Vaccination with leukemia cells expressing cell-surface-associated GM-CSF blocks leukemia induction in immunocompetent mice

Abstract The fundamental basis for immunotherapy of leukemia is that leukemia cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant of leukemia cells. To overcome this immune tolerance, we transduced WEHI-3B mouse monocyte leukemia cells (1) with a transmembrane form of GM-CSF (tmGM-CSF). The tmGM-CSF was constructed using the pDisplay vector for cell-surface targeting (Invitrogen) into the pLOX lentivirus gene transfer vector (2). After infection of WEHI-3B cells with a recombinant lentivirus encoding tmGM-CSF, nearly all the transduced cells expressed tmGM-CSF on the cell-surface, as determined by flow cytometry analysis using anti-GM-CSF. To determine whether vaccination with tmGM-CSF expressing WEHI-3B cells would prevent leukemia formation, immunocompetent BALB/c mice were immunized with lethally-irradiated WEHI-3B cells (106, 3 times 7 day intervals), which express tmGM-CSF, prior to challenging vaccinated mice with WEHI-3B cells (5x104) that express GFP as a marker. 100% of vaccinated mice were protected from leukemia. Non-vaccinated mice succumbed to leukemia within 50–55 days. Vaccination of mice with lethally-irradiated WEHI-3B cells expressing CD40L protected 80% of the mice from leukemia. In contrast, mice immunized with lethally-irradiated WEHI-3B/GFP cells lacking tmGM-CSF were not protected. Mice vaccinated three times at 5,12, 19 days after challenge with WEHI-3B/GFP cells had a significant increase in survival in that 60% of mice were alive and healthy at 16 days (to this date) after all control non-vaccinated mice had died. Similar vaccine studies were performed with BCR-ABL (b3a2)+ 32D cells (106) in immunocompetent C3H/HeJ mice (3). These mice die of leukemia within 35 days. After infection of BCR-ABL+ 32D cells with the lentivirus encoding tmGM-CSF/GFP, tmGM-CSF was expressed on the cell-surface. The C3H/HeJ mice challenged with BCR-ABL+32D/GFP cells (106) showed a significant level of protection by vaccination with lethally-irradiated tmGM-CSF+ 32D BCR-ABL cells (106, 2 times at 7 day intervals); 40% of the vaccinated mice remained healthy; all non-vaccinated mice died of leukemia. There was a significant difference in survival (P=0.03) between the vaccinated and non-vaccinated groups. Interestingly, the spleens of vaccinated C3H/HeJ mice that died of leukemia at the same time as non-vaccinated mice approached normal size whereas non-vaccinated mice had enlarged spleens. Our findings suggest that over-expression of cell-surface tmGM-CSF in leukemia cells can overcome immune tolerance, allowing the immune system to efficiently recognize and destroy the leukemia cells, providing extended survival of vaccinated mice. Because significant protection from death was achieved by vaccination after challenge with leukemia cells, tmGM-CSF expression in leukemia cells has potential as a therapeutic strategy for treatment of leukemia.

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Vaccination with Leukemia Cell Expression Membrane Bound GM-CSF Overcomes the Host Immunosuppression Induced by Leukemia

Blood ◽

10.1182/blood.v112.11.5435.5435 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5435-5435

Author(s):

Di Ling ◽

Ralph B. Arlinghaus ◽

Xiaoyang Ling

Keyword(s):

Cell Surface ◽

Treg Cells ◽

T Cell Response ◽

Leukemia Cells ◽

Cell Mediated Immunity ◽

Immune Recognition ◽

Gm Csf ◽

Treg Population ◽

Membrane Bound ◽

Murine Leukemia

Abstract Immunotherapy of leukemia involves stimulating host-cell mediated immunity by facilitating immune recognition of leukemia cells, which are normally weakly immunogenic. We previously showed that vaccination with membrane bound GM-CSF leukemic cells protects mice from leukemia challenge (Ling et al., Oncogene, 2007). In these studies, after addition of a transmembrane domain to the original GM-CSF coding sequence (tmGM-CSF), the construct was transduced into murine leukemia cells (WEHI-3B), which was shown to be more than 98% on the cell surface. Vaccination with lethally irradiated tmGM-CSF expressing murine leukemia cells prevents leukemia in immunocompetent mice (BALB/c), as 100% of vaccinated BALB/c mice were protected from leukemia (Ling et al, Oncogene 2007). No protection was observed by vaccination of nude mice, indicating that adaptive immunity is involved in the protective response. In the present studies, we extended our original observation and provided evidence to show that leukemic mice undergo immunosuppression and that vaccination with leukemia cells expressing cell surface tmGM-CSF overcomes immunosuppression. Vaccination with lethally irradiated leukemia cells expressing cell surface tmGM-CSF overcame the immunosuppression induced by leukemia development, as normal levels of CD4+/CD25+/Foxp3+ T-regulatory (Treg) cells were maintained in spleens and thymus after challenge with leukemia cells. In contrast, the Treg population was significantly increased in leukemic mice vaccinated with leukemia cells lacking cell surface tmGM-CSF (p<0.001) after leukemia challenge, and these mice had a lower CD8+/Treg cell ratio (p<0.01). The ratio of CD8+/Treg cells was higher in tmGM-CSF/GFP vaccinated mice than in GFP vaccinated mice (p<0.001), which in-turn leads to a more effective CD8+ T-cell response. DC levels were also increased from normal levels in mice vaccinated with tmGM-CSF+ leukemia cells compared to control vaccinations. These results suggest that vaccination with leukemia cells expressing GM-CSF on their cell surface leads to an effective cell-mediated immune response in the vaccinated host by overcoming an impaired host cellular immunity induced up-regulation of Treg cells caused by the leukemia process. This strategy has potential for use in the treatment of various human leukemias.

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Ferritin-Conjugated Antibody for the Demonstration of Isoantigens on the Surface of Murine Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099982 ◽

1968 ◽

Vol 26 ◽

pp. 48-49

Author(s):

T. Aoki ◽

J. Izard ◽

U. Hämmerling ◽

E. de Harven ◽

L. J. Old

Keyword(s):

Cell Surface ◽

Gamma Globulin ◽

Leukemia Cells ◽

Labelling Technique ◽

Direct Labelling ◽

Labelling Method

Although a variety of viral and cellular antigens have been demonstrated by ferritin-labeled antibody, this technique has not been used to locate isoantigens on the surface of nucleated cells. The recognition of several systems of isoantigens on the surface of thymocytes, lymphocytes and leukemia cells of the mouse and the ease with which these cells can be obtained in free suspension led us to consider the ferritin-labelling method to determine the amount and location of these isoantigens on the cell surface. Because of the problems involved in the direct labelling of mouse gamma globulin by ferritin, we have chosen an indirect labelling technique (i.e. ferritin-conjugated rabbit anti mouse γG)to detect localization of mouse isoantibody.

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The distribution of antigen on the surface of RBL-2H3 rat basophilic leukemia cells observed by back-scattered electron imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142992 ◽

1986 ◽

Vol 44 ◽

pp. 274-275

Author(s):

R.F. Stump ◽

J.R. Pfeiffer ◽

JC. Seagrave ◽

D. Huskisson ◽

J.M. Oliver

Keyword(s):

Cell Surface ◽

Dynamic Properties ◽

Leukemia Cells ◽

Antigen Binding ◽

Scattered Electron ◽

Ige Receptor ◽

Receptor Complexes ◽

Rat Basophilic Leukemia Cells ◽

Electron Imaging ◽

Basophilic Leukemia

In RBL-2H3 rat basophilic leukemia cells, antigen binding to cell surface IgE-receptor complexes stimulates the release of inflammatory mediators and initiates a series of membrane and cytoskeletal events including a transformation of the cell surface from a microvillous to a lamellar topography. It is likely that dynamic properties of the IgE receptor contribute to the activation of these responses. Fewtrell and Metzger have established that limited crosslinking of IgE-receptor complexes is essential to trigger secretion. In addition, Baird and colleagues have reported that antigen binding causes a rapid immobilization of IgE-receptor complexes, and we have demonstrated an apparent increase with time in the affinity of IgE-receptor complexes for antigen.

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Immunotherapy of Mouse Leukemia with Monoclonal Antibodies Directed against Type-C Virus Structural Proteins

Tumori Journal ◽

10.1177/030089168407000102 ◽

1984 ◽

Vol 70 (1) ◽

pp. 9-16

Author(s):

Mauro Boiocchi ◽

Piera Mondellini

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Cell Surface ◽

Therapeutic Effect ◽

Structural Proteins ◽

Leukemia Cells ◽

Envelope Glycoproteins ◽

Mouse Leukemia ◽

Type C

The monoclonal antibody A6, isolated during a study on the natural immunoresponse of BALB/c mice against leukemia cells (4), reacts with the envelope glycoproteins gp70 of the MuLV and with the cell surface of the SL2 AKR leukemia. In the present paper, we describe the in vivo immunotherapeutic effect exerted by the A6 monoclonal antibody on the growth of the transplanted leukemia SL2. The greater therapeutic effect observed when the A6 was used with exogenous complement cooperation suggests that the immunotherapeutic activity is mediated by C'-dependent cytotoxicity.

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A radioresistant fraction of acute promyelocytic leukemia cells exhibit CD38 cell-surface antigen and mRNA expression

Oncology Letters ◽

10.3892/ol.2018.8099 ◽

2018 ◽

Author(s):

Satoru Monzen ◽

Mitsuru Chiba ◽

Tatsuya Ueno ◽

Yuki Morino ◽

Kenji Terada ◽

...

Keyword(s):

Cell Surface ◽

Mrna Expression ◽

Acute Promyelocytic Leukemia ◽

Surface Antigen ◽

Promyelocytic Leukemia ◽

Cell Surface Antigen ◽

Leukemia Cells

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EWI-2 modulates lymphocyte integrin α4β1 functions

Blood ◽

10.1182/blood-2003-07-2201 ◽

2004 ◽

Vol 103 (8) ◽

pp. 3013-3019 ◽

Cited By ~ 39

Author(s):

Tatiana V. Kolesnikova ◽

Christopher S. Stipp ◽

Ravi M. Rao ◽

William S. Lane ◽

Francis W. Luscinskas ◽

...

Keyword(s):

Cell Surface ◽

Cell Adhesion Molecule ◽

Apparent Size ◽

Surface Proteins ◽

Size Exclusion ◽

Leukemia Cells ◽

Wild Type ◽

Vascular Cell Adhesion ◽

Exclusion Chromatography ◽

Cell Adhesion Molecule 1

Abstract The most prominent cell-surface integrin α4β1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti–α4β1 integrin antibody-coated beads. By mass spectrometry, this protein was identified as EWI-2, a previously described cell-surface partner for tetraspanin proteins CD9 and CD81. Wild-type EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the α4β1 ligand, vascular cell adhesion molecule-1 (VCAM-1), in shear flow assays. However, EWI-2 markedly impaired spreading and ruffling on VCAM-1. In contrast, a mutant EWI-2 molecule, with a different cytoplasmic tail, neither impaired cell spreading nor associated with α4β1 and CD81. The endogenous wild-type EWI-2–CD81–α4β1 complex was fully soluble, and highly specific as seen by the absence of other MOLT-4 cell-surface proteins. Also, it was relatively small in size (0.5 × 106 Da to 4 × 106 Da), as estimated by size exclusion chromatography. Overexpression of EWI-2 in MOLT-4 cells caused reorganization of cell-surface CD81, increased the extent of CD81-CD81, CD81-α4β1, and α4β1-α4β1 associations, and increased the apparent size of CD81-α4β1 complexes. We suggest that EWI-2–dependent reorganization of α4β1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions. (Blood. 2004;103: 3013-3019)

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Granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) enhances high-dose Ara-C-induced programmed cell death or apoptosis in human myeloid leukemia cells

Blood ◽

10.1182/blood.v80.11.2883.2883 ◽

1992 ◽

Vol 80 (11) ◽

pp. 2883-2890 ◽

Cited By ~ 37

Author(s):

K Bhalla ◽

C Tang ◽

AM Ibrado ◽

S Grant ◽

E Tourkina ◽

...

Keyword(s):

Cell Death ◽

Myeloid Leukemia ◽

Colony Growth ◽

Leukemia Cells ◽

High Dose ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM- CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.

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Treatment of newly diagnosed acute myelogenous leukemia with granulocyte-macrophage colony-stimulating factor (GM-CSF) before and during continuous-infusion high-dose ara-C + daunorubicin: comparison to patients treated without GM-CSF

Blood ◽

10.1182/blood.v79.9.2246.2246 ◽

1992 ◽

Vol 79 (9) ◽

pp. 2246-2255 ◽

Cited By ~ 113

Author(s):

E Estey ◽

PF Thall ◽

H Kantarjian ◽

S O'Brien ◽

CA Koller ◽

...

Keyword(s):

Myeloid Leukemia ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Newly Diagnosed ◽

Gm Csf ◽

Negative Effect ◽

Acute Myelogenous ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

Abstract We gave 56 patients with newly diagnosed acute myelogenous leukemia (AML) granulocyte-macrophage colony-stimulating factor (GM-CSF) 20 or 125 micrograms/m2 once daily subcutaneously before (for up to 8 days or until GM-CSF-related complications developed) and during, or only during (patients presenting with blast counts greater than 50,000 or other leukemia-related complications) ara-C (1.5 g/m2 daily x 4 by continuous infusion) and daunorubicin (45 mg/m2 daily x 3) chemotherapy. Because results seemed independent of GM-CSF schedule, we compared results in these 56 patients with results in 176 patients with newly diagnosed AML given the same dose and schedule of ara-C without GM-CSF (110 patients ara-C alone, 66 patients ara-C + amsacrine or mitoxantrone). Comparison involved fitting a logistic regression model predicting probability of complete remission (CR) and a Cox regression model to predict survival (most patients in all three studies were dead) with treatment included as a covariate in both analyses. After adjusting for other prognostically significant covariates [presence of an antecedent hematologic disorder, an Inv (16), t(8;21), or abnormalities of chromosomes 5 and/or 7, performance status, age, bilirubin], treatment with ara-C + daunorubicin + GM-CSF was predictive of both a lower CR rate and a lower survival probability. There were no treatment-covariate interactions, suggesting that the negative effect of this GM-CSF treatment regime was not an artifact of some imbalance in patient characteristics. The unadjusted Kaplan-Meier hazard rate of the ara-C + daunorubicin + GM-CSF group was not uniquely high during the initial 4 weeks after start of therapy, but was highest among the three treatment groups throughout weeks 5 to 16, suggesting that the negative effect of this treatment was not caused by acute toxicity. Patients who did not enter CR with this treatment tended to have persistent leukemia rather than prolonged marrow aplasia, suggesting that this treatment and, in particular, GM-CSF may increase resistance of myeloid leukemia cells to chemotherapy. To date, relapse rates are similar in all three groups (P = .43) (as are survival rates once patients are in CR) but much of the remission duration data is heavily censored, unlike the survival data. Our results suggest caution in the use of GM-CSF to sensitize myeloid leukemia cells to daunorubicin + ara- C chemotherapy.

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Posttransplant Immunotherapy with a GM-CSF-Based Tumor Vaccine (GVAX®) Following Autologous Stem Cell Transplant (ASCT) for Acute Myeloid Leukemia (AML).

Blood ◽

10.1182/blood.v104.11.441.441 ◽

2004 ◽

Vol 104 (11) ◽

pp. 441-441

Author(s):

Daniel J. DeAngelo ◽

Edwin P. Alyea ◽

Ivan M. Borrello ◽

Hyam I. Levitsky ◽

Wendy Stock ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Leukemia Cell ◽

Delayed Type Hypersensitivity ◽

Leukemia Cells ◽

Autologous Tumor ◽

Cell Transplant ◽

Vaccine Platform ◽

Blood Draw ◽

Gm Csf

Abstract Background: Preclinical models have demonstrated the efficacy of GM-CSF secreting cancer vaccines accompanied by vaccine-primed lymphocyte infusion following ASCT. Methods: Patients ≤ 60 years old with de novo AML (excluding M3) were enrolled. Leukemia cells were harvested at diagnosis followed by induction and consolidation chemotherapy and ASCT. A pretransplant vaccine composed of irradiated autologous leukemia cells mixed with GM-CSF gene-modified K562 cells (CG9962) was given followed by collection of vaccine-primed lymphocytes that were reinfused with the stem cell graft. Posttransplant vaccinations were initiated at week 6 (or upon platelet engraftment) and given every 3 weeks (10x107 tumor cells + 4x107 CG9962 cells) x 8 vaccinations. Results: Leukemia cell harvest was successful in 51/54 patients (blood draw − 22, leukapheresis − 28, bone marrow aspirate − 4). To date, 44/54 patients (81%) have achieved a complete remission (CR) following induction chemotherapy, 27 have received the pretransplant vaccination, and 15 have initiated posttransplant vaccinations. Local vaccine injection site reactions developed in all patients. Delayed-type hypersensitivity (DTH) reactions to irradiated, autologous tumor were induced post vaccination in 4/11. Antibodies reactive against autologous tumor were induced in 5/11 and against CG9962 cells in 10/10. Minimal residual disease (MRD) is being monitored by quantitative analysis of peripheral blood (PB) and bone marrow (BM) for WT1, a leukemia-associated gene, by RT-PCR. All patients had detectable WT1 transcripts in PB and BM at diagnosis. Most patients in CR had persistently detectable WT1 transcripts in BM following induction (92%), consolidation (91%), and ASCT (63%), whereas, clearance of WT-1 in PB was more frequent (49% detectable post induction, 50% post consolidation, and 31% post ASCT). A decrease in WT1 was noted in 67% (12/18 in the BM and 10/15 in PB) following the pretransplant vaccination. Conclusions: Collection of large quantities of autologous leukemia cells for vaccine production is feasible. Reduction in WT-1 transcript levels following the pre-transplant vaccine suggests anti-leukemic activity of this GVAX® vaccine platform.

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