A tri-specific killer engager against mesothelin targets NK cells towards lung cancer.

New treatments are required to enhance current therapies for lung cancer. Mesothelin is a surface protein overexpressed in non-small cell lung cancer (NSCLC) that shows promise as an immunotherapeutic target in phase I clinical trials. However, the immunosuppressive environment in NSCLC may limit efficacy of these therapies. We applied time-of-flight mass cytometry to examine the state of circulating mononuclear cells in fourteen patients undergoing treatment for unresectable lung cancer. Six patients had earlier stage NSCLC (I-IVA) and eight had highly advanced NSCLC (IVB). The advanced NSCLC patients relapsed with greater frequency than the earlier stage patients. Before treatment, patients with very advanced NSCLC had a greater proportion of CD14- myeloid cells than patients with earlier NSCLC. These patients also had fewer circulating natural killer (NK) cells bearing an Fc receptor, CD16, which is crucial to antibody-dependent cellular cytotoxicity. We designed a high affinity tri-specific killer engager (TriKE®) to enhance NK cytotoxicity against mesothelin+ targets in this environment. The TriKE consisted of CD16 and mesothelin binding elements linked together by IL-15. TriKE enhanced proliferation of lung cancer patient NK cells in vitro. Lung cancer lines are refractory to NK cell killing, but the TriKE enhanced cytotoxicity and cytokine production by patient NK cells when challenged with tumor. Importantly, TriKE triggered NK cell responses from patients at all stages of disease and treatment, suggesting TriKE can enhance current therapies. These pre-clinical studies suggest mesothelin-targeted TriKE has the potential to overcome the immunosuppressive environment of NSCLC to treat disease.

NK-Cell-Mediated Targeting of Various Solid Tumors Using a B7-H3 Tri-Specific Killer Engager In Vitro and In Vivo.

We improved the bispecific antibody platform that primarily engages natural killer (NK) cells to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by adding IL-15 as a crosslinker that expands and self-sustains the effector NK cell population. The overall goal was to target B7-H3, an established marker predominantly expressed on cancer cells and minimally expressed on normal cells, and prove that it could target cancer cells in vitro and inhibit tumor growth in vivo. The tri-specific killer engager (TriKETM) was assembled by DNA shuffling and ligation using DNA encoding a camelid anti-CD16 antibody fragment, a wild-type IL-15 moiety, and an anti-B7-H3 scFv (clone 376.96). The expressed and purified cam1615B7H3 protein was tested for in vitro NK cell activity against a variety of tumors and in vivo against a tagged human MA-148 ovarian cancer cell line grafted in NSG mice. cam1615B7H3 showed specific NK cell expansion, high killing activity across a range of B7-H3+ carcinomas, and the ability to mediate growth inhibition of aggressive ovarian cancer in vivo. cam1615B7H3 TriKE improves NK cell function, expansion, targeted cytotoxicity against various types of B7-H3-positive human cancer cell lines, and delivers an anti-cancer effect in vivo in a solid tumor setting.

Targeting pediatric sarcoma with a bispecific ligand immunotoxin targeting urokinase and epidermal growth factor receptors.

Children with high risk sarcoma have a poor prognosis despite surgical resection, irradiation and chemotherapy. Alternative therapies are urgently needed. Urokinase-type plasminogen activator receptor (uPAR) and epidermal growth factor receptor (EGFR) are surface proteins expressed by some pediatric sarcomas. We show for the first time that a de-immunized bispecific ligand toxin, EGFATFKDEL, directed against EGFR and uPAR, successfully targets pediatric sarcoma. Using flow cytometry, we identified a rhabdomyosarcoma (RMS) cell line, RH30, that expresses both uPAR and EGFR, and a Ewing sarcoma (EWS) cell line, TC-71, that expresses only uPAR. We tested the differential sensitivity of these two sarcoma cell lines to toxin-induced killing, using both in vitro assays and an in vivo murine model. We show that pediatric sarcomas are highly sensitive to EGFATFKDEL (at subnanomolar concentrations) in vitro. In vivo, tumor growth was significantly attenuated after treatment with EGFTFKDEL, compared to untreated controls, in both RH30 and TC-71 tumor bearing mice. In addition, we found that simultaneously targeting both receptors in a dual positive cell line was more effective than targeting a single receptor or antigen, resulting in a greater tumor response, including complete tumor regression in an animal model of bulky disease. Our findings provide support for further exploration of bispecific targeting of pediatric sarcomas with bispecific ligand toxins, such as EGFATFKDEL.