OS12.6.A Combination therapy of CAR-NK-cells and anti-PD-1 results in high efficacy against advanced-stage glioblastoma in a syngeneic mouse model and induces protective anti-tumor immunity in vivo

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii15-ii15
Author(s):  
F Strassheimer ◽  
M I Strecker ◽  
T Alekseeva ◽  
J Macas ◽  
M C Demes ◽  
...  
Keyword(s):  
T Cells ◽  
Combination Therapy ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Combined Treatment ◽  
Checkpoint Blockade ◽  
Advanced Stage ◽  
Lymphocyte Infiltration ◽  
Term Survival

Abstract INTRODUCTION Checkpoint inhibitors as well as adoptive cell therapy hold promise for cancer therapy and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB microenvironment (TME) has shown prominent immunosuppressive features, including expression of PD-L1 on tumor cells and increased frequency of FOXP3-positive regulatory T cells. While the surrounding brain is HER2-negative, GB are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy. Previous results from mouse glioma models showed efficacy of CAR-NK cells (NK-92/5.28.z) targeted against HER2 as monotherapy with early stage but not with advanced-stage tumors. MATERIALS AND METHODS The murine glioma cell line GL261 was transfected with human HER2. Tumor cells were implanted either subcutaneously or orthotopically into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined. Lymphocyte infiltration and immunosuppressive TME were characterized via highplex multi-color flow cytometry (FACS Symphony) and IHC (Phenoptics). Furthermore, gene expression profiles of tumor-infiltrating cells were determined via bulk RNAseq (NanoString). RESULTS Combined treatment with NK-92/5.28.z cells and anti-PD-1 checkpoint blockade resulted in synergistic effects, with tumor regression and long-term survival observed even in advanced-stage tumor bearing mice. Analysis of the TME showed changes in lymphocyte infiltration and increased expression of exhaustion markers in tumor and immune upon combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody resulting in an altered TME. Both, PD-1 and Lag-3 expression are highly upregulated on tumor infiltrating T cells. Total infiltrating lymphocytes show a rather cytotoxic phenotype up combination treatment with NK-92/5.28.z cells and anti-PD-1 antibody CONCLUSION Our data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced by combination with checkpoint blockade, resulting in successful treatment of advanced tumors refractory to NK-92/5.28.z monotherapy. Furthermore, the combination therapy induced a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To translate the concept of CAR-NK-cell therapy plus checkpoint inhibition to a clinical setting, we are adding a combination therapy cohort to our ongoing phase I clinical study (CAR2BRAIN; NCT03383978).

scholarly journals P06.12 Combination therapy of CAR-NK-cells and anti-PD-1 antibody results in high efficacy against advanced-stage glioblastoma in a syngeneic mouse model and induces protective anti-tumor immunity in vivo

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A46.2-A47
Author(s):  
F Strassheimer ◽  
MI Strecker ◽  
T Alekseeva ◽  
J Macas ◽  
MC Demes ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Combined Treatment ◽  
Checkpoint Blockade ◽  
Great Promise ◽  
Advanced Stage ◽  
Lymphocyte Infiltration ◽  
Term Survival ◽  
Syngeneic Mouse Model

BackgroundCheckpoint inhibitors as well as adoptive cell therapy hold great promise for cancer therapy and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB tumor microenvironment (TME) has shown prominent immunosuppressive features including expression of PD-L1 on tumor cells and increased frequency of FOX-P3 positive regulatory T cells. While the surrounding brain is HER2-negative, GB tumors are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy. Previous results from mouse glioma models showed efficacy of CAR-NK cells (NK-92/5.28.z) targeted against HER2 as monotherapy with relatively small tumors, but not with advanced late-stage tumors.Materials and MethodsThe murine glioma cell line GL261 was transfected with HER2. Tumor cells were implanted either subcutaneously or orthotopically into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined. Lymphocyte infiltration and immunosuppressive TME were characterized in high-dimensional high-throughput analysis via RNAseq and multiplex IHC.ResultsCombined treatment with NK-92/5.28.z cells and anti-PD-1 checkpoint blockade resulted in synergistic effects with tumor regression and long-term survival even of advanced-stage tumor bearing mice. Analysis of TME showed enhanced cytotoxic lymphocyte infiltration and altered profiles of exhaustion markers in tumor and immune cells, leading to an altered TME after combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody.ConclusionsThese data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced in combination with checkpoint blockade, resulting in successful treatment of advanced tumors refractory to NK-92/5.28.z monotherapy. Furthermore, the combination therapy induces a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To address this question in a clinical setting, we are preparing a combination therapy cohort as part of our ongoing phase I clinical study (CAR2BRAIN; NCT03383978).Disclosure InformationF. Strassheimer: None. M.I. Strecker: None. T. Alekseeva: None. J. Macas: None. M.C. Demes: None. I.C. Mildenberger: None. T. Tonn: None. P.J. Wild: None. L. Sevenich: None. Y. Reiss: None. P.N. Harter: None. K.H. Plate: None. W.S. Wels: None. J.P. Steinbach: None. M.C. Burger: None.

scholarly journals P12.04 Synergistic effects of combination therapy of CAR-NK cells and anti-PD-1 antibody result in high efficacy against advanced stage orthotopic glioblastoma grafts in a syngeneic mouse model and induce protective anti-tumor immunity in vivo

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii60-iii60
Author(s):  
F Strassheimer ◽  
M I Strecker ◽  
C Zhang ◽  
I C Mildenberger ◽  
P N Harter ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Tumor Cells ◽  
Checkpoint Inhibitors ◽  
Adoptive Cell Therapy ◽  
Combined Treatment ◽  
Great Promise ◽  
Advanced Stage ◽  
Lymphocyte Infiltration ◽  
Term Survival ◽  
Syngeneic Mouse Model

Abstract BACKGROUND Checkpoint inhibitors as well as adoptive cell therapy hold great promise for cancer treatment and encouraging treatment responses have already been demonstrated in different cancer indications. Glioblastoma (GB) is the most common and aggressive primary brain tumor. Standard therapy has very limited efficacy in the majority of patients. Analysis of the GB tumor microenvironment (TME) has shown prominent immunosuppressive features including expression of PD-L1 on tumor cells and increased frequency of FOX-P3 positive regulatory T cells. While the surrounding brain is HER2-negative, GB tumors are frequently HER2-positive, suggesting HER2 as a promising target for adoptive immunotherapy. MATERIALS AND METHODS The murine glioma cell line GL261 was transfected with HER2. Tumor cells were orthotopically implanted into C57BL/6 mice and treated either with HER2-specific NK-92/5.28.z cells alone or in combination with an anti-PD-1 antibody. Effects on tumor growth and survival were determined, lymphocyte infiltration and immunosuppressive TME were characterized. RESULTS Combined treatment with NK-92/5.28.z cells and anti-PD-1 antibody resulted in synergistic tumor regression and long-term survival of advanced-stage tumor bearing mice. Analysis of TME showed enhanced cytotoxic lymphocyte infiltration and altered profiles of exhaustion markers in tumor and immune cells. CONCLUSION These data demonstrate that efficacy of NK-92/5.28.z cells can be enhanced by co-therapy with checkpoint inhibitors, resulting in successful treatment of advanced tumors refractory to mono-therapy. Furthermore, this combination therapy induces a cytotoxic rather than immunosuppressive TME, leading to a primed immune system. To address this question in a clinical setting, we are planning a phase I clinical study (CAR2BRAIN-CHECK).

scholarly journals NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 590-590 ◽  
Author(s):  
Alejandra Leivas ◽  
Paula Rio ◽  
Rebeca Mateos ◽  
Mari Liz Paciello ◽  
Almudena Garcia-Ortiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Car T ◽  
Equity Ownership

Abstract Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

scholarly journals IMMU-19. TARGETING GLIOBLASTOMA IMMUNOSUPPRESSION AND TREATMENT RESISTANCE WITH IBRUTINIB IN COMBINATION WITH STEREOTACTIC RADIATION AND IMMUNE CHECKPOINT BLOCKADE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii108-ii108
Author(s):  
Jayeeta Ghose ◽  
Baisakhi Raychaudhuri ◽  
Kevin Liu ◽  
William Jiang ◽  
Pooja Gulati ◽  
...  
Keyword(s):  
T Cells ◽  
Combination Therapy ◽  
Median Survival ◽  
Immune Checkpoint ◽  
Survival Benefit ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Control Group ◽  
Triple Combination ◽  
Triple Combination Therapy

Abstract BACKGROUND Glioblastoma (GBM) is associated with systemic and intratumoral immunosuppression. Part of this immunosuppression is mediated by myeloid derived suppressor cells (MDSCs). Preclinical evidence shows that ibrutinib, a tyrosine kinase inhibitor FDA approved for use in chronic lymphocytic leukemia and known to be CNS penetrant, can decrease MDSC generation and function. Also, focal radiation therapy (RT) synergizes with anti-PD-1 therapy in mouse GBM models. Thus, we aimed to test the combination of these approaches on immune activation and survival in a preclinical immune-intact GBM mouse model. METHODS C57BL/6 mice intracranially implanted with the murine glioma cell line GL261-Luc2 were divided into 8 groups consisting of treatments with ibrutinib, RT (10 Gy SRS), or anti-PD-1 individually or in each combination (along with a no treatment control group). Immune cell subset changes (flow-cytometry) and animal survival (Kaplan-Meier) were assessed (n=10 mice per group). RESULTS Median survival of the following groups including control (28 days), ibrutinib (27 days), RT (30 days) or anti-PD-1 (32 days) showed no significant differences. However, a significant improvement in median survival was seen in mice given combinations of ibrutinib+RT (35 days), ibrutinib+anti-PD-1 (38 days), and triple therapy with ibrutinib+RT+anti-PD-1 (48 days, p < 0.05) compared to controls or single treatment groups. The reproducible survival benefit of triple combination therapy was abrogated in the setting of CD4+ and CD8+ T cell depletion. Contralateral intracranial tumor re-challenge in long-term surviving mice suggested generation of tumor-specific immune memory responses. The immune profile of the tumor microenvironment (TME) showed increased cytotoxic CD8+ T cells and decreased MDSCs and regulatory T cells in the triple combination therapy mice compared to controls. CONCLUSION The combination of ibrutinib, focal RT, and anti-PD-1 immune checkpoint blockade led to a significant survival benefit compared to controls in a preclinical model of GBM.

scholarly journals Pharmacologic marrow purging in murine T cell leukemia

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1656-1661 ◽  
Author(s):  
EA Copelan ◽  
SC Johnson ◽  
MR Grever ◽  
JF Sheridan ◽  
PJ Tutschka
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
In Vitro Assays ◽  
Cell Leukemia ◽  
Term Survival ◽  
Adenosine Deaminase Activity ◽  
Marrow Purging ◽  
Malignant T Cells

Abstract Deoxycoformycin in combination with deoxyadenosine was used to purge 6C3HED malignant T cells from murine marrow in vitro. Adenosine deaminase activity of 6C3HED cells was ablated by incubation with 10(- 6) mol/L deoxycoformycin (dCF). During a 12-hour incubation with 10(-6) mol/L dCF and 10(-4) mol/L deoxyadenosine, tumor cells sequentially accumulated dATP, became depleted of NAD followed by ATP, then died. More than 5 logs of 6C3HED cells were killed as measured by survival of mice injected with treated tumor cells. Identical incubation of 5 x 10(6) marrow cells did not interfere with rescue of syngeneic lethally irradiated mice. Long-term survival was demonstrated in 12 of 14 mice that received marrow that had been contaminated with 5% 6C3HED cells, incubated with deoxycoformycin and deoxyadenosine, then used to rescue lethally irradiated mice. This murine model provides information not available from in vitro assays and may be useful in the development of strategies to purge malignant T cells from marrow.

scholarly journals 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

scholarly journals 151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A159-A159
Author(s):  
Michael Whang ◽  
Ming-Hong Xie ◽  
Kate Jamboretz ◽  
Hadia Lemar ◽  
Chao Guo ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Adoptive Cell Therapy ◽  
Therapeutic Window ◽  
Target Cells ◽  
Healthy Donors ◽  
Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

scholarly journals Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Jun Yang ◽  
Xin Liu ◽  
Susan B. Nyland ◽  
Ranran Zhang ◽  
Lindsay K. Ryland ◽  
...  
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Nk Cells ◽  
Nk Cell ◽  
Cytotoxic T Cells ◽  
Platelet Derived Growth Factor ◽  
Leukemic Cells ◽  
Immunocytochemical Staining ◽  
Term Survival ◽  
Lgl Leukemia

Abstract Large granular lymphocyte (LGL) leukemia results from chronic expansion of cytotoxic T cells or natural killer (NK) cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified platelet-derived growth factor (PDGF) as a key master switch in controlling these survival pathways in T-cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGLs of both T- and NK-cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGLs was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-β transcripts than purified normal CD8+ T cells or NK cells. We observed that phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream protein kinase B (PKB)/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGLs. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long-term survival of leukemic LGLs, may be an important therapeutic strategy.

Determination of Soluble MICA in Serum as a Novel Marker for Tumor Stage and Metastasis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1344-1344
Author(s):  
Helmut R. Salih ◽  
Petra Stieber ◽  
Andrea Peterfi ◽  
Dorothea Nagel ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
T Cells ◽  
Differential Diagnosis ◽  
Nk Cells ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Control Group ◽  
Healthy Individuals ◽  
Benign Disorders

Abstract The human NKG2D ligands (NKG2DL) MICA and MICB have been shown to be expressed on tumors of epithelial and hematopoietic origin in vivo. Recently we reported that MICA is shed from the cell surface of tumor cells and is present in sera of tumor patients (J Immunol169:4098 (2002), Blood102:1389 (2003)). Since the strength of an anti-tumor response by NK cells and CD8 T cells is critically depending on NKG2DL expression levels, down-regulation of MICA-expression on tumor cells represents an immune escape mechanism that diminishes anti-tumor reactivity of NKG2D-bearing lymphocytes. However, no data are yet available regarding the correlation of soluble MICA (sMICA) levels with specific tumor entities, aggressiveness of the disease, and hence the potential implementation of sMICA as novel marker in differential diagnosis and prognosis of cancer. In this study, we determined sMICA levels in sera of 512 individuals including 296 patients with various cancers, 154 patients with benign disorders and 62 healthy individuals. Healthy individuals revealed significantly lower sMICA values (median<30pg/mL) than patients with benign diseases (84pg/mL; p=0.005) and cancer patients (161pg/mL; p<0.0001). In addition, sMICA levels differed significantly between cancer patients and patients with benign disorders (p<0.0001) that represent the most relevant control group for differential diagnosis. In cancer patients, while there was no association between sMICA levels and tumor size (p=0.456), cell differentiation (p=0.271), or lymph node involvement (p=0.674), sMICA correlated significantly with cancer stage and metastasis (p=0.015 and p=0.007, respectively). Our data indicate that release of MICA might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. Thus, determination of sMICA levels provides valuable information for cancer staging, and sMICA in serum seems to be an indicator for systemic manifestation of malignancy rather than for local tumor extent.

In Vitro and In Vivo Effects of CT-011, a Humanized Anti–PD-1 Monoclonal Antibody, in Combination with Rituximab against Human B-Cell Lymphomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 724-724
Author(s):  
Fuliang Chu ◽  
Myriam Foglietta ◽  
Hong Qin ◽  
Rakesh Sharma ◽  
Qing Yi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Tumor Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
In Vivo Effects ◽  
Human B Cell

Abstract Abstract 724 Background: Programmed death (PD)–1 is an inhibitory receptor that impairs the function of activated T-cells and natural killer (NK) cells when engaged by its ligands PD-L1 or PD-L2. We have previously demonstrated that PD-1 is markedly up-regulated in intratumoral and peripheral blood CD4+ and CD8+ T cells in patients with follicular lymphoma (FL), a finding associated with impaired T-cell function, suggesting that PD-1 blockade may improve FL immune control. CT-011, a humanized anti PD-1 monoclonal antibody, was previously studied in a phase I clinical trial in patients with advanced hematological malignancies. CT-011 was well tolerated and induced sustained elevations of CD4+ T cells in the peripheral blood. More importantly, apparent clinical benefit was observed in six patients, including one patient with FL who had large tumor masses that achieved a durable complete remission lasting >14 months. Here, we studied the in vitro and in vivo effects of CT-011 on T-cell and/or NK-cell immune responses against human B-cell lymphoma and the hypothesis that CT-011 may improve tumor control when combined with rituximab, a chimeric anti-CD20 monoclonal antibody for the treatment of human FL. Materials and Methods: To determine the effects of CT-011 on antitumor T cells, intratumoral T cells were isolated from primary FL tumor samples, and cultured with or without autologous tumor cells in the presence or absence of CT-011 or isotype control antibody (50 μg/ml each) for 5 days, and tested for proliferation by 3H thymidine incorporation assay. To determine the effects of CT-011 on NK cells, peripheral blood mononuclear cells (PBMCs) derived from normal donors or patients with FL were cultured in the presence or absence of CT-011 (50 μg/ml) with or without IL-2 for 96 hours and analyzed for expression of various activating receptors including CD16, CD32, CD64, Fas ligand, NKG2D, NKp30, NKp44, and NKp46. The in vivo effects of CT-011 were tested in two B-cell lymphoma xenograft models. Ramos and RL lymphoma tumor cells were injected subcutaneously into nude and SCID mice, respectively, and CT-011 (10 μg/mouse) was injected weekly with or without rituximab starting approximately 7–10 days after tumor inoculation. Results: We observed that CT-011 significantly increased the proliferation of intratumoral T cells in response to autologous tumor cells compared with isotype control antibody. Treatment with CT-011 enhanced the expression of Fas ligand, CD32, CD64, and NKp30 on human NK cells in the presence of IL-2 as compared with PBMCs treated with IL-2 alone or media control. In the RL lymphoma xenograft model in SCID mice, treatment with CT-011 significantly delayed tumor growth (P≤0.05) and improved survival (P≤0.01) compared with control mice injected with saline. In a Ramos lymphoma xenograft model in nude mice, treatment with CT-011 and rituximab eradicated established tumors in a significant proportion of mice (P≤0.05) and markedly improved survival compared with rituximab alone or saline. Conclusions: Taken together, these studies suggest that blockade of PD-1 with CT-011 enhances the function of anti-tumor T-cells and augments the expression of activating receptors on NK cells. Treatment with CT-011 led to improved tumor control against human B-cell lymphoma in xenograft models and the combined use of CT-011 and rituximab was more effective that rituximab alone. These results provide the rationale to test the combination of CT-011 with rituximab in patients with B-cell lymphoma, given that the combination is likely to be complementary and may even be synergistic, leading to enhanced clinical efficacy without increasing toxicity. The development of such approaches that activate both the innate (NK-cells) and adaptive (T-cells) immune systems is likely to minimize the emergence of immune escape variants and improve clinical outcome in patients with lymphoma. A clinical trial evaluating CT-011 in combination with rituximab is planned in patients with relapsed FL. Disclosures: Rodionov: Cure Tech Ltd.: Employment. Rotem-Yehudar:Cure Tech Ltd.: Employment.

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