Preclinical data on Sorrento Therapeutics’ bi-specific antibodies (BsAbs; also known as “dual specificity” antibodies) and antibody drug conjugates (ADCs) against c-Met in breast cancer presented during  the 37th Annual San Antonio Breast Cancer Symposium (SABCS) being held at The Henry B. Gonzalez Convention Center in San Antonio, Texas, December 9 – 13, 2014, confirms that c-Met is a potential therapeutic target for antibody-drug conjugates in breast cancer.

The highlights of the findings, presented during the general poster session by Kouros Motamed, PhD, Vice President of Strategic Alliances & Clinical Communications, Sorrento Therapeutics, Inc., include preclinical data on generation and evaluation of an anti-c-Met/anti-PD-L1 and anti-c-Met/anti-ErbB3 bi-specific, and an anti-c-Met antibody-drug conjugate or ADC as new immunotherapeutic strategies in breast cancer. [1][2]

STI-D0602
The proto-oncogene transmembrane receptor tyrosine kinase c-MET is involved in cellular proliferation, survival, migration, and invasion in normal and tumor cells and plays a key role in malignant transformation of epithelial cells. MET is widely expressed and associated with poor prognosis in a number of cancers, including breast, lung, liver, kidney, and brain cancers.  Over expression of c-MET in breast cancer, with or without gene amplification, has been reported in primary breast cancers and correlates with poor prognosis and adverse survival outcomes. [2] This may make c-MET it a promising target for antibody drug conjugates.

Because inhibition of c-MET signaling, via tyrosine kinase inhibitors (TKIs) or antagonistic antibodies, is usually not sufficient for sustained treatment efficacy, Yanwen Fu, Edwige Gros, Alice Lee and their colleagues at Sorrento Therapeutics believe that antibody drug conjugates offer the promise and potential of delivering more potent anti-tumor activity.

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To support their hypothesis, the researchers generated antibody-drug conjugates containing a proprietary human anti-c-MET antibody (STI-D0602) with either a tubulin inhibitor or a DNA damaging agent. STI-D0602, a fully human antibody (IgG1) selected from Sorrento’s G-MAB antibody library, one of the largest and most diverse fully human antibody libraries in the industry designed to facilitate the rapid identification and selection of highly specific monoclonal antibody therapeutic product candidates, was conjugated with a cytotoxin via site-specific bio-conjugation. The conjugates retained binding affinity and showed potent cell killing in a variety of c-MET-positive cell lines.

Inhibition of MET and HER signaling
Hepatocyte growth factor or HGF is the only known ligand of the MET receptor. The MET/HGF promotes cancer cell migration and invasion. MET/HGF is also implicated in mediating resistance to current anticancer therapies, including radiation. Furthermore, overexpression of MET is associated with poor prognosis in HER2-positive breast cancer patients, which is confirmed by  a shorter progression free survival (PFS) following HER1/HER2-targeted treatment with lapatinib (Tykerb®; GSK). [3]

Ongoing research has show that dual EGFR/MET inhibition is synergistic in EGFR-directed therapy resistance triple negative breast cancer [4][5]. Based on this data, the researchers proposed a combination therapy with inhibitors of MET and HER signaling for a subset of metastatic breast cancer patients with MET- amplified/overexpressed tumors.

The anti-c-Met antibodies inhibited signaling by stimulating c-Met internalization in the absence of receptor activation. Anti-c-Met ADCs retained binding affinity and demonstrated potent cell killing in a variety of TNBC cell lines. STI-D0602-DM1 conjugate showed potent in vivo efficacy without significant toxicity.

Bi-specific Antibodies
Hybridoma technology, first described in the august 1975 edition of Nature by Kohler and Milstein, made targeted antibody therapy a reality.  Therapy with unconjugated or ‘naked’ antibodies depends of host immune effector mechanisms, including antibody-dependent cell-mediated cytotoxicity or ADCC, complement-dependent cytotoxicity or CDC and/or direct anti proliferative effects induced by antibodies binding to a tumor cell.  In clinical studies, ADCC has been demonstrated to significantly enhance the efficacy of various antibodies, including rituximab, trastuzumab, and cetuximab [2]. While therapies with single-agent unconjugated antibodies are benefiting a substantial number of patients,  most of the currently approved therapeutic anti-cancer antibodies are monospecific and therefore only capable of interfering with the biological function of a single molecular target. However, breast cancers mostly involve crosstalk of often synergistic signal transduction pathways.[6][7]

As a result, isolated blockade of a single signal transduction pathway is generally met by escape mechanisms, including  up-regulation of redundant pathways.  This makes monospecific immunotherapy less effective. To contract, numerous strategies have been development for selectively redirecting effector cells/molecules towards tumor cells designed to overcome dose limiting toxicities and to increase efficacy of immunotherapy of cancer.

The majority of these strategies applied look at the specificity of tumor associated antigen recognition of antibodies.  By using either hybridoma fusion, chemical derivatization or molecular biology technology, scientists are able to construct antibodies with dual specificity. The resulting bi-specific antibodies or bsAbs combine the binding specificity of two antibodies in one molecule.  This construct can be used to redirect the cytolytic activity of a number of immune effector cells including cytotoxic T lymphocytes, natural killer cells, neutrophils and monocytes/macrophages to tumor cells, enhancing tumor destruction.[1][8]

What’s more, by targeting two different receptors in combination on the same cell, bsAbs can induce modifications of cell signaling, including the inactivation of proliferation or inflammatory pathways.

In a second poster presented at the San Antonio Breast Cancer Symposium (Bi-specific Antibodies Targeting Signaling Pathway Crosstalk are a New Breast Cancer Immunotherapeutic Strategy” – poster number P2-16-02),  Yanliang Zhang, Edwige Gros, Sarabjit Chagar, and their colleagues describe how using both chemical and molecular biology techniques, they have developed new approaches to generate IgG-like bi-specific antibodies (BsAbs) designed to target either two compensating signal transduction pathways (i.e. HER family members) or a breast cancer specific antigen and an immuno-regulatory molecule (i.e. PD-L1 or PD1).

To generate an anti-c-Met and anti-PD- L1 chemical bi-specific antibody (CBA), the researchers applied a chemical biology method which involves specific hetero-dimerization of two half antibody molecules using bioorthogonal chemistry, a chemical reaction that can occur inside of living systems without interfering with native biochemical processes. Further, applying a molecular biology approach, the scientists also produced anti-c-Met and ErbB3 scFv-Fc bi-specific antibody.

Bi-specific antibodies of many different formats have been developed with superior anti cancer activities. While the idea of bi-specific antibodies is not new, so far, the major obstacles in producing bi-specific antibodies is the so-called chain association issue. which means that 16 different possible combinations for each antibody’s two heavy chains with its two light chains result in 10 different antibodies (all heavy chains can pair with each other and every light chain can bind unspecifically to two regions at the top of the heavy chains). To ensure correct pairing of heavy chains from different antibodies while at the same time preventing formation of unwanted side products the bioengineers at Sorrento applied the so-called “Knob-into-Hole” technology originally invented in the late 1990’s by scientists at Genentech.  Using this process, the company’s bioengineers showed that scFv fragment based approach and conjugation based method are viable methods of generating bi-specific antibodies.

Superior binding
The final result was that anti-c-Met/ErbB3 bi-specfic BA-0702 in scFv-Fc format demonstrated superior binding activity towards tumor cell lines MDA-MA-468 and MCF7 to each parental monospecific IgG1s. Also, BA-0702 showed activity in suppressing HGF induced c-Met phosphorylation in cancer cell line MDA-MB- 231 and also showed much higher cell killing activity (HS578T) than each parental IgG1s when they all were complexed with monomethyl auristatin F (MMAF)-conjugated Protein-G, indicating its potential application in antibody-drug conjugates.

Similarly, Anti-c-Met/PD-L1 chemical bi-specific CBA-0710 retained excellent affinity for their respective cellular target and demonstrated potent in vitro activity.