Moutih Rafei

A Fusion of GMCSF and IL-21 Initiates Hypersignaling Through the IL-21Rα Chain With Immune Activating and Tumoricidal Effects In Vivo

We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Rα-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21s aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-α, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Rα+ lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Rα+ lymphocytes, we tested its cytolytic effect on IL-21Rα+ EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Rα agonist that co-opts IL-21Rα-dependent signaling in a manner permissive for targeted cancer immunotherapy.We hypothesized that fusing granulocyte-macrophage colony-stimulation factor (GMCSF) and interleukin (IL)-21 as a single bifunctional cytokine (hereafter GIFT-21) would lead to synergistic anticancer immune effects because of their respective roles in mediating inflammation. Mechanistic analysis of GIFT-21 found that it leads to IL-21Ralpha-dependent STAT3 hyperactivation while also contemporaneously behaving as a dominant-negative inhibitor of GMCSF-driven STAT5 activation. GIFT-21s aberrant interactions with its cognate receptors on macrophages resulted in production of 30-fold greater amounts of IL-6, TNF-alpha, and MCP-1 when compared to controls. Furthermore, GIFT-21 treatment of primary B and T lymphocytes leads to STAT1-dependent apoptosis of IL-21Ralpha(+) lymphocytes. B16 melanoma cells gene-enhanced to produce GIFT-21 were immune rejected by syngeneic C57Bl/6 mice comparable to the effect of IL-21 alone. However, a significant GIFT-21-driven survival advantage was seen when NOD-SCID mice were implanted with GIFT-21-secreting B16 cells, consistent with a meaningful role of macrophages in tumor rejection. Because GIFT-21 leads to apoptosis of IL-21Ralpha(+) lymphocytes, we tested its cytolytic effect on IL-21Ralpha(+) EL-4 lymphoma tumors implanted in C57Bl/6 mice and could demonstrate a significant increase in survival. These data indicate that GIFT-21 is a novel IL-21Ralpha agonist that co-opts IL-21Ralpha-dependent signaling in a manner permissive for targeted cancer immunotherapy.

Autologous Bone Marrow Stromal Cells Genetically Engineered to Secrete an IGF-I Receptor Decoy Prevent the Growth of Liver Metastases

Liver metastases respond poorly to current therapy and remain a frequent cause of cancer-related mortality. We reported previously that tumor cells expressing a soluble form of the insulin-like growth factor-I receptor (sIGFIR) lost the ability to metastasize to the liver. Here, we sought to develop a novel therapeutic approach for prevention of hepatic metastasis based on sustained in vivo delivery of the soluble receptor by genetically engineered autologous bone marrow stromal cells. We found that when implanted into mice, these cells secreted high plasma levels of sIGFIR and inhibited experimental hepatic metastases of colon and lung carcinoma cells. In hepatic micrometastases, a reduction in intralesional angiogenesis and increased tumor cell apoptosis were observed. The results show that the soluble receptor acted as a decoy to abort insulin-like growth factor-I receptor (IGF-IR) functions during the early stages of metastasis and identify sustained sIGFIR delivery by cell-based vehicles as a potential approach for prevention of hepatic metastasis.

A MCP1 fusokine with CCR2-specific tumoricidal activity.

BackgroundThe CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development.MethodsWe have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent.ResultsWe found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients.ConclusionOur data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2+ malignancies including lymphoid and central nervous system malignancies.

Interleukin-21 promotes thymopoiesis recovery following hematopoietic stem cell transplantation

BackgroundImpaired T cell reconstitution remains a major deterrent in the field of bone marrow (BM) transplantation (BMT) due to pre-conditioning-induced damages inflicted to the thymi of recipient hosts. Given the previously reported thymo-stimulatory property of interleukin (IL)-21, we reasoned that its use post-BMT could have a profound effect on de novo T cell development.MethodsTo evaluate the effect of IL-21 on de novo T cell development in vivo, BM derived from RAG2p-GFP mice was transplanted into LP/J mice. Lymphocyte reconstitution was first assessed using a hematological analyzer and a flow cytometer on collected blood samples. Detailed flow cytometry analysis was then performed on the BM, thymus, and spleen of transplanted animals. Finally, the effect of human IL-21 on thymopoiesis was validated in humanized mice.ResultsUsing a major histocompatibility complex (MHC)-matched allogeneic BMT model, we found that IL-21 administration improves immune reconstitution by triggering the proliferation of BM Lin−Sca1+c-kit+ (LSK) subsets. The pharmacological effect of IL-21 also culminates in the recovery of both hematopoietic (thymocytes) and non-hematopoietic (stromal) cells within the thymi of IL-21-treated recipient animals. Although T cells derived from all transplanted groups proliferate, secrete various cytokines, and express granzyme B similarly in response to T cell receptor (TCR) stimulation, full regeneration of peripheral naïve CD4+ and CD8+ T cells and normal TCRvβ distribution could only be detected in IL-21-treated recipient mice. Astonishingly, none of the recipient mice who underwent IL-21 treatment developed graft-versus-host disease (GVHD) in the MHC-matched allogeneic setting while the graft-versus-tumor (GVT) effect was strongly retained. Inhibition of GVHD onset could also be attributed to the enhanced generation of regulatory B cells (B10) observed in the IL-21, but not PBS, recipient mice. We also tested the thymopoiesis-stimulating property of human IL-21 in NSG mice transplanted with cord blood (CB) and found significant improvement in de novo human CD3+ T cell development.ConclusionsIn sum, our study indicates that IL-21 represents a new class of unforeseen thymopoietin capable of restoring thymic function following BMT.

GIFT15 fusokine to foil immunity’s follies

prosurvival properties [5]. Under these condi‐ tions, it is fair to conceive that all activated lymphomyeloid cells expressing the IL‐15R and exposed to GIFT15 would lose their capacity to trigger immune responses. In light of these results, we tested whether the expression of the fusokine could protect xenografted cells from rejection in immune competent animals. While mice lacking GIFT15 rejected all grafted cells, animals treated with the fusokine have toler‐ ated the xenograft [4]. These results suggest that GIFT15 can act as a suppressor agent of allograft rejection and allows for xenotransplant as well and may present an interesting tool for the treat‐ ment of autoimmune diseases. While studying the pharmacological properties of GIFT15 on unfractionated splenocytes in vitro, we observed that the fusokine had a remarkable and unprece‐ dented effect on naive B cells, converting them into suppressor cells of B‐cell ontogeny (h ereafter GIFT15 Bregs) [6].

Interleukin-21: A New Class of Thymopoietin for Immune Rejuvenation

More than two billion people will reach the age of 65 by 2050. Therefore, infectious disease/cancer-related morbidity and mortality of aged subjects is expected to rise. Several factors including impaired functions of body barriers and changes in microbial colonization contribute in increasing elderly susceptibility to infections and cancer. However, thymic involution, a hallmark of immunosenescence, is undoubtedly the principal component of this prominent problem. Although the thymus remains functional at older age, its pronounced diminished T-cell export rate is insufficient to sustain a competent naive peripheral T-cell pool. Consequently, gradual dwindling in T-cell receptor (TCR) repertoire diversity takes place. As a result, the capacity of the elderly immune system to confer protection against cancer, acute/chronic infections or to respond to vaccination erodes. Therefore, there is an urgent need for the development of novel strategies aimed at: i) providing superior control of infectious diseases and cancer, and ii) improving responsiveness to all forms of immunotherapies.