The described therapeutic tools that specifically target human CCR9+-tumors and have been tried in xenogeneic models are limited to the use of the CCR9-ligand coupled to a cytotoxic agent (CCL25-PE38 fusion protein) (33), the use of ligand-specific antibodies, alone or in combination with etoposide (25), or the mAb 91R that selectively inhibited growth of a human acute T lymphoblastic leukemia (T-ALL) cell line in Rag2?/? xenografts (34). provides resistance to apoptosis the phosphatidylinositide 3-kinase/Akt pathway on several solid tumors (20, 21, 24C30); it activates the JNK1 antiapoptotic pathway in leukemic cells (31) and participates in Notch1-mediated cell proliferation (19). Targeted therapies and immunotherapy have safety advantages over non-specific cytotoxic agents, since they are able to discriminate between normal and tumor cells. Therefore, their use for the treatment of cancer is in constant expansion (32). The described therapeutic tools that specifically target human CCR9+-tumors and have been tried in xenogeneic models are limited to the use of the CCR9-ligand coupled to a cytotoxic agent (CCL25-PE38 fusion protein) (33), the use of ligand-specific antibodies, alone or in combination with etoposide AMG-8718 (25), or the mAb 91R that selectively inhibited growth of a human acute T lymphoblastic leukemia (T-ALL) cell line in Rag2?/? xenografts (34). The first two strategies eliminate tumor cells by targeting the CCL25CCCR9 interaction, whereas the last directly targets the cells expressing CCR9. These data provide evidence of CCR9 as a potential target for cancer immunotherapy. With the aim of selecting other anti-CCR9 mAb with (i) different specificities, (ii) different affinities for CCR9, (iii) provided of different mechanism(s) of action, and (iv) displaying high melting points, new hybridomas were generated and screened. mAbs with these properties could be more convenient to be used for therapeutic purposes. Here, we AMG-8718 report the generation and characterization of 92R, an anti-CCR9 mAb able to selectively inhibit growth of human acute T-ALL cells transplanted into immunodeficient Rag2?/? or NSG mice. This antibody has therapeutic potential for the targeted elimination of CCR9+-tumor cells, used either alone or in combination with other therapies. Materials and Methods Cells and Reagents Human embryonic kidney 293 (HEK-293, CRL-1573) cells and HEK-293 cells stably transfected with the human chemokine receptor CCR9, or the empty vector (pCIneo) were a kind gift of A. Zaballos (CNB-CSIC, Madrid, Spain), cells were cultured as described (3). MOLT-4 (CRL-182) and Jurkat (TIB-152) human T-ALL cell lines were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbeccos modified Eagles medium (Gibco) supplemented with 10% fetal bovine AMG-8718 serum (FBS, Gibco), 2?mM l-glutamine, 50?U/ml penicillin, and 50?g/ml streptomycin (complete medium). Neomycin-resistant stable HEK-293 transfectants were cultured in the presence of 1?mg/ml G418 (Sigma) and periodically tested for CCR9 expression (not shown). Recombinant human CCL25 and CXCL12 were purchased from Peprotech. We used the following antibodies: 3C3 (ATCC HB-12653), 112509, mouse mAb anti-hCCR9 (IgG2a; R&D) and M4, a serum pool generated by immunizing BALB/c mice with three intraperitoneal injections of 107 MOLT-4 cells in PBS (days 1, 25, and 50); sera were collected on day 60. Generation of Human CCR9-Specific mAb Murine 91R and 92R anti-human CCR9 mAb were raised after immunization of BALB/c mice with a gene gun (Bio-Rad) particle-mediated DNA administration of the pCIneo plasmid bearing the human CCR9 cDNA, as previously described (34). Mouse sera were collected 7C10?days (d) after the last boost and tested for specific antibodies by flow cytometry using stably transfected hCCR9-HEK-293 cells, and pCIneo-HEK-293 cells as negative control. Selected mice were boosted intravenously with 107 hCCR9-HEK293 cells 3 and 2?days prior to splenocyte fusion (35). Two weeks post-fusion, culture supernatants were screened by flow cytometry for CCR9-specific antibodies using hCCR9-HEK293 cells. Positive hybridomas were cloned, mAb purified from culture supernatants and antibody isotype determined by enzyme-linked immunosorbent assay (ELISA) (35). Flow Cytometry For staining, 2??105?cells/well were centrifuged in V-bottom 96-well plates and washed with phosphate-buffered saline, pH 7.4 (PBS) supplemented with 0.5% bovine serum albumin (BSA), 1% FBS, and 0.1% sodium azide (PBSst). Non-specific binding of the mAb to the cell surface was blocked by preincubating the cells with 40?g/ml rat IgG (Sigma) in a 100?l final volume (20?min, 4C). Cells were incubated with the primary mAb (30?min, 4C), huCdc7 washed, and the binding was revealed with a secondary FITC- or PE-goat F(ab)2 anti-mouse IgG (H?+?L) antibody (Beckman Coulter; 30?min, 4C). Samples were analyzed on an Epics XL or a Cytomics cytometer (Beckman Coulter). For competition analyses, cells were incubated with 50?l of either the unlabeled antibody or an isotype-matched mAb (10?g/ml, 40?min, 4C), followed by 50?l of an anti-CCR9 biotin-labeled antibody (0.5C2?g/ml, 30?min, 4C). After washing, FITC- or PE-conjugated streptavidin was added (30?min, 4C). Cell.