A novel antibody–drug conjugate (ADC) was synthesized incorporating ferritin-based nanoparticles. An average of three molecules of monoclonal antibody (mAb) Ep1 to the human melanoma-specific antigen CSPG4 were conjugated to a single ferritin cage encapsulating about 50 cisplatin molecules (HFt–Pt–Ep1). The HFt–Pt–Ep1 nanoparticle had an estimated molecular size of about 900 kD and 33 nm, and flow cytometry demonstrated specific binding to a CSPG4⁺ melanoma cell line, but not to a CSPG4⁻ breast carcinoma cell line. As compared to the cisplatin-containing ferritin nanoparticle alone (HFt–Pt), which inhibited thymidine incorporation more efficiently in breast carcinoma than melanoma cells, the mAb-derivatized HFt–Pt–Ep1 nanoparticle had a 25-fold preference for the latter. A similar preference for melanoma was observed upon systemic intravenous administration of HFt–Pt–Ep1 to nude mice xenotransplanted with pre-established, palpable melanoma and breast carcinoma tumors. Thus, we have been able to determine precise combinations and stoichiometric relationships between mAbs and nanoparticle protein cages, whereby the latter lose their tropism for ubiquitously distributed cellular receptors, and acquire instead remarkably lineage-selective binding. HFt–Pt–Ep1 is therefore an interesting model to improve the therapeutic index of antiblastic therapy in a tumor such as melanoma, which at its advanced stages is totally refractory to mono- and combination-chemotherapy.

The nonclassic class I human leukocyte antigen E (HLA-E) molecule engages the inhibitory NKG2A receptor on several cytotoxic effectors, including natural killer (NK) cells. Its tissue distribution was claimed to be wider in normal than in neoplastic tissues, and surface HLA-E was undetectable in most tumor cell lines. Herein, these issues were reinvestigated taking advantage of HLA-E-specific antibodies, immunohistochemistry, and biochemical methods detecting intracellular and surface HLA-E regardless of conformation. Contrary to published evidence, HLA-E was detected in a few normal epithelia and in a large fraction (approximately 1/3) of solid tumors, including those derived from HLA-E-negative/low-normal counterparts. Remarkably, HLA-E was detected in 30 of 30 tumor cell lines representative of major lymphoid and nonlymphoid lineages, and in 11 of 11, it was surface-expressed, although in a conformation poorly reactive with commonly used antibodies. Coexpression of HLA-E and HLA class I ligand donors was not required for surface expression but was associated with NKG2A-mediated protection from lysis by the cytotoxic cell line NKL and polyclonal NK cells from healthy donors, as demonstrated by antibody-mediated relief of protection in 10% to 20% of the tested target-effector combinations. NKG2A-mediated protection of additional targets became evident on NK effector blocking with antibodies to activating receptors (DNAM-1, natural cytotoxicity receptors, and NKG2D). Thus, initial evidence that the long-elusive HLA-E molecule is enhanced by malignant transformation and is functional in tumor cells is presented here, although its importance and precise functional role remain to be addressed in the context of a general understanding of the NK ligand-receptor network.

The endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-I peptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4 and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response and depends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed, high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NK inhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in its two-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express high levels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target for cancer immunotherapy. Cancer Res; 71(5); 1597–606. ©2011 AACR.