Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro

Background:Regenerating islet-derived gene family member 4 (Reg4), a well-investigated growth factor in the regenerative pancreas, has recently been reported to be highly associated with a majority of gastrointestinal cancers. Pathological hyper-expression or artificial over-expression of Reg4 causes acceleration of tumor growth, migration, and resistance to chemotherapeutic 5-Fluorouracil (5-FU). Until now, no method has been successfully established for eliminating the effects of Reg4 protein.Methods:This study reports the production of an engineered immunoglobin, a single-chain variable fragment (scFv-Reg4), to specifically bind Reg4 and block the bioactivity. The complementary-determining regions (CDRs) against Reg4 were assigned using MOE and ZDOCK servers. The binding affinity (KD) was determined by bio-layer interferometry (BLI). MKN45 and AGS cell proliferation was determined by Thiazolyl blue tetrazolium bromide (MTT) method and the cell apoptosis was detected by flow cytometry assay.Results:The KD of scFv-Reg4 to Reg4 was determined to be 1.91×10-8. In MKN45 and AGS cell lines, scFv- Reg4 depressed Reg4-stimulated cell proliferation and the inhibitory rates were 27.7±1.5% and 17.3±2.6%, respectively. Furthermore, scFv significantly enhanced 5-FU-induced cell death, from 23.0±1.0% to 28.4±1.2% in MKN45 and 28.2±0.7% to 36.6±0.6% in AGS cells. Treatment with scFv alone could lyse cancer cells to a certain extent, but no significance has been observed.Conclusion:The single-chain antibody (scFv-Reg4) significantly inhibited gastric cancer cell proliferation and synergistically enhanced the lethal effect of 5-FU. Thus, traditional chemo-/radio- therapeutics supplemented with scFv-Reg4 may provide advances in the strategy for gastrointestinal cancer treatment.

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Development of a bispecific immune engager using a recombinant malaria protein

Cell Death and Disease ◽

10.1038/s41419-021-03611-0 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Mie A. Nordmaj ◽

Morgan E. Roberts ◽

Emilie S. Sachse ◽

Robert Dagil ◽

Anne Poder Andersen ◽

...

Keyword(s):

Cancer Cells ◽

Immune Evasion ◽

Blood Cells ◽

Xenograft Model ◽

Cancer Targeting ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Conjugation System ◽

Immune Mediated

AbstractAs an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

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In vitro antioxidant treatment recovers proliferative responses of anergic CD4+ lymphocytes from human immunodeficiency virus-infected individuals

Blood ◽

10.1182/blood.v87.11.4746.bloodjournal87114746 ◽

1996 ◽

Vol 87 (11) ◽

pp. 4746-4753 ◽

Cited By ~ 30

Author(s):

A Cayota ◽

F Vuillier ◽

G Gonzalez ◽

G Dighiero

Keyword(s):

Human Immunodeficiency Virus ◽

Cell Death ◽

Programmed Cell Death ◽

Redox Status ◽

Therapeutic Strategies ◽

Antioxidant Treatment ◽

Immunodeficiency Virus ◽

Cd4 Lymphocytes ◽

Cellular Thiol

Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3- mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N- acetyl-L-cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death.

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A novel mechanism for imatinib mesylate–induced cell death of BCR-ABL–positive human leukemic cells: caspase-independent, necrosis-like programmed cell death mediated by serine protease activity

Blood ◽

10.1182/blood-2003-05-1605 ◽

2004 ◽

Vol 103 (6) ◽

pp. 2299-2307 ◽

Cited By ~ 73

Author(s):

Masayuki Okada ◽

Souichi Adachi ◽

Tsuyoshi Imai ◽

Ken-ichiro Watanabe ◽

Shin-ya Toyokuni ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Serine Protease ◽

Imatinib Mesylate ◽

Protease Activity ◽

Kinase Inhibitor ◽

Leukemic Cells ◽

Endonuclease G ◽

Serine Protease Activity ◽

Human Leukemic Cells

Abstract Caspase-independent programmed cell death can exhibit either an apoptosis-like or a necrosis-like morphology. The ABL kinase inhibitor, imatinib mesylate, has been reported to induce apoptosis of BCR-ABL–positive cells in a caspase-dependent fashion. We investigated whether caspases alone were the mediators of imatinib mesylate–induced cell death. In contrast to previous reports, we found that a broad caspase inhibitor, zVAD-fmk, failed to prevent the death of imatinib mesylate–treated BCR-ABL–positive human leukemic cells. Moreover, zVAD-fmk–preincubated, imatinib mesylate–treated cells exhibited a necrosis-like morphology characterized by cellular pyknosis, cytoplasmic vacuolization, and the absence of nuclear signs of apoptosis. These cells manifested a loss of the mitochondrial transmembrane potential, indicating the mitochondrial involvement in this caspase-independent necrosis. We excluded the participation of several mitochondrial factors possibly involved in caspase-independent cell death such as apoptosis-inducing factor, endonuclease G, and reactive oxygen species. However, we observed the mitochondrial release of the serine protease Omi/HtrA2 into the cytosol of the cells treated with imatinib mesylate or zVAD-fmk plus imatinib mesylate. Furthermore, serine protease inhibitors prevented the caspase-independent necrosis. Taken together, our results suggest that imatinib mesylate induces a caspase-independent, necrosis-like programmed cell death mediated by the serine protease activity of Omi/HtrA2.

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Broad-Spectrum Antiviral Activity of 3D8, a Nucleic Acid-Hydrolyzing Single-Chain Variable Fragment (scFv), Targeting SARS-CoV-2 and Multiple Coronaviruses In Vitro

Viruses ◽

10.3390/v13040650 ◽

2021 ◽

Vol 13 (4) ◽

pp. 650

Author(s):

Gunsup Lee ◽

Shailesh Budhathoki ◽

Geum-Young Lee ◽

Kwang-ji Oh ◽

Yeon Kyoung Ham ◽

...

Keyword(s):

Nucleic Acid ◽

Antiviral Activity ◽

Broad Spectrum ◽

Recombinant Antibody ◽

Therapeutic Effects ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Diarrhea Virus ◽

3D8 Scfv

The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

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Transcutaneous penetration of a single-chain variable fragment (scFv) compared to a full-size antibody: potential tool for atopic dermatitis (AD) treatment

Allergy Asthma & Clinical Immunology ◽

10.1186/s13223-021-00574-x ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Audrey Baylet ◽

Raoul Vyumvuhore ◽

Marine Laclaverie ◽

Laëtitia Marchand ◽

Carine Mainzer ◽

...

Keyword(s):

Atopic Dermatitis ◽

Ex Vivo ◽

Topical Therapy ◽

Human Keratinocytes ◽

Raman Microspectroscopy ◽

Interleukin 4 ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Administration Routes

AbstractCurrently, several biologics are used for the treatment of cutaneous pathologies such as atopic dermatitis (AD), psoriasis or skin cancers. The main administration routes are subcutaneous and intravenous injections. However, little is known about antibody penetration through the skin. The aim was to study the transcutaneous penetration of a reduced-size antibody as a single-chain variable fragment (scFv) compared to a whole antibody (Ab) and to determine its capacity to neutralize an inflammatory cytokine involved in AD such as human interleukin-4 (hIL-4). Transcutaneous penetration was evaluated by ex vivo studies on tape-stripped pig ear skin. ScFv and Ab visualization through the skin was measured by Raman microspectroscopy. In addition, hIL-4 neutralization was studied in vitro using HEK-Blue™ IL-4/IL-13 cells and normal human keratinocytes (NHKs). After 24 h of application, analysis by Raman microspectroscopy showed that scFv penetrated into the upper dermis while Ab remained on the stratum corneum. In addition, the anti-hIL4 scFv showed very efficient and dose-dependent hIL-4 neutralization. Thus, scFv penetrates through to the upper papillary dermis while Ab mostly remains on the surface, the anti-hIL4 scFv also neutralizes its target effectively suggesting its potential use as topical therapy for AD.

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Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2

Journal of Experimental Medicine ◽

10.1084/jem.20112741 ◽

2012 ◽

Vol 209 (6) ◽

pp. 1201-1217 ◽

Cited By ~ 442

Author(s):

Tadashi Yokosuka ◽

Masako Takamatsu ◽

Wakana Kobayashi-Imanishi ◽

Akiko Hashimoto-Tane ◽

Miyuki Azuma ◽

...

Keyword(s):

Cell Death ◽

T Cell ◽

Programmed Cell Death ◽

T Cell Activation ◽

Cell Activation ◽

Tyrosine Phosphatase ◽

Cell Receptor ◽

Programmed Cell Death 1

Programmed cell death 1 (PD-1) is a negative costimulatory receptor critical for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a molecular mechanism of PD-1–mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently associated with the phosphatase SHP2 (Src homology 2 domain–containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addition to PD-1 clustering, PD-1–TCR colocalization within microclusters is required for efficient PD-1–mediated suppression. This inhibitory mechanism also functions in PD-1hi T cells generated in vivo and can be overridden by a neutralizing anti–PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.

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OCT4&SOX2-specific cytotoxic T lymphocytes plus programmed cell death protein 1 inhibitor presented with synergistic effect on killing lung cancer stem-like cells in vitro and treating drug-resistant lung cancer mice in vivo

Journal of Cellular Physiology ◽

10.1002/jcp.27423 ◽

2018 ◽

Vol 234 (5) ◽

pp. 6758-6768 ◽

Cited By ~ 4

Author(s):

Xueyan Zhang ◽

Fang Hu ◽

Changhui Li ◽

Xiaoxuan Zheng ◽

Bo Zhang ◽

...

Keyword(s):

Lung Cancer ◽

Cell Death ◽

T Lymphocytes ◽

Programmed Cell Death ◽

Synergistic Effect ◽

Cytotoxic T Lymphocytes ◽

Drug Resistant ◽

Cell Death Protein

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Phosphorylation of the mitochondrial triphosphate tunnel metalloenzyme TTM1 regulates programmed cell death in senescence

10.1101/2020.10.06.328278 ◽

2020 ◽

Author(s):

Purva Karia ◽

Keiko Yoshioka ◽

Wolfgang Moeder

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Map Kinases ◽

Mitochondrial Protein ◽

Mitochondrial Outer Membrane ◽

Mitogen Activated Protein ◽

Animal Growth ◽

Triphosphate Tunnel Metalloenzyme

ABSTRACTThe role of mitochondria in programmed cell death (PCD) during animal growth and development is well documented, but much less is known for plants. We previously showed that the Arabidopsis thaliana triphosphate tunnel metalloenzyme (TTM) proteins TTM1 and TTM2 are tail-anchored proteins that localize in the mitochondrial outer membrane and participate in PCD during senescence and immunity, respectively. Here, we show that TTM1 is specifically involved in senescence induced by abscisic acid (ABA). Moreover, phosphorylation of TTM1 by multiple mitogen-activated protein kinases (MAPKs) regulates its function and turnover. A combination of proteomics and in vitro kinase assays revealed three major phosphorylation sites of TTM1 (S10, S437, and S490), which are phosphorylated upon perception of senescence cues such as ABA and prolonged darkness. S437 is phosphorylated by the MAP kinases MPK3 and MPK4, and S437 phosphorylation is essential for TTM1 function in senescence. These MPKs, together with three additional MAP kinases (MPK1, MPK7, and MPK6), phosphorylate S10 and S490, marking TTM1 for protein turnover, which likely prevents uncontrolled cell death. Taken together, our results show that multiple MPKs regulate the function and turnover of the mitochondrial protein TTM1 during senescence-related PCD, revealing a novel link between mitochondria and PCD.SummaryEmail addresses: [email protected]

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