(534b) Engineering Bispecific Antibodies for Targeted Inhibition of Tumor Metastasis

Introduction: Metastasis is responsible for 90% of deaths from solid tumors. However, current FDA cancer treatments are focused on preventing tumor cell proliferation rather than migration, and the mechanisms driving tumor metastasis remain unclear. Recently, our team reported a synergistic signaling pathway involving the interleukin-6 (IL-6) cytokine and the interleukin-8 (IL-8) chemokine that promotes tumor metastasis. Inhibition of the IL-6 and IL-8 receptors (IL-6R and IL-8R) using a combination of tocilizumab, an anti-IL-6R antibody which is clinically approved for treatment of rheumatoid arthritis, and reparixin, a small molecule drug targeting IL-8R that is currently in phase II clinical trials for treatment of breast cancer, effectively reduces metastasis in mouse models of cancer. However, translation of an antibody/small molecule combination therapy to the clinic is complicated by logistical challenges, such as dosing ratio optimization and increased regulatory hurdles. Moreover, small molecule drugs such as reparixin face significant clinical challenges in terms of specificity. Compared with monoclonal antibodies and small molecule therapies, bispecific antibodies (BsAbs) have higher affinity, potency, and selectivity. In addition, the dual-targeting BsAb approach is also more robust against acquired resistance due to reduced likelihood of mutational escape. In this study, we engineered BsAbs that simultaneously target the IL-6 and IL-8 receptors for targeted inhibition of cancer metastasis. We particularly focused on highly invasive cancer types, such as triple negative breast cancer (TNBC) and pancreatic duct adenocarcinoma (PDAC).

Materials and Methods: We designed two BsAbs (BS1 and BS2) based on the human immunoglobulin G (IgG) platform (Figure 1A) using the variable regions of an anti-IL-6R and an anti-IL-8R antibody. BS1 and BS2 were recombinantly expressed in a mammalian cell expression system. We characterized their binding affinities to IL-6R and IL-8R, and determined the degree of inhibition of the IL-6/IL-6R and IL-8/IL-8R interactions on IL-6R- and IL-8R- expressing cell lines via flow cytometry. Blockade of IL-6 signaling was tested on HepG2 cells by flow cytometry, and blockade of IL-8 was evaluated using HTLA cells in the PRESTO-TANGO assay system. We also interrogated the effects of BsAbs on the migration of metastatic cancer cells in a 3D collagen matrix model. Finally, we tested the efficacy of BsAbs in inhibiting cancer metastasis in mouse tumor xenograft models.

Results and Discussion: We successfully produced BS1 and BS2 with high purity (>99%), as demonstrated via SDS-PAGE analysis (Figure 1B). Both BS1 and BS2 bind IL-6R and IL-8R, and competitively inhibit the IL-6/IL-6R and IL-8/IL-8R interaction. BS1 has a weaker binding affinity and less potently competes with ligands compared to BS2 due to the monovalent versus tetravalent construction of the respective BsAbs. We also demonstrated that BS1 and BS2 inhibit IL-6 and IL-8 signaling with similar potency to the parent monoclonal antibodies. Both BsAbs greatly reduced motility, persistence, and diffusivity of cancer cell migration in 3D collagen matrices, and they were significantly more effective at inhibiting tumor cell migration than combination treatment with the parent monoclonal antibodies or with tocilizumab and reparixin. Finally, BS1 and BS2 achieved >75% inhibition of tumor metastasis in orthotopic mouse xenograft models of TNBC at remarkably low doses, illustrating the significant therapeutic advantage for our bispecific approach.

Conclusions: Our engineered BsAbs simultaneously bind IL-6R and IL-8R and effectively block the IL-6/IL-8 axis to inhibit tumor metastasis. These antibodies present a novel treatment option for highly invasive cancers and promise to improve treatment efficacy, minimize harmful side effects, and synergize with other anti-cancer agents. Moreover, our novel BsAb designs will provide new insights into the development of bispecific constructs for future therapies targeting a broad range of diseases.