Development Of Therapeutic Antibody Drugs Against Tumor

Therapeutic antibody drugs such as monoclonal antibodies and antibody-drug conjugate (ADC) are widely used for cancer therapy because of the high specificity and multiple anti-tumor mechanisms, In present study, we developed two different formats of therapeutic antibody drugs, human single-domain antibody SD1targeting mesothelin and HN3-PE38immunotoxin targeting GPC3, for cancer therapy.1. A full human single domain antibody elicits potent anti-tumor activity both in vitro and in vivo.Mesothelin is highly expressed in multiple forms of magliances, and shows great promise for clinical development against solid cancers. Antibodies against mesothelin have been shown to act via immunotoxin-based inhibition of tumor growth and induction of antibody-dependent cellular cytotoxicity (ADCC). However, none of the current antibodies against mesothelin demonstrate additional anti-tumor activity such as complement-dependent cytotoxicity (CDC), which considered as an important additional mechanism of therapeutic antibodies against tumors. To improve current antibody therapy against mesothelin, we decided to develop antibody with additional function:(1) We used phage display antibody engineering technology and synthetic peptide screening to identify SD1, a human single-domain antibody to mesothelin which recognizes a conformational epitope at the C-terminal end (residues539-588) of mesothelin close to the cell surface.(2) To investigate SD1as a potential therapeutic agent, we generated a recombinant human Fc (SD1-hFc) fusion protein. SD1-hFc can specifically bind to mesothelin positive cancer cell lines. Interestingly, the SD1-hFc protein exhibits strong CDC activity, in addition to ADCC, against mesothelin-expressing tumor cells. (3) Furthermore, SD1-hFc causes growth inhibition of human tumor xenografts in nude mice as a single agent via both CDC and ADCC activity. SD1is the first human single-domain antibody targeting mesothelin-expressing tumors, shows potential as a cancer therapeutic candidate, and may improve current antibody therapy targeting mesothelin-expressing tumors.2. Combination treatment of antibody-toxin conjugate targeting glypican-3and irinotecan induces regression of established hepatocellular carcinoma in miceGlypican-3(GPC3) is70kDa glycoprotein that is attaeched to cell membrane by GPI anchor. GPC3is hightly expressed in hepatocellular carcinoma (HCC), but not normal liver tissues and could be a a potential target in ADC for HCC therapy. However, antibodies that bind GPC3, by themselves, have not shown strong cytotoxic effect against tumors. Attaching potent cytotoxic drugs or toxins may improve the clinical utility GPC3-targeted therapies. Nevertheless, it has not been established that GPC3can be a target of antibody-drug conjugate. Here, we investigated the GPC3as a potiential candidate for ADC against HCC. Then we generation and analysis of a unique antibody-toxin conjugate targeting HCC.We foun that:(1) GPC3is highly expressed in HCC cell lines especially on HepG2and Hep3B (>105sites per cell) and can be fast internalized from cell sureface, indicating GPC3can be potential target for ADC against HCC.(2) An engineered human antibody domain (HN3) was fused to a clinically used form of Pseudomonas exotoxin (PE38). The HN3-PE38immunotoxin was highly cytotoxic to GPC3-bearing human HCC cell lines but was not cytotoxic to target-negative cells. Intravenous administration of HN3-PE38in nude mice with established subcutaneous HepG2and Hep3B HCC tumors caused considerable anti-tumor activity, especially in HepG2tumor model, as a singe agent, HN3-PE38causes Hep3B tumor shrinkage.(3) In vivo durg screening identified Irinotecan as a potent chmotherapeutical drug agains HCC and could be a potential candidate to enhance activity of immunotoxin.(4) Combination treatment of HN3-PE38and Irinotecan synergistically induce regression of HCC in mice. Furthermore, with the increased injection time of Irinotecan, the combination treatment could complete eradicate the HepG2tumors in nude mice. This shows for the first time that GPC3is a target of immunotoxins or probably other antibody drug conjugate that act inside cells and suggests a previously underscribed approach to treat liver cancer.Taken together, in present study, we developed two different antibody drugs targeting two different tumor antigens and could potentially improve the current therapy against mesothelin or GPC3positive cancers.