used alone and in combination with chemotherapeutic agents in SCID mice bearing human lymphoma xenografts. The in vivo antitumor effects of galiximab used alone and in combination with fludarabine or doxorubicin were determined in solid and disseminated human B-lymphoma tumors grown in SCID mice. Galiximab monotherapy in vivo demonstrated significant antitumor activity in a Raji lymphoma solid tumor model and in an SKW disseminated lymphoma tumor model. There was significant inhibition in tumor growth and prolongation of survival. In vitro, galiximab sensitized Raji cells to apoptosis by both fludarabine and doxorubicin. Tumor growth inhibition was significantly enhanced when the mice were treated with the combination of galiximab and fludarabine. These findings support the potential clinical application of galiximab in combination with chemotherapeutic drugs for the treatment of CD80-expressing hematological malignancies.

Development of an Fn14 agonistic antibody as an anti-tumor agent.

TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity.

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.