scholarly journals Autologous transplantation of bone marrow purged in vitro with anti-CD7- (WT1-) ricin A immunotoxin in T-cell lymphoblastic leukemia and lymphoma

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 1152-1158 ◽  
Author(s):  
FW Preijers ◽  
T De Witte ◽  
JM Wessels ◽  
GC De Gast ◽  
E Van Leeuwen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
High Risk ◽  
Tumor Cells ◽  
Lymphoblastic Leukemia ◽  
T Cell Response ◽  
Growth Potential ◽  
Ricin A ◽  
Immunologic Reconstitution

Abstract Seven patients with high-risk acute T-cell lymphoblastic leukemia (T- ALL) and six with T cell lymphoma (T-LL) were treated with autologous bone marrow transplantation (ABMT) after in vitro purging of their bone marrow with WT1 (CD7)-ricin A-chain immunotoxin. CD7 expression on the tumor cells showed large variations between the individual patients and was highly related to the specific cytotoxicity of WT1-ricin A. Incubation of bone marrow with up to 10(-8)mol/L WT1-ricin A in the presence of 6 mmol/L NH4Cl did not compromise the growth potential of the hematopoietic progenitors CFU-GM, CFU-GEMM, and BFU-E. Hematologic engraftment (greater than 10(9) leukocytes/L) occurred within a normal time period (median, 17 days). Seven patients are alive and in complete remission (CR) at 48+, 44+, 40+, 26+, 11+, 7+, and 6+ months after ABMT. Four patients relapsed within 6 months after ABMT. Two of them had the lowest CD7 expression on their tumor cells, the other two were transplanted in CR2 and CR3. Two patients died from transplantation related infections. The immunologic reconstitution was delayed, although the numbers of T cells reached normal levels within 1 month. The number of CD7+ cells remained low up to 1 year after transplantation. The T4/T8-ratio was decreased for at least 6 months. The T-cell response to mitogens recovered to normal levels after 1 year. This study shows that ABMT with WT1-ricin A purged bone marrow in high-risk T-cell malignancies results in a complete hematopoietic and a delayed immunologic reconstitution. The actuarial relapse free survival is 61% at 3 years.

scholarly journals Autologous transplantation of bone marrow purged in vitro with anti-CD7- (WT1-) ricin A immunotoxin in T-cell lymphoblastic leukemia and lymphoma

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 1152-1158
Author(s):  
FW Preijers ◽  
T De Witte ◽  
JM Wessels ◽  
GC De Gast ◽  
E Van Leeuwen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
High Risk ◽  
Tumor Cells ◽  
Lymphoblastic Leukemia ◽  
T Cell Response ◽  
Growth Potential ◽  
Ricin A ◽  
Immunologic Reconstitution

Seven patients with high-risk acute T-cell lymphoblastic leukemia (T- ALL) and six with T cell lymphoma (T-LL) were treated with autologous bone marrow transplantation (ABMT) after in vitro purging of their bone marrow with WT1 (CD7)-ricin A-chain immunotoxin. CD7 expression on the tumor cells showed large variations between the individual patients and was highly related to the specific cytotoxicity of WT1-ricin A. Incubation of bone marrow with up to 10(-8)mol/L WT1-ricin A in the presence of 6 mmol/L NH4Cl did not compromise the growth potential of the hematopoietic progenitors CFU-GM, CFU-GEMM, and BFU-E. Hematologic engraftment (greater than 10(9) leukocytes/L) occurred within a normal time period (median, 17 days). Seven patients are alive and in complete remission (CR) at 48+, 44+, 40+, 26+, 11+, 7+, and 6+ months after ABMT. Four patients relapsed within 6 months after ABMT. Two of them had the lowest CD7 expression on their tumor cells, the other two were transplanted in CR2 and CR3. Two patients died from transplantation related infections. The immunologic reconstitution was delayed, although the numbers of T cells reached normal levels within 1 month. The number of CD7+ cells remained low up to 1 year after transplantation. The T4/T8-ratio was decreased for at least 6 months. The T-cell response to mitogens recovered to normal levels after 1 year. This study shows that ABMT with WT1-ricin A purged bone marrow in high-risk T-cell malignancies results in a complete hematopoietic and a delayed immunologic reconstitution. The actuarial relapse free survival is 61% at 3 years.

12 Development of an in vitro assay to assess bispecific T cell engager using T cells from CD3e humanized mice

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A12-A12
Author(s):  
Jun Zhou ◽  
Shuang Zhu ◽  
Hongjuan Zhang ◽  
Lei Zheng ◽  
Mingfa Zang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Lymphoblastic Leukemia ◽  
Humanized Mice ◽  
Activation Markers ◽  
In Vivo Models ◽  
Vitro Assay ◽  
Bite Antibody

BackgroundBispecific T cell engagers (BiTE) is a fast-growing class of immunotherapies. They are bispecific antibody that bind to T cell-surface protein (for example, CD3e) and a specific tumor associate antigen (TAA) on tumor cells, by which to redirect T cells against tumor cells in a MHC-independent manner. A successful example in the clinical is Blinatumomab, a BiTE antibody against CD3/CD19 approved in 2014 to treat acute lymphoblastic leukemia. Currently, many CD3-based BiTE are in clinical trials, including BCMAxCD3, Her2xCD3, CEAxCD3, and PSMAxCD3. To evaluate the efficacy of BiTE in vitro, human peripheral blood monocyte cells (hPBMC) are commonly being used as a source of T cells to co-culture with tumor cells. The disadvantage of using hPBMC is donor-to-donor variability and the availability of the original donor if a study needs to be repeated.MethodsTo overcome this, we proposed to replace hPBMC with T cells from human CD3e (hCD3) genetically engineered mouse models mice (GEMM) for in in vitro coculture assay. T cells were isolated from hCD3 GEMM mice using negative selection mouse T cell isolation kit. Conventional tumor cell lines or luciferase-engineered patient-derived-xenograft (PDX)-derived organoids (PDXO) expressing specific antigens are co-cultured with hCD3 T cells in 96-well plates in the presence of BiTE antibody.ResultsWe measured the killing of tumor cells using either flow cytometry or luciferase activity as readouts. To analyze tumor-reactivity of T cells to cancer cell line or organoids, IFN-gamma in the culture medium was measured and activation markers on T cells was assessed.ConclusionsOur data showed the feasibility of using humanized mice T cells as a replacement for hPBMCs to assess BiTE antibody in vitro. We are further validating the application of murine hCD3 T cells for in vivo models to test bispecific T cell engagers.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3693-3701 ◽  
Author(s):  
Ypke V. J. M. van Oosterhout ◽  
Liesbeth van Emst ◽  
Anton V. M. B. Schattenberg ◽  
Wil J. M. Tax ◽  
Dirk J. Ruiter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Graft Versus Host Disease ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Host Disease ◽  
Graft Versus Host ◽  
Ricin A

Abstract This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10−8 mol/L (about 1.8 μg/mL). Because most natural killer (NK) cells are CD7+, NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m2 IT combination, administered intravenously at 48-hour intervals. The T1/2 was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells.

scholarly journals The immunosuppressant rapamycin blocks in vitro responses to hematopoietic cytokines and inhibits recovering but not steady-state hematopoiesis in vivo

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1543-1552 ◽  
Author(s):  
VF Quesniaux ◽  
S Wehrli ◽  
C Steiner ◽  
J Joergensen ◽  
HJ Schuurman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
T Cell ◽  
T Cell Response ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Hematopoietic Recovery ◽  
Stimulating Factor

Abstract The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.

scholarly journals Hypothesis: Engrafted Human Bone Marrow and Blood Cells in Culture

Blood ◽  
1972 ◽  
Vol 40 (5) ◽  
pp. 754-758 ◽  
Author(s):  
Hans W. von Heyden ◽  
George E. Moore
Keyword(s):  
Bone Marrow ◽  
Epstein Barr Virus ◽  
Lymphoblastic Leukemia ◽  
Growth Potential ◽  
Barr Virus ◽  
Before And After ◽  
Immunosuppressed Patients ◽  
Epstein Barr ◽  
Lymphoid Cell Lines

Abstract Lymphoblasts appearing in immunosuppressed patients after bone marrow transfusion are compared to those that can be established in vitro as permanent lymphoid cell lines. It is suggested that Epstein-Barr virus (EBV) could be responsible for the recurrent "lymphoblastic leukemia" in these patients and that the transplanted cells may be a clone of nonmalignant cells that has become capable of growing without normal restraints. It is important that in future patients the transplanted cells be characterized as to morphology, chromosome constitution, relative clonability and transplantability, the presence of EBV, T or B cell-like traits, and their growth potential in immunosuppressed heterologous hosts. The antibody titer to EBV should be measured before and after leukocyte transfusion.

scholarly journals Development of the T-ALLiPSC-based Therapeutic Cancer Vaccines for T-cell acute Lymphoblastic Leukemia

2021 ◽  
Author(s):  
Zhu Li ◽  
Xuemei Chen ◽  
Luning Liu ◽  
Meiling Zhou ◽  
Guangqian Zhou ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocyte ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Vaccines ◽  
Therapeutic Cancer Vaccine ◽  
Whole Cell Lysate ◽  
Cell Acute Lymphoblastic Leukemia

Abstract PurposeThe T-cell acute lymphoblastic leukemia (T-ALL) is a kind of hematological malignancy in children. Despite the significant improvement in the cure rate of T-ALL upon treatment with chemotherapy regimens, steroids, and allotransplantation there are relapses. This study focuses on the tumor-specific therapeutic vaccines derived from the induced pluripotent stem cells (iPSC) to address the issue of T-ALL recurrence.MethodsPatient-derived tumor cells were reprogrammed into the iPSCs and the RNA-seq data of the T-ALL-iPSCs and H-iPSCs were analyzed. In vitro, the whole cell lysate antigens of iPSCs were prepared to induce the dendritic cells (DC) maturation, which in turn stimulated the tumor-specific T cells to kill the T-ALL tumor cells (Jurkat, CCRF-CEM, MOLT-4).ResultsBoth T-ALL-iPSCs and H-iPSCs were highly related to the tumor-related genes. The transcriptome analysis showed the T-ALL-iPSCs to be similar to the T-ALL tumor cells. The cytotoxic T lymphocyte (CTL) stimulated by the DC-loaded T-ALL-iPSC-derived antigens showed specific cytotoxicity against the T-ALL cells in vitro.ConclusionsThe T-ALL-iPSC-based therapeutic cancer vaccine can elicit a specific anti-tumor effect on T-ALL.

scholarly journals IMMU-15. PROTEOGENOMIC DISCOVERY OF NOVEL TUMOR PEPTIDES AS NEOANTIGENS FOR PERSONALIZED T CELL IMMUNOTHERAPY IN MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii362-iii363
Author(s):  
Samuel Rivero-Hinojosa ◽  
Melanie Grant ◽  
Aswini Panigrahi ◽  
Huizhen Zhang ◽  
Veronika Caisova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Tumor Cells ◽  
T Cell Response ◽  
Mass Spectrometry Data ◽  
Target Antigen ◽  
T Cell Therapy ◽  
Cd8 Cells ◽  
Normal Tissues

Abstract T cell immunotherapies are promising new tools to combat high-risk subgroups of medulloblastoma without increasing the late effects burden. The ideal target antigen of an effective antitumor T-cell response is abundantly expressed by tumor cells but not by normal tissues, in order to limit off-target effects. Tumors translate a host of highly novel transcripts that are the result of aberrations in tumor DNA and the unmasking of alternative or novel exons. We developed a novel proteogenomic approach to identify tumor-restricted peptides and used them to select and expand T cells capable of mounting a tumor-specific cytotoxic immune response. Using RNA-seq and WGS data, we created personalized custom searchable databases containing predicted novel proteins from somatic mutations, novel junctions and fusion transcripts from 56 medulloblastoma tumors. By searching these databases with raw mass spectrometry data from paired medulloblastoma tumors, we identified tens of neoantigen peptides arising from the translation of tumor-specific transcripts; novel isoforms being the predominant source. We tested these peptides for their ability to select and expand autologous polyclonal populations of T cells and tested the immunogenicity of each individual peptide. Flow cytometry revealed populations of CD4+ and CD8+ cells with an activation profile marked by IFN-γ and TNF-α. Immunosuppressive marker profiles were also characterized. Using cytotoxicity assays, we demonstrated that tumor specific T cells can eliminate neoantigen bearing tumor cells. Thus, proteogenomics can identify immunogenic tumor specific peptides that can be used to create a personalized, targeted T cell therapy for children with high risk medulloblastoma.

scholarly journals Development of a CD123xCD3 Bispecific Antibody (JNJ-63709178) for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2824-2824 ◽  
Author(s):  
François Gaudet ◽  
Jennifer F Nemeth ◽  
Ronan McDaid ◽  
Yingzhe Li ◽  
Benjamin Harman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Stock Options ◽  
Cell Activation ◽  
Bispecific Antibody ◽  
Tumor Regression

Abstract AML is a cancer of the myeloid lineage that is characterized by the accumulation of abnormal white blood cells in the bone marrow and blood. Existing therapies do not lead to cures, partially due to their inability to eliminate residual leukemic stem cells (LSCs) in the bone marrow. T-cell redirection has been shown to be an effective method of treatment for hematologic malignancies (eg, blinatumomab) and represents an attractive approach to treat AML. CD123 (α-chain of the interleukin-3 receptor) has been shown to be expressed on the surface of AML blasts and LSCs. To eradicate CD123+ cells, we developed a bispecific antibody (JNJ-63709178) using the Genmab DuoBody® technology that can bind both CD123 on tumor cells and CD3 on T cells. JNJ-63709178 is a humanized IgG4 bispecific antibody with silenced Fc function. This antibody is able to recruit T cells to CD123-expressing tumor cells and induce the killing of these tumor cells in vitro (MOLM-13, OCI-AML5 and KG-1; EC50 = 0.51-0.91 nM). In contrast, this antibody does not kill CD123- cell lines, demonstrating the specificity of cytotoxicity. Consistently, the degree of cell killing correlated with the level of T-cell activation (CD69 and CD25) and cytokine release (TGF-β and TNF-α). Control bispecific antibodies containing a null arm (viral epitope) paired with a CD123 arm (CD123xnull) or a CD3 arm (nullxCD3) did not induce cytotoxicity or T-cell activation in the assays tested. JNJ-63709178 had no effect on T-cell activation when incubated with T cells alone. In AML murine xenograft models, JNJ-63709178 was able to suppress tumor growth and induce tumor regression (MOLM-13 and KG-1, respectively) in the presence of human peripheral blood mononuclear cells (PBMCs) or T cells. Tumor regression correlated with the infiltration of T cells in the tumor and the expression of T-cell activation markers such as CD25, PD1 and TIM3. Furthermore, this antibody was able to induce the killing of primary CD123+ cancer cells from the blood of patients with AML without the need to supplement with fresh T cells (EC50 = 0.83 nM). These results indicate that JNJ-63709178 can potently and specifically kill CD123+ cancer cells in vitro, in vivo and ex vivo. Pharmacokinetic studies in cynomolgus monkeys support twice weekly dosing for human studies. JNJ-63709178 is currently being investigated in a Phase 1 clinical trial in relapsed and refractory AML (ClinicalTrials.gov ID: NCT02715011). Disclosures Gaudet: Janssen Pharmaceuticals R&D: Employment, Other: Stock options, Patents & Royalties: pending, not yet issued. Nemeth:Janssen Pharmaceuticals R&D: Employment, Other: stock, Patents & Royalties: patent pending. McDaid:Janssen Pharmaceuticals Research and Development: Employment. Li:Janssen: Employment. Harman:Janssen Pharmaceuticals R&D: Employment. Millar:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Teplyakov:Janssen Pharmaceuticals R&D: Employment. Wheeler:Janssen Pharmaceuticals R&D: Employment. Luo:Janssen Pharmaceuticals R&D: Employment. Tam:Janssen Pharmaceuticals R&D: Employment, Other: stocks, Research Funding. Wu:Janssen Pharmaceuticals R&D: Employment. Chen:Janssen Pharmaceuticals R&D: Employment. Rudnick:Janssen Pharmaceuticals R&D: Employment. Chu:Janssen Pharmaceuticals R&D: Employment. Hughes:Janssen Pharmaceuticals R&D: Employment. Luistro:Janssen: Employment. Chin:Janssen: Employment. Babich:Janssen: Employment. Kalota:Janssen Pharmaceuticals R&D: Employment, Other: stock. Singh:Janssen Pharmaceuticals R&D: Employment, Other: stock options. Salvati:Janssen Pharmaceuticals R&D: Employment, Other: stock options, Patents & Royalties: patent. Elsayed:Janssen: Employment, Other: stock options. Attar:Janssen: Employment.

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