Abstract
Abstract Milatuzumab (hLL1, Immunomedics, Inc.) is a humanized anti-CD74 monoclonal IgG1 antibody (mAb) currently in phase I-II clinical trials as a therapeutic mAb for CD74-expressing non-Hodgkin lymphoma, chronic lymphocytic leukemia, and multiple myeloma. Since CD74 is also expressed by normal antigen-presenting cells (APCs), including DCs, we investigated the potential application of the Fab fragment of hLL1 as a novel targeting vector in a cancer vaccine. Methods: A fusion protein, hLL1-Fab-A3B3, was constructed by fusing the Fab fragment of milatuzumab to the A3B3 domain of CEA. The binding profiles and cytotoxic potenial of hLL1-Fab-A3B3 on APC subsets in human peripheral blood mononuclear cells (PBMCs) were assessed by flow cytometry; the impact of hLL1-Fab-A3B3 on APC function was assessed by allogeneic mixed leukocyte reaction (MLR) through CFSE-based cell proliferation assay. The cross-presentation of CEA by DCs was evaluated by three rounds of weekly stimulation of autologous T cells with DCs loaded with hLL1-Fab-A3B3 or CEA protein, followed by measuring CEA-specific CD8+ T cells with HLA-A*0201-restricted CAP-1 pentamer (YLSGANLNL). Results: Both bivalent milatuzumab and monvalent hLL1-Fab-A3B3 bound efficiently to human APC subsets, which include BDCA-1+ myeloid DCs type 1 (mDC1), BDCA-2+ plasmacytoid DCs (pDCs), BDCA-3+ myeloid DCs type 2 (mDC2), B lymphocytes, and monocytes, but neither of the two bound to T cells. While milatuzumab selectively reduced mDC1 (by 60.8%, P=0.003, n=6 donors) and mDC2 (by 53.8%, P=0.0026, n=7 donors) in PBMCs, presumably mediated by the presence of Fc, or a necessity for divalent milatuzumab binding, hLL1-Fab-A3B3 did not have this effect, nor did it affect the viability of T cells and other APC subsets (pDCs, monocytes, and B cells). The allogeneic MLR was significantly inhibited by milatuzumab (P<0.0001), but not by hLL1-Fab-A3B3 treatment (P=0.6644). After three rounds of weekly stimulation of autologous T cells, DCs loaded with hLL1-Fab-A3B3 were more efficient in inducing CEA-specific CD8+ T cells than DCs loaded with CEA, suggesting that the hLL1-Fab enhances the cross-presentation of CEA by DCs. Conclusion: hLL1-Fab is a promising, novel targeting antibody for use as a cancer vaccine, because it enhances the cross-presentation of antigens without significant cytotoxicity and functional alteration on APCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 755. doi:10.1158/1538-7445.AM2011-755
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