Differential expression of CCR8 in tumors versus normal tissue allows specific depletion of tumor-infiltrating T regulatory cells by GS-1811, a novel Fc-optimized anti-CCR8 antibody.

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.

Ulocuplumab (BMS-936564 / MDX1338): a fully human anti-CXCR4 antibody induces cell death in chronic lymphocytic leukemia mediated through a reactive oxygen species-dependent pathway.

The CXCR4 receptor (Chemokine C-X-C motif receptor 4) is highly expressed in different hematological malignancies including chronic lymphocytic leukemia (CLL). The CXCR4 ligand (CXCL12) stimulates CXCR4 promoting cell survival and proliferation, and may contribute to the tropism of leukemia cells towards lymphoid tissues. Therefore, strategies targeting CXCR4 may constitute an effective therapeutic approach for CLL. To address that question, we studied the effect of Ulocuplumab (BMS-936564), a fully human IgG4 anti-CXCR4 antibody, using a stroma--CLL cells co-culture model. We found that Ulocuplumab (BMS-936564) inhibited CXCL12 mediated CXCR4 activation-migration of CLL cells at nanomolar concentrations. This effect was comparable to AMD3100 (Plerixafor--Mozobil), a small molecule CXCR4 inhibitor. However, Ulocuplumab (BMS-936564) but not AMD3100 induced apoptosis in CLL at nanomolar concentrations in the presence or absence of stromal cell support. This pro-apoptotic effect was independent of CLL high-risk prognostic markers, was associated with production of reactive oxygen species and did not require caspase activation. Overall, these findings are evidence that Ulocuplumab (BMS-936564) has biological activity in CLL, highlight the relevance of the CXCR4-CXCL12 pathway as a therapeutic target in CLL, and provide biological rationale for ongoing clinical trials in CLL and other hematological malignancies.

BMS-936564/MDX-1338: a fully human anti-CXCR4 antibody induces apoptosis in vitro and shows antitumor activity in vivo in hematologic malignancies.

PURPOSE: CXCR4 has been identified as a prognostic marker for acute myeloid leukemia (AML) and other malignancies. We describe the development and characterization of a fully human antibody to CXCR4 and its application for therapy of AML, non-Hodgkin lymphoma (NHL), chronic lymphoid leukemia (CLL), and multiple myeloma. EXPERIMENTAL DESIGN: Human transgenic mice were immunized with CXCR4-expressing cells, and antibodies reactive with CXCR4 were analyzed for apoptosis induction and ability to interfere with CXCL12-induced migration and calcium flux. In vivo efficacy was determined in multiple AML, NHL, and multiple myeloma xenograft tumors in severe combined immunodeficient mice. RESULTS: BMS-936564/MDX-1338 is a fully human IgG(4) monoclonal antibody that specifically recognizes human CXCR4. In vitro studies show that MDX-1338 binds to CXCR4-expressing cells with low nanomolar affinity, blocks CXCL12 binding to CXCR4-expressing cells, and inhibits CXCL12-induced migration and calcium flux with low nanomolar EC(50) values. When given as monotherapy, MDX-1338 exhibits antitumor activity in established tumors including AML, NHL, and multiple myeloma xenograft models. In addition, we show that MDX-1338 induced apoptosis on a panel of cell lines and propose that antibody-induced apoptosis is one of the mechanisms of tumor growth inhibition. CONCLUSIONS: BMS-936564/MDX-1338 is a potent CXCR4 antagonist which is efficacious as monotherapy in tumor-bearing mice and is currently in phase I for the treatment of relapsed/refractory AML, NHL, CLL, and multiple myeloma.

A nonfucosylated human antibody to CD19 with potent B-cell depletive activity for therapy of B-cell malignancies.

A human anti-CD19 antibody was expressed in fucosyltransferase-deficient CHO cells to generate nonfucosylated MDX-1342. Binding of MDX-1342 to human CD19-expressing cells was similar to its fucosylated parental antibody. However, MDX-1342 exhibited increased affinity for FcγRIIIa-Phe158 and FcγRIIIa-Val158 receptors as well as enhanced effector cell function, as demonstrated by increased potency and efficacy in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. MDX-1342 showed dose-dependent improvement in survival using a murine B-cell lymphoma model in which Ramos cells were administered systemically. In addition, low nanomolar binding to cynomolgus monkey CD19 and increased affinity for cynomolgus monkey FcγRIIIa was observed. In vivo administration of MDX-1342 in cynomolgus monkeys revealed potent B-cell depletion, suggesting its potential utility as a B-lymphocyte depletive therapy for malignancies and autoimmune indications.

In vitro and in vivo characterization of MDX-1401 for therapy of malignant lymphoma.

PURPOSE: This study was undertaken to evaluate the effects of MDX-1401, a nonfucosylated fully human monoclonal antibody that binds to human CD30, and to determine whether it exhibits greater in vitro and in vivo activity than its parental antibody. EXPERIMENTAL DESIGN: Assays measuring antibody binding to CD30-expressing cells and FcgammaRIIIa (CD16) transfectants as well as antibody-dependent cellular cytotoxicity (ADCC) were conducted. Antitumor activity was determined using a Karpas-299 systemic model. RESULTS: The binding of MDX-1401 to CD30 antigen was identical to fucose-containing parental anti-CD30 antibody (MDX-060). In contrast, MDX-1401 showed increased binding affinity to FcgammaRIIIa-transfected cells resulting in increased effector function. MDX-1401 greatly improved ADCC activity as evidenced by a decrease in half-maximal effective concentration (EC(50)) and an increase in maximum cell lysis when compared with MDX-060. Increased ADCC activity was observed among a panel of cell lines, including one with very low CD30 antigen expression in which parental antibody failed to induce any detectable ADCC. MDX-1401 activity with all FcgammaRIIIa polymorphic variants, including less active Phe/Phe158 and Phe/Val158 effector cells, was shown. Furthermore, MDX-1401 was efficacious in inhibiting tumor growth in CD30(+) lymphoma xenografts. CONCLUSIONS: The low doses of antibody required for ADCC activity irrespective of donor genotype, the ability to mediate ADCC in target cells expressing low levels of CD30, and increased in vivo efficacy support the development of MDX-1401 for treatment of malignant lymphoma.

Linker for activation of T cells, zeta-associated protein-70, and Src homology 2 domain-containing leukocyte protein-76 are required for TCR-induced microtubule-organizing center polarization.

Engagement of the T cell with Ag on an APC results in a series of immediate signaling events emanating from the stimulation of the TCR. These events include the induced phosphorylation of a number of cellular proteins with a subsequent increase in intracellular calcium and the restructuring of the microtubule and actin cytoskeleton within the T cell. This restructuring of the cytoskeleton culminates in the polarization of the T cell's secretory apparatus toward the engaging APC, allowing the T cell to direct secretion of cytokines toward the appropriate APC. This polarization can be monitored by analyzing the position of the microtubule-organizing center (MTOC), as it moves toward the interface of the T cell and APC. The requirements for MTOC polarization were examined at a single-cell level by studying the interaction of a Jurkat cell line expressing a fluorescently labeled MTOC with Staphylococcal enterotoxin superantigen-bound Raji B cell line, which served as the APC. We found that repolarization of the MTOC substantially followed fluxes in calcium. We also used immobilized anti-TCR mAb and Jurkat signaling mutants, defective in TCR-induced calcium increases, to determine whether signaling components that are necessary for a calcium response also play a role in MTOC polarization. We found that zeta-associated protein-70 as well as its substrate adaptor proteins linker for activation of T cells and Src homology 2 domain-containing leukocyte protein-76 are required for MTOC polarization. Moreover, our studies revealed that a calcium-dependent event not requiring calcineurin or calcium/calmodulin-dependent kinase is required for TCR-induced polarization of the MTOC.