scholarly journals Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 627-635 ◽  
Author(s):  
RC Stong ◽  
F Uckun ◽  
RJ Youle ◽  
JH Kersey ◽  
DA Vallera
Keyword(s):  
Protein Synthesis ◽  
T Cell ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Transplantation ◽  
Leukemic Cells ◽  
Target Antigen ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Kinetics Of ◽  
Kinetics Of Protein Synthesis

Abstract The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four- IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.

scholarly journals Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 627-635 ◽  
Author(s):  
RC Stong ◽  
F Uckun ◽  
RJ Youle ◽  
JH Kersey ◽  
DA Vallera
Keyword(s):  
Protein Synthesis ◽  
T Cell ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Transplantation ◽  
Leukemic Cells ◽  
Target Antigen ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Kinetics Of ◽  
Kinetics Of Protein Synthesis

The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four- IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.

Over-Expression of TRAIL on Donor T Cells Enhances GVT and Suppresses Gvhd Via Elimination of Alloreactive T Cells and Host APC

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 817-817 ◽  
Author(s):  
Arnab Ghosh ◽  
Yildirim Dogan ◽  
Amanda M. Holland ◽  
Odette M Smith ◽  
Lauren F. Young ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autologous Transplantation ◽  
Genetically Engineered ◽  
Cell Group ◽  
Target Cells ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Over Expression ◽  
Donor T Cells

Abstract Abstract 817 Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p<0.01, 100% survival in TRAIL+ T cell group) (Fig 1A, also shown at the annual meeting last year). Precursor (pre)T cells have the benefit of regenerating the T cell compartment without causing GVHD and being available for “off the shelf” use. We generated TRAIL+ preT cells from transduced B6 hematopoietic stem cells and expanded them using the OP9-DL1 co-culture system. Adoptive transfer of B6 TRAIL+ preT cells into syngeneic-transplanted BALB/c mice could reconstitute the T cell compartment with TRAIL-expressing T cells and caused enhanced antitumor activity (p<0.05) compared to mock (GFP)-transduced controls. Interestingly, in addition to enhanced GVT, the recipients treated with TRAIL+ T cells had significantly less GVHD lethality and morbidity (Fig1B). This was observed across multiple GVHD models (B6 ^ CBF1, B6^ BALB/c and B10.BR^ B6). To explore the factors contributing to TRAIL-mediated suppression of GVHD, we used animals deficient in DR5 (DR5ko) in our models of GVHD. We found that GVHD suppressive effects of TRAIL were lost when hosts were DR5ko or when DR5ko TRAIL+ T cells were adoptively transferred indicating that TRAIL+ T cells suppress GVHD by targeting both host and donor compartments. We observed a higher DR5 expression in host MHC-IIhi antigen presenting cells (APC) following total body irradiation, suggesting that TRAIL+ donor T cells could potently eliminate host APC, resulting in less GVHD. Further, on transferring wild type T cells into irradiated hosts, we found that alloreactive CD25+ T cells had a significantly higher DR5 expression compared to CD25− T cells. This indicates that TRAIL+ T cells can specifically target the alloreactive CD25+ T cells in order to suppress GVHD. Collectively, our data demonstrate that donor T cells genetically engineered to express TRAIL can enhance GVT effects and suppress the development of lethal GVHD in recipients of allo-HSCT. Our data suggests that his suppression of GVHD is mediated by the elimination the alloreactive donor T cells and the elimination of GVHD-promoting residual APC. Furthermore, we demonstrated that allogeneic ex vivo generated preT cells expressing TRAIL could mediate a strong protection against tumor challenge in syngeneic HSCT recipients. TRAIL over-expression thus represents a potential off the shelf approach to enhancing GVT in both allogeneic and autologous transplantation. Despite elimination of alloreactive donor T cells, TRAIL+ T cells demonstrated enhanced GVT by directly targeting DR5+ tumors in the absence of alloreactivity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Optimal elimination of leukemic T cells from human bone marrow with T101-ricin A-chain immunotoxin

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 289-297 ◽  
Author(s):  
P Casellas ◽  
X Canat ◽  
AA Fauser ◽  
O Gros ◽  
G Laurent ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Autologous Transplantation ◽  
Leukemic Cells ◽  
Ricin A Chain ◽  
Hematopoietic Stem ◽  
Human T Cell ◽  
A Chain ◽  
Ricin A

Abstract In view of bone marrow purging before autologous transplantation in T cell malignancies, an anti-human T cell immunotoxin (IT) has been prepared by coupling ricin A-chain to the monoclonal antibody T101 that binds the T1 differentiation antigen expressed by T lymphocytes as well as by T cell-derived hematologic malignancies. Using a sensitive and reliable clonogenic assay, optimal conditions were defined for the elimination of clonogenic human T leukemic cells among bone marrow cells. Maximal cytoreduction was obtained with IT at a dose of 2 micrograms/mL in the presence of 10 mmol/L NH4Cl. This treatment led to the reduction of more than six orders of magnitude of T101-positive clonogenic leukemic cells, with no harm to T101-negative cells. Moreover, we observed no toxicity of IT to human hematopoietic stem cells (CFU-GEMMT) derived from bone marrow of healthy volunteers. Thus, pretreatment of bone marrow samples with IT plus NH4Cl offers a safe, simple, reliable, and highly efficient means to eliminate undesirable leukemic T cells from the graft.

scholarly journals Optimal elimination of leukemic T cells from human bone marrow with T101-ricin A-chain immunotoxin

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 289-297
Author(s):  
P Casellas ◽  
X Canat ◽  
AA Fauser ◽  
O Gros ◽  
G Laurent ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Autologous Transplantation ◽  
Leukemic Cells ◽  
Ricin A Chain ◽  
Hematopoietic Stem ◽  
Human T Cell ◽  
A Chain ◽  
Ricin A

In view of bone marrow purging before autologous transplantation in T cell malignancies, an anti-human T cell immunotoxin (IT) has been prepared by coupling ricin A-chain to the monoclonal antibody T101 that binds the T1 differentiation antigen expressed by T lymphocytes as well as by T cell-derived hematologic malignancies. Using a sensitive and reliable clonogenic assay, optimal conditions were defined for the elimination of clonogenic human T leukemic cells among bone marrow cells. Maximal cytoreduction was obtained with IT at a dose of 2 micrograms/mL in the presence of 10 mmol/L NH4Cl. This treatment led to the reduction of more than six orders of magnitude of T101-positive clonogenic leukemic cells, with no harm to T101-negative cells. Moreover, we observed no toxicity of IT to human hematopoietic stem cells (CFU-GEMMT) derived from bone marrow of healthy volunteers. Thus, pretreatment of bone marrow samples with IT plus NH4Cl offers a safe, simple, reliable, and highly efficient means to eliminate undesirable leukemic T cells from the graft.

Kinetics of Protein Synthesis in Enucleate Frog Oocytes

Science ◽  
1968 ◽  
Vol 160 (3832) ◽  
pp. 1115-1117 ◽  
Author(s):  
R. E. Ecker ◽  
L. D. Smith ◽  
S. Subtelny
Keyword(s):  
Protein Synthesis ◽  
Kinetics Of ◽  
Kinetics Of Protein Synthesis

scholarly journals Decomposition of time-dependent fluorescence signals reveals codon-specific kinetics of protein synthesis

2018 ◽  
Vol 46 (22) ◽  
pp. e130-e130 ◽  
Author(s):  
Nadin Haase ◽  
Wolf Holtkamp ◽  
Reinhard Lipowsky ◽  
Marina Rodnina ◽  
Sophia Rudorf
Keyword(s):  
Protein Synthesis ◽  
Time Dependent ◽  
Kinetics Of ◽  
Kinetics Of Protein Synthesis

scholarly journals Robust, But Transient Expression of Adeno-Associated Virus-Transduced Genes During Human T Lymphopoiesis

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4854-4864 ◽  
Author(s):  
Jason P. Gardner ◽  
Haihong Zhu ◽  
Peter C. Colosi ◽  
Gary J. Kurtzman ◽  
David T. Scadden
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Marker Gene ◽  
T Cell Differentiation ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
The Impact ◽  
T Lymphopoiesis

Abstract Recombinant adeno-associated viruses (rAAV) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. We evaluated rAAV vector containing a LacZ reporter gene under the control of a cytomegalovirus (CMV) promoter in the context of primary human CD34+CD2− progenitor cells induced to undergo T-cell differentiation using an in vitro T-lymphopoiesis system. Target cells from either adult bone marrow or umbilical cord blood were efficiently transduced, and 71% to 79% CD2+ cells expressed a LacZ marker gene mRNA and produced LacZ-encoded protein after exposure to rAAV-CMV-LacZ. The impact of transgene expression on the differentiation of T cells was assessed by sequential quantitation of immunophenotypic subsets of virus-exposed cells and no alteration was noted compared with control. The durability of transgene expression was assessed and found to decay by day 35 with kinetics dependent on the multiplicity of infection. In addition, vector DNA was absent from CD4 or CD8 subselected CD3+ cells by DNA-polymerase chain reaction. These data suggest that rAAV vectors may result in robust transgene expression in primitive cells undergoing T-cell lineage commitment without toxicity or alteration in the pattern of T-cell differentiation. However, expression is transient and integration of the transgene unlikely. Recombinant AAV vectors are potentially valuable gene transfer tools for the genetic manipulation of events during T-cell ontogony but their potential in gene therapy strategies for diseases such as acquired immunodeficiency syndrome is limited.

scholarly journals Chimeric antigen receptor T cell therapies for acute myeloid leukemia

2020 ◽  
Vol 14 (6) ◽  
pp. 701-710
Author(s):  
Bin Gu ◽  
Jianhong Chu ◽  
Depei Wu
Keyword(s):  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Antigen Receptor ◽  
Target Antigen ◽  
Hematopoietic Stem ◽  
Car T ◽  
Acute Myeloid

AbstractChimeric antigen receptor T cell (CAR T) therapies have achieved unprecedented efficacy in B-cell tumors, prompting scientists and doctors to exploit this strategy to treat other tumor types. Acute myeloid leukemia (AML) is a group of heterogeneous myeloid malignancies. Relapse remains the main cause of treatment failure, especially for patients with intermediate or high risk stratification. Allogeneic hematopoietic stem cell transplantation could be an effective therapy because of the graft-versus-leukemia effect, which unfortunately puts the patient at risk of serious complications, such as graft-versus-host disease. Although the identification of an ideal target antigen for AML is challenging, CAR T therapy remains a highly promising strategy for AML patients, particularly for those who are ineligible to receive a transplantation or have positive minimal residual disease. In this review, we focus on the most recent and promising advances in CAR T therapies for AML.

scholarly journals Purging murine leukemic marrow with alkyl-lysophospholipids

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1288-1291 ◽  
Author(s):  
L Glasser ◽  
LB Somberg ◽  
WR Vogler
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cytotoxic Effect ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Myelomonocytic Leukemia ◽  
In Vitro Treatment

Abstract Autologous bone marrow transplantation is potentially curative in the treatment of acute leukemia if residual leukemic cells in the marrow can be eliminated prior to transplantation. We studied the purging effects of a synthetic alkyl-lysophospholipid (ALP) on marrow containing leukemic cells from a transplantable myelomonocytic leukemia (WEHI-3B) in BALB/c mice. Simulated remission bone marrow containing 2% leukemic cells treated in vitro with 20 and 100 micrograms/mL of ET-18- OCH3 (1-octadecyl-2-methyl-sn-glycerol-3-phosphocholine) significantly prolonged survival of lethally irradiated transplanted recipients. At a dose of 100 micrograms/mL, 88% of the mice survived for the duration of the experiment (approximately five months). Autopsies showed that 25% of these survivors had microscopic evidence of leukemia. Thus, in vitro treatment of marrow eliminated leukemic blasts and spared sufficient normal stem cells to allow hematologic reconstitution. The effect of ET- 18-OCH3 is not entirely selective for leukemic cells. A spleen colony assay showed that ALP has some cytotoxic effect on normal hematopoietic stem cells.

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