scholarly journals NOVEL IMMUNOTHERAPEUTIC TARGETED GRANZYME DELIVERY SYSTEMS IN TREATMENT OF MALIGNANT TUMORS

2021 ◽  
Vol 20 (2) ◽  
pp. 31-41
Author(s):  
I. V. Yarosh ◽  
V. A. Misyurin ◽  
I. I. Krasnyuk
Keyword(s):  
Cell Death ◽  
Target Cell ◽  
Serine Proteases ◽  
Malignant Tumors ◽  
Granzyme B ◽  
Killer Cell ◽  
Cascade Reactions ◽  
Target Cells ◽  
Cell Property ◽  
Infected Cells

Cytotoxicity is the main human killer cell property. The cytotoxicity reaction of human killer cells is achieved through a complex of molecules, including perforins, granzyme, cathepsin and others. However, only one molecule is enough for target cell death: granzyme. Other molecules are intended for granzyme activation and its delivery to the target cell cytoplasm. Granzymes are a whole family of serine proteases that perform their function in the human body as integral cytolytic effectors during programmed cell death of cancer and pathogen-infected cells. Secreted mainly by cytotoxic T-lymphocytes and NK-cells, granzymes initiate apoptosis via caspase-dependent and caspase-independent pathways. These natural properties make granzymes one of the most promising human enzymes for use in the development of targeted therapeutic strategies in the treatment of various types of cancer.The most promising is granzyme B, because it has the most powerful effector properties. Due to the initiation of cascade reactions that activate apoptosis, granzyme is attractive as a basis for the development of medicines applicable in clinical oncology. At this time, several approaches have been developed for delivering granzyme molecules to tumor cells and facilitating its penetration through the cell membrane. Moreover, some solutions are proposed to overcome the resistance of target cells to granzyme-mediated apoptosis. These approaches are discussed in this review.The purpose of this review was to systematize information on the use of granzyme B as a nanostructured drug delivery system in the treatment of solid and hematological malignancies. In addition, this review discusses ways to overcome the resistance of granzyme penetration into target cells.

Entry and Trafficking of Granzyme B in Target Cells During Granzyme B-Perforin–Mediated Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1044-1054 ◽  
Author(s):  
Michael J. Pinkoski ◽  
Marita Hobman ◽  
Jeffrey A. Heibein ◽  
Kevin Tomaselli ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Indirect Immunofluorescence ◽  
Target Cell ◽  
Granzyme B ◽  
Fluorescein Isothiocyanate ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Direct Visualization ◽  
New Model ◽  
American Society

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin–induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death. © 1998 by The American Society of Hematology.

Entry and Trafficking of Granzyme B in Target Cells During Granzyme B-Perforin–Mediated Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 1044-1054 ◽  
Author(s):  
Michael J. Pinkoski ◽  
Marita Hobman ◽  
Jeffrey A. Heibein ◽  
Kevin Tomaselli ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Indirect Immunofluorescence ◽  
Target Cell ◽  
Granzyme B ◽  
Fluorescein Isothiocyanate ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Direct Visualization ◽  
New Model ◽  
American Society

Abstract In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin–induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death. © 1998 by The American Society of Hematology.

scholarly journals The Human Cytomegalovirus Protein UL16 Mediates Increased Resistance to Natural Killer Cell Cytotoxicity through Resistance to Cytolytic Proteins

2003 ◽  
Vol 77 (8) ◽  
pp. 4539-4545 ◽  
Author(s):  
Jenny Odeberg ◽  
Helena Browne ◽  
Sunil Metkar ◽  
Christopher J. Froelich ◽  
Lars Brandén ◽  
...  
Keyword(s):  
Nk Cells ◽  
Human Cytomegalovirus ◽  
Deletion Mutant ◽  
Nk Cell ◽  
Granzyme B ◽  
Killer Cell ◽  
Target Cells ◽  
Natural Killer Cell Cytotoxicity ◽  
Hcmv Strain ◽  
Infected Cells

ABSTRACT Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

scholarly journals Granzyme B-induced Cell Death Involves Induction of p53 Tumor Suppressor Gene and Its Activation in Tumor Target Cells

2007 ◽  
Vol 282 (45) ◽  
pp. 32991-32999 ◽  
Author(s):  
Franck Meslin ◽  
Jerome Thiery ◽  
Catherine Richon ◽  
Abdelali Jalil ◽  
Salem Chouaib
Keyword(s):  
Cell Death ◽  
Tumor Suppressor ◽  
Tumor Suppressor Gene ◽  
Granzyme B ◽  
Suppressor Gene ◽  
Target Cells ◽  
Tumor Target ◽  
P53 Tumor Suppressor ◽  
P53 Tumor Suppressor Gene

Natural killer cell education does not affect the magnitude of granzyme B delivery to target cells by antibody-dependent cellular cytotoxicity

AIDS ◽  
2015 ◽  
Vol 29 (12) ◽  
pp. 1433-1443 ◽  
Author(s):  
Gamze Isitman ◽  
Irene Lisovsky ◽  
Alexandra Tremblay-McLean ◽  
Matthew S. Parsons ◽  
Naglaa H. Shoukry ◽  
...  
Keyword(s):  
Natural Killer Cell ◽  
Natural Killer ◽  
Granzyme B ◽  
Killer Cell ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

Fas-based d1OS-mediated cytotoxicity requires macromolecular synthesis for effector cell activation but not for target cell death

1994 ◽  
Vol 345 (1313) ◽  
pp. 303-309 ◽  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Cell Surface ◽  
Target Cell ◽  
Cell Activation ◽  
Effector Cell ◽  
Apoptotic Cell ◽  
Target Cells ◽  
Macromolecular Synthesis ◽  
Effector Cells

Two main mechanisms seem at play in T cell-mediated cytotoxicity, a process in which target cell death often follows an apoptotic cell death pattern. One of these involves Fas at the target cell surface and a Fas ligand at the effector cell surface. This allowed us to reinvestigate the long-standing question of macromolecular synthesis requirement in T cell-mediated cytotoxicity, using the dlOS model cell line which is cytotoxic apparently only via the Fas molecularly defined mechanism. We showed, first, that induction of cytotoxic activity of effector cells, obtained by preincubating these effector cells with a phorbol ester and a calcium ionophore, could be inhibited by macromolecular synthesis inhibitors (cycloheximide, actinomycin D, DRB). We then investigated whether macromolecular synthesis was required, when effector and target cells were mixed, to obtain target cell death. Preincubating already activated effector cells for 30 min with macromolecular synthesis inhibitors, then adding target cells and performing the 51 Cr release cytotoxicity test in the presence of these inhibitors, did not significantly decrease subsequent target cell death, indicating that already activated effector cells could kill without further requirement for macromolecular synthesis. In addition, target cell preincubation for up to 3 h in the presence of one of these inhibitors did not decrease cell death. The high sensitivity of mouse thymocytes to this type of cytotoxicity enabled us to devise the following experiment. As previously shown by others, thymocyte death induced by dexamethasone (DEX) could be blocked by coincubation with cycloheximide (CHX). Such DEX-treated CHX-rescued thymocytes, the survival of which was an internal control of efficiency of protein synthesis inhibition, were then subjected to effector cells in the presence of CHX, and were shown to die. Thus, there is no requirement for macromolecular synthesis at the target cell level in this variety of apoptotic cell death. Altogether, in this defined mechanism of T cell-mediated cytotoxicity, macromolecular synthesis is required for dlOS effector cell activation, but not for lysis by already activated effector cells nor for target cell death.

scholarly journals The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity.

1995 ◽  
Vol 181 (5) ◽  
pp. 1905-1909 ◽  
Author(s):  
H Nakajima ◽  
P Golstein ◽  
P A Henkart
Keyword(s):  
Cell Death ◽  
Target Cell ◽  
Cytotoxic T Lymphocyte ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Cell Nuclei ◽  
Effector Cells ◽  
Cell Death Pathways ◽  
Mast Cell Tumors ◽  
Nuclear Damage

The requirement for target cell nuclei in the two apoptotic death pathways used by cytotoxic lymphocytes was tested using model effector systems in which the granzyme and Fas pathways of target damage are isolated. Mast cell tumors expressing granzymes A and B in addition to cytolysin/perforin lysed tumor target cells about 10-fold more efficiently than comparable effector cells without granzymes. Enucleated cytoplast targets derived from these cells were also lysed with a similar 10-fold effect of granzymes. In contrast to cytoplasts, effector granzyme expression did not influence lysis of red cell targets. The Fas pathway was assessed using the selected cytotoxic T lymphocyte hybridoma subline d11S, which lysed target cells expressing Fas but not those lacking Fas. Similarly, cytoplasts derived from Fas+ but not Fas- cells were also readily lysed by these effector cells. Thus, neither the nucleus itself nor the characteristic apoptotic nuclear damage associated with the two major cell death pathways used by cytotoxic lymphocytes are required for cell death per se.

Target cell death triggered by cytotoxic T lymphocytes: a target cell mutant distinguishes passive pore formation and active cell suicide mechanisms

1994 ◽  
Vol 14 (1) ◽  
pp. 427-436
Author(s):  
D S Ucker ◽  
J D Wilson ◽  
L D Hebshi
Keyword(s):  
Cell Death ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Target Cell ◽  
Active Cell ◽  
Death Process ◽  
Target Cells ◽  
Antigenic Stimulus ◽  
Cell Suicide

The role of the target cell in its own death mediated by cytotoxic T lymphocytes (CTL) has been controversial. The ability of the pore-forming granule components of CTL to induce target cell death directly has been taken to suggest an essentially passive role for the target. This view of CTL-mediated killing ascribes to the target the single role of providing an antigenic stimulus to the CTL; this signal results in the vectoral degranulation and secretion of pore-forming elements onto the target. On the other hand, by a number of criteria, target cell death triggered by CTL appears fundamentally different from death resulting from membrane damage and osmotic lysis. CTL-triggered target cell death involves primary internal lesions of the target cell that reflect a physiological cell death process. Orderly nuclear disintegration, including lamin phosphorylation and solubilization, chromatin condensation, and genome digestion, are among the earliest events, preceding the loss of plasma membrane integrity. We have tested directly the involvement of the target cell in its own death by examining whether we could isolate mutants of target cells that have retained the ability to be recognized by and provide an antigenic stimulus to CTL while having lost the capacity to respond by dying. Here, we describe one such mutant, BW87. We have used this CTL-resistant mutant to analyze the mechanisms of CTL-triggered target cell death under a variety of conditions. The identification of a mutable target cell element essential for the cell death response to CTL provides genetic evidence that target cell death reflects an active cell suicide process similar to other physiological cell deaths.

Murine NK Cells Require Activation-Dependent Expression of Granzyme B and Perforin To Become Potent Cytotoxic Effectors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 920-920
Author(s):  
Todd A. Fehniger ◽  
Sheng F. Cai ◽  
Xuefang Cao ◽  
Andrew J. Bredemeyer ◽  
Rachel M. Presti ◽  
...  
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Target Cell ◽  
Post Infection ◽  
Nk Cell ◽  
Granzyme B ◽  
Poly I:C ◽  
Wild Type

Abstract NK cells predominantly utilize the granule exocytosis pathway to kill virus-infected and malignant target cells. Current paradigms suggest that resting NK cells have pre-formed granules containing granzymes A, B, and perforin and are ready to kill targets immediately upon proper recognition by NK receptors. Here, we report that resting murine NK cells in the spleen exhibit poor cytotoxicity (5.4±1.6% target cell death, 20:1 E:T ratio and 4 hour incubation), compared with cytokine-activated (IL-15, 48 hours) splenic NK cells (59.7±10.6% target cell death), against the RMAS tumor cell line in vitro as measured by a flow-based killing assay. In addition, using intracellular flow cytometric analysis with monoclonal antibodies specific for granzymes A, B, and perforin, we find that resting murine NK cells express abundant granzyme A (86.2±1.9% positive), but little or no granzyme B (4.4±5.4% positive) or perforin (2.6±1.8% positive). Activation of murine NK cells with IL-15 induces robust expression of both perforin (59.1±2.0% positive) and granzyme B (91.5±7.9% positive), which correlates with increased cytotoxicity. Further, granzyme B cluster −/− (26±6.7% target cell death) and perforin −/− (5.7±1.3% target cell death) NK cells have poor cytotoxicity in vitro despite IL-15 activation. Poly I:C simulates RNA virus infection and activates NK cell cytotoxicity in vivo through TLR3 and cytokine cascades. NK cell granzyme B and perforin expression is induced in vivo 24 hours after poly I:C injection, correlating with increased in vitro NK killing of tumor targets. In wild type mice infected with murine cytomegalovirus (MCMV), NK cell expression of both perforin (83.5±4.9% positive) and granzyme B (89.3±2.1% positive) is upregulated in the spleen, peaking 2–4 days post-infection and returning to baseline by 8 days post-infection. In addition, MCMV titers are significantly elevated at day 3 post-infection in both granzyme B cluster −/− (P<0.01) and perforin −/− (P<0.01) mice, compared to wild type mice. Moreover, survival following MCMV infection was significantly lower in granzyme B cluster −/− and perforin −/− mice, compared with wild type mice (P<0.001, see survival curve). Thus, our findings show that murine NK cells require the activation of granzyme B and perforin to become potent cytotoxic effectors. We also demonstrate for the first time that granzyme B is critical for early host defense against MCMV. These findings explain the long-standing observation that murine NK cells require prior activation for potent natural killing of tumor targets in vitro. Further, this requirement for activation-dependent granzyme B and perforin expression in NK cells may influence outcomes in murine models of innate immune anti-tumor and anti-viral responses. Figure Figure

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