Differential killing of CD56-expressing cells by drug-conjugated human antibodies targeting membrane-distal and membrane-proximal non-overlapping epitopes

Abstract

CD56 (NCAM, neural cell adhesion molecule) is over-expressed in many tumor types, including neuroblastoma, multiple myeloma, small cell lung cancer, ovarian cancer, acute myeloid leukemia, NK-T lymphoma, neuroendocrine cancer and pancreatic cancer. Using phage display, we identified 2 highaffinity anti-CD56 human monoclonal antibodies (mAbs), m900 and m906, which bound to spatially separated non-overlapping epitopes with similar affinity (equilibrium dissociation constant 2.9 and 4.5 nM, respectively). m900 bound to the membrane proximal fibronectin type III-like domains, whereas m906 bound to the N-terminal IgG-like domains. m906 induced significant down-regulation of CD56 in 4 neuroblastoma cell lines tested, while m900-induced downregulation of CD56 was much lower. Antibodydrug conjugates (ADCs) made by conjugation with a highly potent pyrrolobenzodiazepine dimer (PBD) exhibited killing activity that correlated with CD56 down-regulation, and to some extent with in vivo binding ability of the antibodies. The m906PBD ADC was much more potent than m900PBD, likely due to higher CD56-mediated downregulation and stronger binding to cells. Treatment with m906PBD ADC resulted in very potent cytotoxicity (IC50: 0.05-1.7 pM). These results suggest a novel approach for targeting CD56-expressing neuroblastoma cells. Further studies in animal models and in humans are needed to find whether these antibodies and their drug conjugates are promising candidate therapeutics.

One-domain CD4 Fused to Human Anti-CD16 Antibody Domain Mediates Effective Killing of HIV-1-Infected Cells

“…NK cells were enriched from human PBMC by using the NK cell isolation kit II in the negative selection modes (Miltenyi Biotec). Purified NK cells were detected by incubation with biotinylated anti-CD56 antibody (m909, developed by our group 42 ) followed by streptavidin-FITC and PE conjugated mouse anti-human CD16 antibody. Binding to NK cells was measured by incubating 10 6 NK cells in 200 μL PBS containing 0.1% bovine serum albumin (BSA) (PBSA) with ~10 nM of BiKEs for 30 min at room temperature.…”

CAMKV Is a Candidate Immunotherapeutic Target in MYCN Amplified Neuroblastoma

“…Finally, the membrane localization of CAMKV was confirmed in MYCN amplified NGP neuroblastoma cells by immunofluorescence ( Figure 5C). We used NCAM1 (CD56), an established cell surface molecule expressed in neuroblastoma as a marker for the plasma membrane (32,46) and found NCAM1 and CAMKV proteins colocalize on the plasma membrane of NGP cells. Together, these data show that CAMKV is a membrane-bound protein in MYCN amplified neuroblastoma with the potential to be targeted using a CAMKV-specific biologic.…”

From Anthramycin to Pyrrolobenzodiazepine (PBD)‐Containing Antibody–Drug Conjugates (ADCs)

“…Feng and co-workers at the NCI have recently reported studies involving a PBD-based CD56-targeted ADC. [120] CD56 is over-expressed in a number of different cancer types including multiple myeloma, acute myeloid leukaemia and pancreatic cancer. Using a phage display technique, two high-affinity anti-CD56 monoclonal antibodies were initially identified, M900 and M906, that bind to membrane proximal fibronectin Type III-like domains and the N-terminal IgGlike domain, respectively.…”