CC chemokine receptor 4 (CCR4) has attracted much attention as a promising therapeutic drug target for CCR4(+) tumor cells and Tregs. CCR4 is expressed on some tumor cells such as T-cell acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), adult peripheral T cell lymphoma (PTCL) and cutaneous T cell lymphoma (CTCL). CCR4 is also expressed on majority of Tregs, mainly effector Tregs. In this study we have successfully developed three versions of diphtheria-toxin based anti-human CCR4 immunotoxins (monovalent, bivalent and single-chain fold-back diabody). Binding analysis by flow cytometry showed that all three versions of the anti-human CCR4 immunotoxins bound to the human CCR4(+) tumor cell line as well as CCR4(+) human PBMC. The bivalent isoform bound stronger than its monovalent counterpart and the single-chain foldback diabody isoform was the strongest among the three versions. In vitro efficacy analysis demonstrated that the bivalent isoform was 20 fold more potent in inhibiting cellular proliferation and protein synthesis in human CCR4(+) tumor cells compared to the monovalent anti-human CCR4 immunotoxin. The single-chain fold-back diabody isoform was 10 fold more potent than its bivalent counterpart and 200 fold more potent than its monovalent counterpart. The in vivo efficacy was assessed using a human CCR4(+) tumor-bearing mouse model. The immunotoxin significantly prolonged the survival of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with human CCR4(+) acute lymphoblastic leukemia cells compared with the control group. This novel anti-human CCR4 immunotoxin is a promising drug candidate for targeting human CCR4(+) tumor cells and Tregs in vivo.
Ontak® is a FDA-approved diphtheria toxin-based recombinant fusion toxin for treatment of human CD25+ cutaneous T cell lymphoma (CTCL). However, it has been discontinued clinically due to the production issue related to the bacterial expression system with difficult purification. Recently we have developed monovalent and bivalent human IL-2 fusion toxins targeting human CD25+ cells using advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system. In vitro efficacy characterization using human CD25+ HUT102/6TG cells demonstrated that both monovalent and bivalent isoforms are potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform. In this study, we further assessed the in vivo efficacy of the human IL-2 fusion toxins using human CD25+ HUT102/6TG tumor-bearing NSG mouse model. The data demonstrated that both monovalent and bivalent human IL-2 fusion toxins significantly prolonged the survival of the human CD25+ tumor-bearing NSG mice in a dose-dependent manner. Then we further assessed the residual tumor cells from the HUT102/6TG tumor-bearing NSG mice using the residual tumor cell bearing NSG mouse model. The results demonstrated that the residual tumor cells were still sensitive to the continual treatment with the human IL-2 fusion toxin. This yeast-expressed human IL-2 fusion toxin will be a promising candidate to replace the clinically discontinued Ontak®.
Regulatory T cells (Treg) play an important role in modulating the immune response and has attracted increasing attention in diverse fields such as cancer treatment, transplantation and autoimmune diseases. CC chemokine receptor 4 (CCR4) is expressed on the majority of Tregs, especially on effector Tregs. Recently we have developed a diphtheria-toxin based anti-human CCR4 immunotoxin for depleting CCR4+ cells in vivo. In this study, we demonstrated that the anti-human CCR4 immunotoxin bound and depleted monkey CCR4+ cells in vitro. We also demonstrated that the immunotoxin bound to the CCR4+Foxp3+ monkey Tregs in vitro. In vivo studies performed in two naive cynomolgus monkeys revealed 78–89% CCR4+Foxp3+ Treg depletion in peripheral blood lasting approximately 10 days. In lymph nodes, 89–96% CCR4+Foxp3+ Tregs were depleted. No effect was observed in other cell populations including CD8+ T cells, other CD4+ T cells, B cells and NK cells. To our knowledge, this is the first agent that effectively depleted non-human primate (NHP) Tregs. This immunotoxin has potential to deplete effector Tregs for combined cancer treatment.
CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19+ tumors. We have developed a diphtheria toxin‐based anti‐human CD19 immunotoxin for targeting human CD19+ tumors. We have constructed three isoforms of the CD19 immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 immunotoxins was assessed using human CD19+ JeKo‐1 tumor‐bearing NOD/SCID IL‐2 receptor γ−/− (NSG) mouse model. In these animals, CD19 immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19+ tumors.
Galectin-3 (Gal-3), a β-galactoside-binding lectin that is expressed in mammalian cells, is known to modulate several biological functions such as cell-cell adhesion, macrophage activation, angiogenesis, metastasis, and fibrosis. The goal of this study was to evaluate the ability of Gal-3 depletion apheresis using an adsorption column with immobilized anti-Gal-3-antibody to reduce inflammation induced by Complete Freund's Adjuvant injection in a skin inflammation porcine model. Here, we report that plasma perfusion by apheresis through a Gal-3 binding immuno-affinity column reduces plasma Gal-3 levels to below limits of quantitative detection, and results in significant decrease in skin inflammation, including degree and duration of inflammatory lesions. Human plasma was tested ex vivo and found to be efficiently depleted using the anti-Gal-3 affinity column. This study demonstrates the potential of Gal-3 depletion apheresis as a therapeutic method for inflammation-mediated disease, supporting continued research in this area for clinical application.
Regulatory T cells (Tregs) are known to play an important role in immunoregulation and have been shown to facilitate induction of transplantation tolerance. Chemokine (C-C motif) receptor 4 (CCR4) is expressed on the surface of effector Tregs involved in controlling alloimmune and autoimmune responses. Recently we have developed a novel diphtheria-toxin based anti-human CCR4 immunotoxin for depleting CCR4+ cells in vivo. In this study, we have demonstrated that the anti-human CCR4 immunotoxin bound to porcine lymphocytes including CD4+FoxP3+ Tregs. Anti-human CCR4 immunotoxin effectively depleted CCR4+ Foxp3+ porcine Tregs in vivo. We observed depletion of up to 70–85% of the CCR4+Foxp3+ porcine Tregs in the peripheral blood and 85–91% in the lymph nodes following the anti-human CCR4 immunotoxin treatment in Massachusetts General Hospital (MGH) miniature swine. The depletion lasted for about one week with no significant reduction observed within CCR4− cell populations including CD8α+ T cells, CCR4−CD4+ T cells and B cells. In summary, anti-human CCR4 immunotoxin effectively depleted CCR4+Foxp3+ porcine Tregs in both peripheral blood and lymph nodes.
Background Establishment of transplantable tumors in clinically relevant large animals allows translational studies of novel cancer therapeutics. Methods Here we describe the establishment, characterization, and serial transplantation of a naturally occurring B-cell lymphoma derived from a unique, highly inbred sub-line of Massachusetts General Hospital (MGH) major histocompatibility complex (MHC)-defined miniature swine. Results The lymphoblastic cell line (LCL) originated from peripheral blood of a 2.5 year old female swine leukocyte antigen (SLA)dd-inbred miniature swine breeder demonstrating clinical signs of malignancy. Flow cytometric phenotypic analysis of subclones derived from the original cell line revealed surface markers commonly expressed in a B-cell lineage neoplasm. A subclone of the original LCL was transplanted into mildly-conditioned histocompatible miniature swine and immunocompromised NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Tissue and blood samples harvested 2 weeks following subcutaneous and intravenous injection in a highly inbred SLAdd pig were cultured for tumor growth and phenotypic analysis before serial transfer into NSG mice. Evidence of tumor growth in vivo was found in all tumor cell recipients. In vitro growth characteristics and surface phenotype were comparable between the original and serially transplanted tumor cell lines. Conclusions These results indicate the feasibility of developing a large-animal transplantable tumor model using cells derived from spontaneously occurring hematologic malignancies within the highly inbred miniature swine herd.
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