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Purpose: Dual-targeted therapy has been shown to be a promising treatment option in recurrent and/or refractory B-cell non-Hodgkin's lymphoma (B-NHL). We generated radioimmunoconjugates (RICs) comprising either a novel humanized anti-CD22 monoclonal antibody, huRFB4, or rituximab, and the low-energy β-emitter Lu. Both RICs were evaluated as single agents in a human Burkitt's lymphoma xenograft mouse model. To increase the therapeutic efficacy of the anti-CD22 RIC, combination therapy with unlabelled anti-CD20 rituximab was explored. Methods: The binding activity of CHX-A″-DTPA-conjugated antibodies to target cells was analysed by flow cytometry. To assess tumour targeting of Lu-labelled antibodies, in vivo biodistribution experiments were performed. For radioimmunotherapy (RIT) studies, non-obese diabetic recombination activating gene-1 (NOD-Rag1) interleukin-2 receptor common gamma chain (IL2rγ ) null mice (NRG mice) were xenografted subcutaneously with Raji Burkitt's lymphoma cells. Lu-conjugated antibodies were administered at a single dose of 9.5 MBq per mouse. For dual-targeted therapy, rituximab was injected at weekly intervals (0.5 - 1.0 mg). Tumour accumulation of RICs was monitored by planar scintigraphy. Results: Conjugation of CHX-A'-DTPA resulted in highly stable RICs with excellent antigen-binding properties. Biodistribution experiments revealed higher tumour uptake of the Lu-labelled anti-CD22 IgG than of Lu-labelled rituximab. Treatment with Lu-conjugated huRFB4 resulted in increased tumour growth inhibition and significantly longer survival than treatment with Lu-conjugated rituximab. The therapeutic efficacy of the anti-CD22 RIC could be markedly enhanced by combination with unlabelled rituximab. Conclusion: These findings suggest that dual targeting with Lu-based CD22-specific RIT in combination with rituximab is a promising new treatment option for refractory B-NHL. [ABSTRACT FROM AUTHOR]

γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNÎ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical evaluation. [ABSTRACT FROM AUTHOR]

NK cells play an important role in tumor immunosurveillance and largely contribute to the therapeutic success of anti-tumor antibodies like Rituximab. Here, we studied the role of the TNF family member 4-1BB ligand (4-1BBL) during the interaction of NK cells with chronic lymphocytic leukemia (CLL) cells. 4-1BBL was highly expressed on patient B-CLL cells in all 56 investigated cases. Signaling via 4-1BBL following interaction with 4-1BB, which was detected on NK cells of CLL patients but not healthy individuals, led to the release of immunoregulatory cytokines including TNF by CLL cells. CLL patient sera contained elevated levels of TNF and induced 4-1BB upregulation on NK cells, which in turn impaired direct and Rituximab-induced NK-cell reactivity against 4-1BBL-expressing targets. NK-cell reactivity was not only enhanced by blocking the interaction of NK cell-expressed 4-1BB with 4-1BBL expressed by CLL cells, but also by preventing 4-1BB upregulation on NK cells via neutralization of TNF in patient serum with Infliximab. Our data indicate that 4-1BBL mediates NK-cell immunosubversion in CLL, and thus might contribute to the reportedly compromised efficacy of Rituximab to induce NK-cell reactivity in the disease, and that TNF neutralization may serve to enhance the efficacy of Rituximab treatment in CLL. [ABSTRACT FROM AUTHOR]

The TNF family member receptor activator for NF-KB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibltory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-y production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutral-ization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients. [ABSTRACT FROM AUTHOR]

γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical evaluation.