Safety levels of systemic IL-12 induced by cDNA expression as a cancer therapeutic

Immunotherapy ◽  
2021 ◽  
Author(s):  
Constanza Savid-Frontera ◽  
Maria E Viano ◽  
Natalia S Baez ◽  
Della Reynolds ◽  
Mariana Matellon ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Systemic Effects ◽  
Mouse Tumor ◽  
Protein Levels ◽  
Improve Treatment ◽  
Tumor Bearing ◽  
Ifn Γ ◽  
Secondary Lymphoid Organs ◽  
Mouse Tumor Models

Aim: The aim of this work is to utilize a gene expression procedure to safely express systemic IL-12 and evaluate its effects in mouse tumor models. Materials & methods: Secondary lymphoid organs and tumors from EL4 and B16 tumor-bearing mice were analyzed by supervised and unsupervised methods. Results: IL-12 cDNA induced systemic IL-12 protein levels lower than the tolerated dose in patients. Control of tumor growth was observed in subcutaneous B16 and EL4 tumors. Systemic IL-12 expression induced a higher frequency of both total tumor-infiltrated CD45+ cells and proliferative IFN-γ+CD8+ T cells along with a lower frequency of CD4+FOXP3+ and CD11b+Gr-1+ cells. Conclusion: This approach characterizes the systemic effects of IL-12, helping to improve treatment of metastases or solid tumors.

scholarly journals 843 Development and engineering of human sialidase for degradation of immunosuppressive sialoglycans to treat cancer

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A884-A884
Author(s):  
Li Peng ◽  
Lizhi Cao ◽  
Sujata Nerle ◽  
Robert LeBlanc ◽  
Abhishek Das ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Cells ◽  
First Generation ◽  
Single Agent ◽  
Mouse Tumor ◽  
Cytokine Release ◽  
Tumor Models ◽  
Cell Immunity ◽  
Mouse Tumor Models

BackgroundSialoglycans, a type of glycans with a terminal sialic acid, have emerged as a critical glyco-immune checkpoint that impairs antitumor response by inhibiting innate and adaptive immunity. Upregulation of sialoglycans on tumors has been observed for decades and correlates with poor clinical outcomes across many tumor types. We previously showed that targeted desialylation of tumors using a bifunctional sialidase x antibody molecule, consisting of sialidase and a tumor-associated antigen (TAA)-targeting antibody, has led to robust single-agent efficacy in mouse tumor models. In addition to tumor cells, most immune cells present substantially more abundant sialoglycans than non-hematological healthy cells, which may also contribute to immunosuppression. Therefore, we studied the impact of immune cell desialylation and evaluated the therapeutic potential of a newly developed sialidase-Fc fusion (Bi-Sialidase), which lacks a TAA-targeting moiety and consists of engineered human neuraminidase 2 (Neu2) and human IgG1 Fc region, in preclinical mouse tumor models.MethodsThe first generation Neu2 variant was further optimized to improve titers and stability to constructed Bi-Sialidase. Bi-Sialidase’s desialylation potency and impact on immune responses were studied in vitro using various human immune functional assays, including T-cell activation, allogeneic mixed lymphocyte reaction, antibody-dependent cellular cytotoxicity, macrophages polarization/activation, neutrophil activation, and peripheral blood mononuclear cell (PBMC) cytokine release assays. We evaluated its antitumor efficacy in mouse tumor models. Bi-Sialidase’s safety profile was characterized by conducting rat and non-human primate (NHP) toxicology studies.ResultsThe optimized Bi-Sialidase achieved a titer of 2.5 g/L from a 15-day fed-batch Chinese hamster ovary cell culture; in contrast, the wild-type and first-generation Neu2 had no production or a low titer (<0.1 g/L) under similar conditions, respectively. We demonstrated that Bi-Sialidase led to dose-dependent desialylation of immune cells and potentiated T-cell immunity, without impacting NK, macrophage, or neutrophil activation by desialylating immune cells. Activated and exhausted T cells upregulated surface sialoglycans and Bi-Sialidase-mediated desialylation reinvigorated exhausted-like T cells as measured by IFNg production. Bi-Sialidase treatment also enhanced DC priming and activation of naïve T cells by desialylating both T cells and DCs. Furthermore, Bi-Sialidase showed single-agent antitumor activity in multiple mouse tumor models, including MC38, CT26, A20, and B16F10. Importantly, Bi-Sialidase did not cause cytokine release in human PBMC assays and was tolerated to up to 100 mg/kg in rats and NHPs, demonstrating a wide safety margin.ConclusionsBi-Sialidase with an optimized Neu2 offers a novel immunomodulatory approach to enhancing T-cell immunity by desialylating immunosuppressive sialoglycans for cancer treatment.

scholarly journals Early Response of CD8+ T Cells in COVID-19 Patients

2021 ◽  
Vol 11 (12) ◽  
pp. 1291
Author(s):  
Deni Ramljak ◽  
Martina Vukoja ◽  
Marina Curlin ◽  
Katarina Vukojevic ◽  
Maja Barbaric ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
T Cells ◽  
Mrna Expression ◽  
Cd8 T Cells ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Effector Memory ◽  
Healthy Controls ◽  
Ifn Γ

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

Interleukin-12 Gene Expression into Acute Myeloid Leukemia-Derived Dendritic Cells Overcomes T-Cell Functional Impairment Induced by Leukemic Microenvironment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1816-1816
Author(s):  
Antonio Curti ◽  
Simona Pandolfi ◽  
Michela Aluigi ◽  
Alessandro Isidori ◽  
Isabella Alessandrini ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Dendritic Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Interleukin 12 ◽  
Functional Features ◽  
Ifn Γ ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) cells are poorly immunogenic and release soluble factors inhibiting T-cell function. AML-derived dendritic cells (AML-DCs) have better antigen presentation capacity than leukemic blasts but share with AML cells some immunosuppressive features. In this study, we show that AML-DCs generated from CD14− AML samples (which represent 80% of total AML patients) are defective in IL-12 production. We, then, transfected CD14−-derived AML-DCs with IL-12 gene through the novel non-viral method nucleofection. IL-12 gene-nucleofected AML-DCs produce significant amount of IL-12 while maintain leukemia-specific karyotype, DC-like phenotype and function. In presence of the supernatant from the human leukemic cell line K562, allogeneic T-cell proliferation and interferon (IFN)-γ production induced by mock-transduced AML-DCs are significantly reduced. This effect is mainly directed on T cells, since AML-DC phenotype and cytokine production are not affected by leukemic supernatant. However, when stimulated by IL-12-producing AML-DCs, T cells produce higher concentrations of IFN-γ, thus maintaining a Th1 cytokine profile. In conclusion, IL-12 gene can be expressed into AML-DCs defective in endogenous IL-12 production by using a novel non-viral method which does not modify their phenotypical, cytogenetic and functional features. IL-12 gene expression into AML-DC counteracts the inhibitory effect of leukemic microenvironment on T lymphocytes

T-bet Over-Expression in T Cells from Patients with Aplastic Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 832-832
Author(s):  
Elena E. Solomou ◽  
Keyvan Keyvanfar ◽  
Elaine M. Sloand ◽  
Barbara Weinstein ◽  
Olga Nunez ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Hematopoietic Stem ◽  
T Box ◽  
Protein Levels ◽  
Proximal Site ◽  
Ifn Γ ◽  
Bet Protein ◽  
Normal Controls

Abstract Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by immune-mediated destruction of hematopoietic stem cells. T cells from patients with AA overproduce IFN-γ, a cytokine that inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. In T cells, regulation of IFN-γ production occurs primarily at the level of transcription. A proximal site of the IFN-γ gene (−75 to −45bp of the IFN-γ promoter) is a binding site for different transcription factors including NFAT, AP-1, ATF, CREB, and T-bet. T-bet is a member of the T-box family of transcription factors, this family contains a highly conserved DNA binding domain, the T-box, that binds to a specific sequence in the promoter of different genes, including the IFN-γ promoter. T-bet is found in Th1 but not in Th2 cells and is the key regulator of Th1 development and function (Rengarajan et al., Immunol Today2000; 21: 479). The inducible expression of T-bet is mediated in part by Itk kinase (Sjabo et al., Science2005; 307: 430). In the present study, we examined T-bet protein levels in T cells from patients with AA. Samples from 17 of 20 patients examined (85%) by immunoblot showed increased T-bet protein levels in unstimulated T cells compared to normal controls (p = 0.0001). Normal controls showed undetectable T-bet protein levels in unstimulated T cells but T-bet expression was induced after 24 hrs of stimulation with PMA and ionomycin. In electrophoretical mobility shift assays, we observed increased T-bet binding to the proximal site of the IFN-γ promoter in T cells from patients with AA; no binding was detected in unstimulated T cells from healthy controls, but binding was present after stimulation for at least 24 hrs. T-bet protein levels correlated with disease activity. Patients with increased T-bet protein levels showed increased intracellular IFN-g levels compared to controls, as detected by flow cytometry (p&lt;0.05). Patients that expressed increased T-bet protein levels also showed increased levels of the Itk kinase (p=0.02). We examined if other kinases that lie downstream of Itk in the signal transduction activation cascade in T cells affected the inducible expression of T-bet. In normal T cells, rottlerin, a PKC-theta (PKC-𝛉) inhibitor, decreased T-bet protein levels by 50%; in AA T cells, rottlerin also decreased T-bet protein levels and IFN-γ intracellular levels by 50%. Our results suggest that the increased IFN-γ levels observed in AA are the result of activation of transcription of the IFN-γ gene by the regulator T-bet. Blocking of transcription of the IFN-γ gene by kinase inhibitors might represent a therapuetic strategy for AA and other human T cell-mediated autoimmune diseases.

SAP (SH2D1A), the Immunomodulator Deficient in X-Linked Lymphoproliferative Syndrome (XLP), Is Profoundly Decreased in Aplastic Anemia: An Immunologic Link between Constitutional and Acquired Bone Marrow Failure.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1141-1141
Author(s):  
Elena E. Solomou ◽  
Valeria Visconte ◽  
Federica Gibellini ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bone Marrow Failure ◽  
Protein Levels ◽  
Ifn Γ ◽  
Th1 Polarization

Abstract Ligation of the signaling lymphocyte activation molecule (SLAM), a member of the immunoglobulin superfamily expressed in T and B cells, results in T cell activation and Th1 cytokine production. SAP is a small cytoplasmic protein expressed in T- and NK cells that controls the activation signals mediated by SLAM. On T cell activation, SAP binds to Fyn kinase; Fyn is activated and phosphorylates tyrosine residues on SLAM; phosphorylation results in the formation of a complex that selectively down-regulates co-stimulatory signals in activated T cells, resulting in inhibition of IFN-γ production. Thus SAP acts as a natural suppressor of SLAM-mediated T cell activation, and, in the absence of SAP, T cells are constitutively activated and overproduce IFN-γ. Mutations in the SAP gene lead to abnormal T cell activation and enhanced Th1 cytokine production in mouse models and in humans: about half of patients with X-linked lympoproliferative disease (XLP) have functionally disabling SAP mutations. Acquired aplastic anemia (AA) is a bone marrow failure syndrome in which hematopoietic cell destruction is effected by cytotoxic T cells and type 1 cytokines. We have recently shown that T cells from patients with AA have increased protein levels of T-bet, resulting in IFN-γ overproduction (Solomou EE et al, Blood2006; 107:3983). IFN-γ inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. We examined SAP expression as an explanation for aberrant T cell activation and extreme Th1 polarization. SAP protein expression on immunoblot was very low to absent in unstimulated T cells from 16 of 20 AA patients examined, as compared to normal levels of expression in equivalent numbers of healthy donors (p&lt;0.001). No significant differences were detected in Fyn and SLAM protein levels between AA and controls. SAP mRNA levels were also significantly decreased in T cells from those AA patients with low SAP protein levels, as determined by RT-PCR. Peripheral blood DNA samples from 18 patients with AA were analyzed for SAP mutations: three novel intronic mutations, not present in controls, were identified among 7 unrelated patients: one mutation was in the promoter region of SAP (position 106, C to T; 3 patients), and two mutations in the intron-exon junction between exons 1 and 2 (position 38975, C toT; 3 patients) and 3 and 4 (position 62771, C to A; 1 patient). IFN-γ, as measured by ELISA, in three patients with undetectable SAP protein levels was significantly increased compared to healthy controls (n=5, p&lt;0.001). Increased IFN-γ levels and Th1 polarization in AA can in part be explained by functional SAP deficiency. SAP-deficient T cells in AA would be unable to block co-stimulatory signals, leading to an activated T cell phenotype and ultimately hematopoietic cell destruction and bone marrow failure. The SAP-deficient phenotype in T cells from patients with aplastic anemia may be secondary to subtle genetic alteration in the gene’s regulation (abnormal promoter binding sites or epigenetic modulation due to mutations in introns) or as yet unidentified aberrant upstream pathways (Ets-1 and Ets-2, the transcription factors that regulate SAP expression).

CD11b− Donor Dendritic Cells Enhance Donor T-Cells’ Th1 Polarization and Graft Versus Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1252-1252 ◽  
Author(s):  
Jian-Ming Li ◽  
Kataryna A. Darlak ◽  
Ying Lu ◽  
Cynthia Giver ◽  
Wayne Harris ◽  
...  
Keyword(s):  
T Cells ◽  
Serum Levels ◽  
Hematopoietic Stem ◽  
Treatment Groups ◽  
Post Transplant ◽  
Tumor Bearing ◽  
Donor T Cells ◽  
Ifn Γ ◽  
Th1 Cytokines

Abstract Background: Based on a clinical association of donor plasmacytoid dendritic cell (DC) content with leukemia relapses after allogeneic BMT (Waller, Blood 2001), we have previously reported that CD11b− donor DC added to a graft containing FACS-purified hematopoietic stem cells (HSC) and T-cells enhanced interferon-γ (IFN-γ) production and GvL activity in MHC-mismatched allogeneic transplant mouse models (Li, Blood 2007). Objective: In this study, we studied the mechanisms whereby donor DC in the graft modulate donor T-cell activity and the graft-versus-leukemia (GvL) effect in MiHA (C3H.SW → C57BL/6J)- and MHC (C57BL/6J → B10.BR)- mismatched models of allogeneic hematopoietic stem cell transplantation (HSCT). Methods: Mice irradiated to 11 Gy received 5 × 104 log-phase viable MMB3.19 myeloid lymphoma cells via intraperitoneal injection or intravenous injection of 1 x 105 LBRM T-cell lymphoma cells one day before transplant. Allografts consisted of 5 × 104 FACS-purified donor BM CD11b− DC or CD11b+ DC plus 3 × 103 FACS-purified c-kit+ sca-1+ lineage− hematopoietic stem cells (HSC) in combination with either 3 × 105 T-cells, 3 × 105 CD8+ T-cells or no additional T-cells transplanted via tail vein. Graft-versus-host disease (GvHD) clinical scores (based on body weight loss, posture, skin, fur texture, activity) were recorded twice weekly in non-tumor bearing recipients. In vitro proliferation and cytotoxic activity of donor-derived T-cells against tumor targets was assessed by CFSE staining and a caspase flow cytometry assay (CyToxiLux PLUS) using donor T-cells harvested from recipients on day 34 and day 82 post transplant. Serum and intracellular Th1 cytokines (IFN-γ, IL-2, and TNF-α) and Th2 cytokines (IL-4, IL-5, and IL-10) from recipients’ peripheral blood and spleens day 3 and day 10 post-transplant was measured by ELISA and flow cytometry. IFN-γ direct killing of leukemia cells was tested by in vitro IFN-γ exposure. Results: In non-tumor bearing mice, recipients of all combinations of donor DC subsets, with and without donor T-cells had equivalent survival (75% – 85%) at 3 months post transplant without significant clinical signs of GvHD. Transplantation of tumor cells to recipients of HSC alone, HSC plus donor T-cells, or HSC plus T-cells and CD11b+ DC in the MiHA- and the MHC-mismatched transplant models led to 0% or 5% 3 month survival, respectively. Strikingly, tumor-bearing mice transplanted with CD11b− DC had significantly enhanced 3 month survival (35% in the MiHA-mismatched model and 45% in the MHCmismatched model) without increased GvHD (p&lt;0.001). There was no significant difference in survival between mice that received HSC plus CD11b− DC and a mixture of CD4+ and CD8+ donor T-cells versus mice that received HSC plus CD11b− DC and only CD8+ donor T-cells. Donor T-cells harvested from recipients of CD11b− DC 34 days after transplant in the MiHA-mismatched model as well as 82 days after transplant in the MHC-mismatched model displayed increased cell proliferation following co-culture with irradiated hosttype splenocytes as a source of alloantigen compared with donor T-cells harvested from recipients of CD11b+ DC or recipients of HSC plus T-cells without donor DC. Leukemia cell killing was greater following incubation of purified donor T-cells recovered from recipients of CD11b− DC with tumor targets compared to T-cells recovered from other treatment groups. Recipients of CD11b− DC had higher serum levels of Th1 cytokines IFN-γ and IL-2 and higher number of Th1 positive donor T-cells compared with recipients of other treatment groups. In contrast, recipients of CD11b+ DC had higher serum levels of Th2 cytokines IL-4, IL-5, and IL-10 and higher number of Th2 positive donor T-cells. IFN-γ added to in vitro cultures with MMB3.19, and LBRM, had no direct cell killing effect. Conclusion: CD11b− donor DC enhanced Th1 polarization of donor T-cells and GvL without increasing GvHD. Donor CD8+ T-cells mediated tumor killing effect. CD11b+ donor DC enhanced Th2 polarization of donor CD4+ T-cells and led to limited GvHD and GvL.

PD-1/PD-1-Ligand Interaction Contributes to Immunosuppressive Microenvironment of Hodgkin Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 379-379
Author(s):  
Ryo Yamamoto ◽  
Momoko Nishikori ◽  
Toshio Kitawaki ◽  
Tomomi Sakai ◽  
Masakatsu Hishizawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Signaling System ◽  
Peripheral T Cells ◽  
Ifn Γ ◽  
Immunosuppressive Microenvironment

Abstract Programmed death-1 (PD-1), a member of the CD28 costimulatory receptor superfamily, inhibits T cell activity by providing a second signal to T cells in conjunction with signaling through the T-cell receptor. PD-1/PD-1 ligand (PD-L) signaling system is indicated to be involved in the functional impairment of T cells such as in chronic viral infection or tumor immune evasion. We hypothesized that this signaling system is also involved in the pathogenesis of Hodgkin lymphoma (HL). We examined expression of B7-H1 and B7-DC, two known PD-Ls, in lymphoid cell lines using RT-PCR and flow cytometry. They were expressed in HL and several T-cell lines, whereas most B-NHL lines lacked their expression. Immunohistochemical staining of HL tissues demonstrated that PD-Ls were also expressed in primary H/RS cells. As gene expression of B7-H1 and B7-DC was increased in Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell lines, we examined the effect of EBV latent membrane proteins on their gene regulation. By luciferase reporter assay, both LMP1 and LMP2A were shown to enhance promoter activity of B7-H1 and B7-DC genes. This finding implies that in cases of EBV-positive HL, latent membrane proteins may help H/RS cells escape from host immune surveillance by upregulating PD-L gene expression. We next analyzed PD-1 expression of tumor-infiltrating T cells of HL tissue samples by flow cytometry, and found that PD-1+ cells were elevated markedly in these cells. As HL patients are well recognized as having defective cellular immunity, we compared PD-1 expression level in peripheral blood T cells of HL patients with those of healthy volunteers and B-NHL patients. PD-1 was significantly elevated in peripheral T cells of HL patients compared to the other two groups. PD-1+ T cells were highest in patients with active disease, and tended to decline along with treatment. Although regulatory T cells are reported to play a part in the pathogenesis of HL, FOXP3+ T cells were not significantly elevated in peripheral T cells of HL patients, and PD-1+ T cells did not overlap with these regulatory population. To elucidate whether the PD-1/PD-L signaling pathway is functional in the immunosuppressive microenvironment of HL, we finally examined the effect of blockade of this pathway. After culturing bulk HL tumor cells with anti-PD-L blocking antibodies, IFN-γ production was measured by ELISA. Blockade of PD-Ls augmented IFN-γ production of HL-infiltrating T cells. We concluded that anti-tumor activity of HL-infiltrating T cells was inhibited via the PD-1/PD-L pathway, and this inhibition could be successfully relieved by PD-L blockade. Taken together, our observations indicate that “T-cell exhaustion” is essential to the pathogenesis of HL, and tumor-infiltrating T cells around H/RS cells seem to be kept in balance by this inhibitory signaling. Our findings provide a potentially effective and clinically applicable strategy for the immunotherapy of HL.

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