The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4-and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.
Summary Adjuvants, including antibodies to tumour necrosis factor receptor superfamily members, augment immune responses. One member of this family, glucocorticoid‐induced tumour necrosis factor receptor (GITR), is expressed at low levels on naive/resting T cells, B cells and macrophages, but at higher levels on T regulatory cells. The aim of this study was to determine the ability of a rat anti‐mouse GITR monoclonal antibody, 2F8, to stimulate murine humoral and cellular immunity in a prime boost model with particular attention to posology and antigen‐specific effects. 2F8 enhanced the humoral immune response to ovalbumin and haemagglutinin (HA) compared with controls and this enhancement was equal to or greater than that obtained in mice dosed with standard adjuvants. 2F8 F(ab′)2 fragments were as effective as intact antibody in boosting humoral immunity, indicating that FcR‐mediated cross‐linking of 2F8 is not required for efficacy. Moreover, the enhanced response was durable and antigen specific. Administration of 2F8 shifted the immune response towards a T helper type 1 response with significant enhancement of immunoglobulin G2a‐ and G2b‐specific anti‐HA antibodies, as well as enhanced cellular immunity as measured by ELISPOT. 2F8‐treated mice also generated significantly more neutralizing antibodies to HA than control mice. Our findings show that anti‐GITR is a robust, versatile adjuvant that, unlike commonly used adjuvants that primarily enhance humoral immunity, enhances both humoral and cellular immunity. These results support the continued development of anti‐GITR for such indications as haematological and solid tumours, chronic viral infections, and as a vaccine adjuvant.
Purpose The aim of this study was to explore the β-emitting lutetium-177 labelled anti-CD37 antibody NNV003 ( 177 Lu-NNV003, Humalutin®) for the treatment of non-Hodgkin’s lymphoma in in vitro studies and in animal models. Methods Cytotoxicity of 177 Lu-NNV003 was measured in REC-1 (mantle cell lymphoma) and DOHH-2 (diffuse large B cell lymphoma) cell lines. Biodistribution was studied in mice bearing subcutaneous DOHH-2 or MEC-2 (chronic lymphocytic leukaemia) xenografts. The therapeutic effect of a single injection of 177 Lu-NNV003 was measured in mice intravenously or subcutaneously injected with REC-1 cells. Haematological and histopathological assessments were used to evaluate the toxic effect of 177 Lu-NNV003. The immunotherapeutic effect of NNV003 was assessed by measuring binding to Fcγ receptors, activation of ADCC and ADCP. NNV003’s immunogenicity potential was assessed using in silico immunogenicity prediction tools. Results 177 Lu-NNV003 showed an activity dependent antiproliferative effect in all cell lines. Maximum tumour uptake in vivo was 45% of injected activity/g in MEC-2 tumours and 15% injected activity/g in DOHH-2 tumours. In mice injected intravenously with REC-1 cells, 177 Lu-NNV003 (50–100 MBq/kg) improved survival compared to control groups ( p < 0.02). In mice with subcutaneous REC-1 xenografts, 500 MBq/kg 177 Lu-NNV003 extended survival compared to the control treatments ( p < 0.005). Transient haematological toxicity was observed in all mice treated with radioactivity. NNV003 induced ADCC and ADCP and was predicted to have a lower immunogenicity potential than its murine counterpart. Conclusion 177 Lu-NNV003 had a significant anti-tumour effect and a favourable toxicity profile. These results warrant further clinical testing in patients with CD37-expressing B cell malignancies. Electronic supplementary material The online version of this article (10.1007/s00259-019-04417-1) contains supplementary material, which is available to authorized users.
Treatment of mice with NHL xenografts with Lu-lilotomab synergistically increased tumour suppression of subsequent anti-CD20 immunotherapy and improved survival. If the same effect is confirmed in a recently started clinical study, it could change the way radioimmunotherapy and CD20 immunotherapy would be used in the future.
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