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The NAD-hydrolyzing ecto-enzyme CD38 is overexpressed by multiple myeloma and other hematological malignancies. We recently generated CD38-specific nanobodies, single immunoglobulin variable domains derived from heavy-chain antibodies naturally occurring in llamas. Nanobodies exhibit high solubility and stability, allowing easy reformatting into recombinant fusion proteins. Here we explore the utility of CD38-specific nanobodies as ligands for nanobody-based chimeric antigen receptors (Nb-CARs). We cloned retroviral expression vectors for CD38-specific Nb-CARs. The human natural killer cell line NK-92 was transduced to stably express these Nb-CARs. As target cells we used CD38-expressing as well as CRISPR/Cas9-generated CD38-deficient tumor cell lines (CA-46, LP-1, and Daudi) transduced with firefly luciferase. With these effector and target cells we established luminescence and flow-cytometry CAR-dependent cellular cytotoxicity assays (CARDCCs). Finally, the cytotoxic efficacy of Nb-CAR NK-92 cells was tested on primary patient-derived CD38-expressing multiple myeloma cells. NK-92 cells expressing CD38-specific Nb-CARs specifically lysed CD38-expressing but not CD38-deficient tumor cell lines. Moreover, the Nb-CAR-NK cells effectively depleted CD38-expressing multiple myeloma cells in primary human bone marrow samples. Our results demonstrate efficacy of Nb-CARs in vitro. The potential clinical efficacy of Nb-CARs in vivo remains to be evaluated.

Gunter Schuch, Sebastian Kobold, Carsten BokemeyerDepartment of Oncology, Hematology, and Bone Marrow Transplantation with Section of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, GermanyAbstract: In recent years, the monoclonal epidermal growth factor receptor (EGFR)-targeting antibody cetuximab was introduced into systemic therapy of colorectal cancer and gained an established role in the treatment of this disease. Cetuximab was shown to be active as a single agent in chemorefractory metastatic disease as well as in combination with varying chemotherapies. Recently, randomized trials demonstrated the activity of cetuximab combinations in the first-line setting of metastatic colorectal cancer. Interestingly, the activity of cetuximab was restricted to patients with KRAS wildtype tumors, as was seen with panitumumab, another EGFR antibody. While 60%–70% of tumors harbor KRAS wildtype genes, 30%–40% of tumors express oncogenic KRAS with mutations in codons 12 and 13 causing constitutive activation of signaling cascades downstream of EGFR and resistance to EGFR blockade. Since proof of KRAS wildtype status became a prerequisite for cetuximab treatment, KRAS testing is being established throughout the world. Future trials will address the question which part of the KRAS wildtype cohort will benefit from EGFR inhibition and how to identify those patients. Additionally, new strategies for treatment of KRAS mutated tumors are strongly needed. Recent developments and future strategies will be summarized.Keywords: cetuximab, colorectal cancer, KRAS