Changhua Zhou

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells

Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

Single domain antibody-based bispecific antibody induces potent specific anti-tumor activity

ABSTRACT Bispecific antibodies have emerged as powerful therapeutic agents given their high specificity and ability to induce a potent immune response. Various bispecific antibody formats have been designed and studied regarding their applications in cancer therapy, though associated with issues of short half-life or manufacturing difficulties. Herein, a novel bispecific antibody, SS-Fc, was constructed by pairing 2 single-domain antibodies, anti-CD16 and anti-CEA, which were fused with CH3 “knobs into holes” mutations individually. SS-Fc was expressed and purified from E.coli. In vitro and in vivo experiments confirmed that SS-Fc can form a heterodimeric bispecific antibody when expressed and purified from E. coli. By engaging natural killer (NK) cells through an anti-CD16 single domain antibody, the SS-Fc bispecific antibody exhibited potent in vitro and in vivo cytotoxicity against cancer cells with carcinoembryonic antigen (CEA) expression. Thus, SS-Fc represents a novel bispecific antibody format that can be applied to a wide range of both discovery and clinical applications.

A novel bispecific antibody, BiSS, with potent anti-cancer activities.

ABSTRACT One of the most active fields in cancer immunotherapy is the study of bispecific antibodies, which engage immune cells to kill cancer cells. However, a variety of issues are associated with most of current bispecific antibody formats. In this study, we present a novel bispecific antibody, BiSS (Bispecific antibody with Single domain, Single domain antibodies), which was constructed by linking 2 single domain antibodies, anti-CEA and anti-CD16, in tandem. Unlike most other bispecific antibodies, the BiSS antibody can be expressed and purified from E.coli in large quantities. By recruiting natural killer cells (NK cells) to CEA-positive cancer cells, BiSS led to cancer cell death in vitro. In xenograft models, the BiSS protein blocked cancer progression. The data suggested that the single domain-based bispecific antibody BiSS was functional and can be potentially applied to a broad range of immunotherapies.

BiHC, a T-Cell–Engaging Bispecific Recombinant Antibody, Has Potent Cytotoxic Activity Against Her2 Tumor Cells

Among different cancer immunotherapy approaches, bispecific antibodies (BsAbs) are of great interest due to their ability to recruit immune cells to kill tumor cells directly. Various BsAbs against Her2 tumor cells have been proposed with potent cytotoxic activities. However, most of these formats require extensive processing to obtain heterodimeric bispecific antibodies. In this study, we describe a bispecific antibody, BiHC (bispecific Her2-CD3 antibody), constructed with a single-domain anti-Her2 and a single-chain Fv (variable fragment) of anti-CD3 in an IgG-like format. In contrast to most IgG-like BsAbs, the two arms in BiHC have different molecular weights, making it easier to separate hetero- or homodimers. BiHC can be expressed in Escherichia coli and purified via Protein A affinity chromatography. The purified BiHC can recruit T cells and induce specific cytotoxicity of Her2-expressing tumor cells in vitro. The BiHC can also efficiently inhibit the tumor growth in vivo. Thus, BiHC is a promising candidate for the treatment of Her2-positive cancers.

Single domain based bispecific antibody, Muc1-Bi-1, and its humanized form, Muc1-Bi-2, induce potent cancer cell killing in muc1 positive tumor cells

Muc1 is one of the most studied tumor antigens. However, antibodies or antibody-toxin conjugates against Muc1 have not shown significant efficacy for tumors with Muc1 overexpression. In this study, we employed bispecific antibody approach to target Muc1 positive tumor cells. A novel bispecific antibody, Muc1-Bi-1, was constructed by linking single domain antibodies, anti-Muc1-VHH and anti-CD16-VHH. Muc1-Bi-2, the humanized form of Muc1-Bi-1, was also constructed by grafting. Both Muc1-Bi bispecific antibodies can be efficiently expressed and purified from bacteria. In vitro, the Muc1-Bi bispecific antibodies can recruit Natural Killer (NK) cells to drive potent and specific cell killing of Muc1-overexpressing tumor cells. In xenograft model, the Muc1-Bi bispecific antibodies can suppress tumor growth in the presence of human peripheral blood mononuclear cells (PBMC). These data suggested that the single domain based Muc1-Bi may provide a valid strategy for targeting tumors with Muc1 overexpression.

Prodrug AST-003 Improves the Therapeutic Index of the Multi-Targeted Tyrosine Kinase Inhibitor Sunitinib.

Patients have responded well to the multi-targeted tyrosine kinase inhibitor (TKI) Sunitinib in the clinic. But the severe toxic side effects associated with Sunitinib limit its therapeutic index. To improve the therapeutic index of Sunitinib, a prodrug strategy was employed to modify Sunitinib. The inactive prodrug AST-003 can be converted to Sunitinib in vitro and in vivo. Compared with Sunitinib, AST-003 has unique biochemical, cellular and pharmacokinetic properties with improved tolerability in mice and yield higher efficacy in tumor xenograft models. This prodrug strategy may constitute a novel paradigm to improve the therapeutic index of Sunitinib and other TKI or anti-angiogenesis drugs in general.

A targeted transforming growth factor-beta (TGF-β) blocker, TTB, inhibits tumor growth and metastasis

Transforming growth factor beta (TGF-β) promotes cancer growth in late stage cancers. To inhibit the TGF-β pathway, we investigated a tumor-targeting TGF-β receptor blocker, TTB, and its role in tumor progress. The targeted TTB comprised of the extracellular domain of the TGF-β receptor II, the endoglin domain of TGF-β receptor III, and the human immuno-globin IgG1 constant fragment (Fc). To enhance tumor microenvironment targeting, a RGD peptide was fused at the N-terminal of TTB. The targeted TTB exhibited potent TGF-β neutralization activities, and inhibited cancer cell migration and invasion as well as colony formation. In xenograft models, the TTB had potent tumor inhibition activities. The TTB also attenuated the TGF-β1-induced Smad2 phosphorylation and epithelial to mesenchymal transformation (EMT), and suppressed breast cancer metastasis. Thus, the TTB is an effective TGF-β blocker with a potential for blocking excessive TGF-β induced pathogenesis in vivo.

A HER2 bispecific antibody can be efficiently expressed in Escherichia coli with potent cytotoxicity

Bispecific antibodies have been actively studied for cancer therapy due to their potent cytotoxicity against tumor cells. A number of bispecific antibody formats have exhibited strong tumor cytotoxicity in vitro and in vivo. However, effective production of bispecific antibodies remains challenging for the majority of bispecific antibody formats. In the present study, a bispecific antibody was designed that links a conventional antigen-binding fragment (Fab) against cluster of differentiation 3 antigen (CD3) to a camel single domain antibody (VHH) against human epidermal growth factor receptor 2 (HER2). This bispecific antibody may be secreted and purified efficiently from Escherichia coli culture medium. The purified bispecific antibody is able to trigger T cell-mediated HER2-specific cytotoxicity in vitro and in vivo. The data gathered in the present study suggest that this bispecific format may be applied to other tumor antigens to produce bispecific antibodies more efficiently.

A Bispecific Antibody Based on Pertuzumab Fab Has Potent Antitumor Activity

Human epidermal growth factor receptor 2 (HER2) is frequently overexpressed and activated in metastatic breast cancers. Monoclonal antibodies targeting Her2, such as trastuzumab and pertuzumab, have become important targeted therapies for patients with HER2-positive breast cancer. Both trastuzumab and pertuzumab can reduce Her2 positive tumor burden by inhibiting Her2 signaling and inducing ADCC activities (antibody dependent cell-mediated cytotoxicity). In this study, we have generated a bispecific antibody, Her2(Per)-S-Fab, by linking the pertuzumab Fab to an anti-CD16 single domain antibody. The Her2(Per)-S-Fab can be expressed and purified efficiently from Escherichia coli. In vitro and in vivo experiments showed Her2(Per)-S-Fab had potent cytotoxicity against Her2-positive tumor cells. Thus, Her2(Per)-S-Fab may provide an alternative to treat Her2-positive cancer patients.