Trident cold atmospheric plasma blocks three cancer survival pathways to overcome therapy resistance.

Therapy resistance is responsible for most cancer-related death and is mediated by the unique ability of cancer cells to leverage metabolic conditions, signaling molecules, redox status, and other pathways for their survival. Interestingly, many cancer survival pathways are susceptible to disturbances in cellular reactive oxygen species (ROS) and may therefore be disrupted by exogenous ROS. Here, we explore whether trident cold atmospheric plasma (Tri-CAP), a gas discharge with exceptionally low-level ROS, could inhibit multiple cancer survival pathways together in a murine cell line model of therapy-resistant chronic myeloid leukemia (CML). We show that Tri-CAP simultaneously disrupts three cancer survival pathways of redox deregulation, glycolysis, and proliferative AKT/mTOR/HIF-1α signaling in this cancer model. Significantly, Tri-CAP blockade induces a very high rate of apoptotic death in CML cell lines and in primary CD34+ hematopoietic stem and progenitor cells from CML patients, both harboring the therapy-resistant T315I mutation. In contrast, nonmalignant controls are minimally affected by Tri-CAP, suggesting it selectively targets resistant cancer cells. We further demonstrate that Tri-CAP elicits similar lethality in human melanoma, breast cancer, and CML cells with disparate, resistant mechanisms and that it both reduces tumor formation in two mouse models and improves survival of tumor-bearing mice. For use in patients, administration of Tri-CAP may be extracorporeal for hematopoietic stem cell transplantation therapy, transdermal, or through its activated solution for infusion therapy. Collectively, our results suggest that Tri-CAP represents a potent strategy for disrupting cancer survival pathways and overcoming therapy resistance in a variety of malignancies.

Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment.

The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

Recipient lymphocyte infusion in MHC-matched bone marrow chimeras induces a limited lymphohematopoietic host-versus-graft reactivity but a significant antileukemic effect mediated by CD8+ T cells and natural killer cells.

BACKGROUND: Challenge of MHC-mismatched murine bone marrow chimeras with recipient-type lymphocytes (recipient lymphocyte infusion) produces antileukemic responses in association with rejection of donor chimerism. In contrast, MHC-matched chimeras resist eradication of donor chimerism by recipient lymphocyte infusion. Here, we investigated lymphohematopoietic host-versus-graft reactivity and antileukemic responses in the MHC-matched setting, which is reminiscent of the majority of clinical transplants. DESIGN AND METHODS: We challenged C3H→AKR radiation chimeras with AKR-type splenocytes (i.e. recipient lymphocyte infusion) and BW5147.3 leukemia cells. We studied the kinetics of chimerism using flowcytometry and the mechanisms involved in antileukemic effects using in vivo antibody-mediated depletion of CD8(+) T and NK cells, and intracellular cytokine staining. RESULTS: Whereas control chimeras showed progressive evolution towards high-level donor T-cell chimerism, recipient lymphocyte infusion chimeras showed a limited reduction of donor chimerism with delayed onset and long-term preservation of lower-level mixed chimerism. Recipient lymphocyte infusion chimeras nevertheless showed a significant survival benefit after leukemia challenge. In vivo antibody-mediated depletion experiments showed that both CD8(+) T cells and NK cells contribute to the antileukemic effect. Consistent with a role for NK cells, the proportion of IFN-γ producing NK cells in recipient lymphocyte infusion chimeras was significantly higher than in control chimeras. CONCLUSIONS: In the MHC-matched setting, recipient lymphocyte infusion elicits lymphohematopoietic host-versus-graft reactivity that is limited but sufficient to provide an antileukemic effect, and this is dependent on CD8(+) T cells and NK cells. The data indicate that NK cells are activated as a bystander phenomenon during lymphohematopoietic T-cell alloreactivity and thus support a novel type of NK involvement in anti-tumor responses after post-transplant adoptive cell therapy.

Leukemias induced by altered TRK-signaling are sensitive to mTOR inhibitors in preclinical models.

Rapamycin is a potent allosteric mTORC1 inhibitor with clinical applications as an anticancer agent. However, only a fraction of cancer patients responds to the drug, and no biomarkers are available to predict tumor sensitivity. Recently, we and others have obtained evidence for potential involvement of tropomyosin-related kinase (TRK) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) in leukemia. In the present study, we tested the therapeutic effect of Rapamycin and its analog RAD001 on altered TRK-induced leukemia in a murine model. Daily treatment with Rapamycin (2 mg/kg) or RAD001 (1 mg/kg) significantly prolonged the survival of treated animals (n = 40) compared with the placebo group. Consistently, both mTOR and S6 proteins were strongly dephosphorylated in vitro and in vivo after treatment with Rapamycin or RAD001. However, Rapamycin did not completely inhibit mTORC1-dependent phosphorylation of 4E-BP1. With exception of one mouse showing slight reactivation of Akt after treatment, no reactivation of MAPK or Akt pathways was observed in other resistant tumors. Interestingly, leukemic cells isolated from a Rapamycin-resistant mouse were still highly sensitive to Rapamycin in vitro. Our findings suggest that altered TRK signaling may be a good predictor of tumor sensitivity to mTOR inhibition and that pathways other than MAPK and Akt exist that may trigger resistance of leukemic cells to Rapamycin in vivo.

(-)-Epigallocatechin-3-gallate induces apoptosis and differentiation in leukaemia by targeting reactive oxygen species and PIN1.

(-)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

Differential disease restriction of Moloney and Friend murine leukemia viruses by the mouse Rmcf gene is governed by the viral long terminal repeat.

Neonatal CxD2 (Rmcfr) and Balb/c (Rmcfs) mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibited approximately equivalent time course and pathology for disease. CxD2 mice showed only slightly reduced presence of Moloney mink cell focus-forming virus (M-MCF) provirus as seen by Southern blot analysis compared to Balb/c mice. This lack of restriction for disease and spread of MCF was in sharp contrast to that seen for CxD2 mice inoculated with Friend murine leukemia virus (F-MuLV), where incidence of disease and propagation of MCFs were severely restricted, as previously reported. Inoculation of CxD2 mice with FM-MuLV, a recombinant F-MuLV virus containing M-MuLV LTR sequences (U3 and R), resulted in T cell disease of time course equal to that seen in Balb/c mice; there also was little restriction for propagation of MCFs. This indicated that presence of the M-MuLV long terminal repeat (LTR) was sufficient for propagation of MCFs in CxD2 mice. Differing restriction for F-MuLV vs. M-MuLV in CxD2 mice was explained on the basis of different "MCF propagator cells" for the two viruses. It was suggested that cells propagating F-MCF (e.g., erythroid progenitors) are blocked by endogenous MCF-like gp70env protein, whereas cells propagating M-MCF (e.g., lymphoid) do not express this protein on their surface. F-MuLV disease in CxD2 mice was greatly accelerated when neonates were inoculated with a F-MuLV/F-MCF pseudotypic mixture. However, F-MCF provirus was not detectable or only barely detectable in F-MuLV/F-MCF-induced tumors, suggesting that F-MCF acted indirectly in induction of these tumors.

Augmentation of the anti-tumor therapeutic efficacy of long-term cultured T lymphocytes by in vivo administration of purified interleukin 2.

Spleen cells from C57BL/6 mice immunized in vivo with a syngeneic Friend virus-induced leukemia, FBL-3, were specifically activated by culture for 7 d with FBL-3, then nonspecifically induced to proliferate in vitro for 12 d by addition of supernatants from concanavalin A-stimulated lymphocytes containing interleukin 2 (IL-2). Such long-term cultured T lymphocytes have previously been shown to specifically lyse FBL-3 and to mediate specific adoptive therapy of advanced disseminated FBL-3 when used as an adjunct to cyclophosphamide (CY) in adoptive chemoimmunotherapy. Because the cultured cells are dependent upon IL-2 for proliferation and survival in vitro, their efficacy in vivo is potentially limited by the availability of endogenous IL-2. Thus, the aim of the current study was to determine whether exogenously administered purified IL-2 could augment the in vivo efficacy of long-term cultured T lymphocytes. Purified IL-2 alone or as an adjunct to CY as ineffective in tumor therapy. However, IL-2 was extremely effective in augmenting the efficacy of IL-2-dependent long-term cultured T lymphocytes in adoptive chemoimmunotherapy. The mechanism by which IL-2 functions in vivo is presumably by promoting in vivo growth and/or survival of adoptively transferred cells. This assumption was supported by the findings that IL-2 did not enhance the modest therapeutic efficacy of irradiated long-term cultured cells that were incapable of proliferating in the host and was ineffective in augmenting the in vivo efficacy of noncultured immune cells that are not immediately dependent upon exogenous IL-2 for survival.

Dietary effect of Antrodia Camphorate extracts on immune responses in WEHI-3 leukemia BALB/c mice.

Antrodia camphorata has been recognized to be a traditional Chinese medicine for abdominal pain, diarrhea, and to protect against hepatitis virus infection. Several ingredients derived from A. camphorata possess various pharmacological and biological activities such as antioxidant and anticancer. In this study, its ability to promote immune responses and to exhibited antileukemia activity in WEHI-3 leukemia BALB/c mice were investigated. The results indicated A. camphorata significantly prolonged the survival rate and prevented the body weight loss in leukemia mice. Four mg/kg of A. camphorata treatment significantly decreased the weight of the spleen. Both doses (2 and 4 mg/kg) of A. camphorata did not affect Mac-3 marker in leukocytes. However, the 4 mg/kg of A. camphorata decreased the levels of CD11b and both doses of treatment increased CD3 and CD19. With lipopolysaccharide stimulation, the 4 mg/kg of A. camphorata promoted the significant proliferation of leukocytes; but with concanavalin A stimulation, both doses promoted the significant proliferation of leukocytes. YAC-1 target cells were killed by NK cells from the mice after treatment with A. camphorata at 4 mg/kg in target cells at a ratio of 50:1. The percentage of macrophages with phagocyted at A. camphorata treatment increased, and these effects were in dose-dependent manners.

A high-throughput screen indicates gemcitabine and JAK inhibitors may be useful for treating pediatric AML.

Improvement in survival has been achieved for children and adolescents with AML but is largely attributed to enhanced supportive care as opposed to the development of better treatment regimens. High risk subtypes continue to have poor outcomes with event free survival rates <40% despite the use of high intensity chemotherapy in combination with hematopoietic stem cell transplant. Here we combine high-throughput screening, intracellular accumulation assays, and in vivo efficacy studies to identify therapeutic strategies for pediatric AML. We report therapeutics not currently used to treat AML, gemcitabine and cabazitaxel, have broad anti-leukemic activity across subtypes and are more effective relative to the AML standard of care, cytarabine, both in vitro and in vivo. JAK inhibitors are selective for acute megakaryoblastic leukemia and significantly prolong survival in multiple preclinical models. Our approach provides advances in the development of treatment strategies for pediatric AML.

Dual treatment with FLT3 inhibitor SU11657 and doxorubicin increases survival of leukemic mice.

FLT3 is mutated in roughly 30% of human AML. We used our model of APL with activated FLT3 to assess the effectiveness of chemotherapy in combination with SU11657, an inhibitor of FLT3. We found that median survival of untreated and doxorubicin-treated mice was not significantly different. While SU11657 alone increased median of survival to 55 days (P=0.01), dual therapy increased median survival to 62 days (P=0.003) when compared to controls. Neither agent alone or in combination increased survival of control mice. These results suggest that the use of targeted therapeutics can overcome resistance to traditional chemotherapies in AML.

Liposome-incorporated Grb2 antisense oligodeoxynucleotide increases the survival of mice bearing bcr-abl-positive leukemia xenografts.

We previously demonstrated that liposome-incorporated antisense oligodeoxynucleotide specific for the grb2 mRNA (L-Grb2) inhibited Grb2 protein expression and the proliferation of bcr-abl-positive leukemia cell lines. To determine whether L-Grb2 has the potential of being a therapeutic modality against bcr-abl-positive leukemia, we studied the tissue distribution of L-Grb2 in normal mice before studying its effects in mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 was widely distributed in the body. The highest tissue concentrations of L-Grb2 were found in the spleen and liver, which are the organs where the tumor mass of bcr-abl-positive leukemia is mainly found. At 4 h post-injection, the amount of L-Grb2 detected per g of tissue was 64 microg in spleen and 50 microg in liver. Intravenous injection of bcr-abl-positive 32D mouse leukemia cells into radiated NOD/scid mice caused a lethal leukemia syndrome; we determined whether L-Grb2 could prolong the survival of mice bearing such xenografts. One day after leukemia cell inoculation, mice received twice weekly intravenous injections of L-Grb2. At an injection dose of 15 mg of L-Grb2 per kg of mouse body weight, 80% of mice treated with L-Grb2 survived to 48 days (end of study) whereas 0% of mice treated with the same dose of liposomal control oligonucleotide survived; the mean survival duration of these groups was 44 and 20 days, respectively. Our data indicate that L-Grb2 prolonged the survival of mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 may be used as a novel cancer therapeutic modality.

Relation between development of leukemia and duration of N-nitroso-N-ethylurea treatment in DONRYU rats.

N-Nitroso-N-ethylurea (NEU; CAS: 759-73-9) is a strong leukemogen that induces erythroblastic leukemia in inbred DONRYU rats. In the present experiments, relationships between development of leukemia, duration of NEU treatment, and sequential changes in the hematopoietic organs during carcinogen administration were examined. In experiment 1, groups of rats were given a 400-ppm NEU solution for 0, 2, 4, 6, 8, or 10 weeks, and the resultant incidence of leukemias was 0, 26, 40, 75, 95, and 100%, respectively. Of the various types of leukemia, the erythroblastic type was observed in 0, 0, 20, 40, 95, and 90% of rats, respectively. The average latent period showed an inverse correlation with the duration of NEU treatment. In experiment 2 the animals were divided into carcinogen-treated and control groups, and rats were sacrificed periodically for histopathologic examination. In the experimental group, the bone marrow became hypoplastic soon after commencement of NEU treatment and at the 6th week became severely aplastic, thereafter recovering slightly. At the 10th week, 2 rats out of 5 examined were leukemic. Relationships between incidence of leukemia, duration of NEU treatment, and sequential changes of the bone marrow during carcinogen administration are discussed.

Potentiation of anticancer agents by amphotericin B.

Amphotericin B (AmB) has been shown to potentiate the cytotoxicity of several different anticancer agents in AKR mice. The extent of potentiation, as assessed quantitatively by a spleen colony assay, varied from a factor of approximately 3 for 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) to over 100 for a number of alkylating agents and to over 1,000 for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Most phase-specific agents showed little potentiation of cytotoxicity by AmB. A dose-response effect was noted with both BCNU and adriamycin, with increased potentiation being observed with increasing doses of the anticancer agents. The effect on increase in life-span was also studied and for most of the combinations it correlated with the potentiating effects disclosed by the spleen colony assay. Such a correlation was not found for AmB combined with either BCNU or vincristine.

Activity of 2-fluoro-5-methylarabinofuranosyluracil against mouse leukemias sensitive to and resistant to 1-beta-D-arabinofuranosylcytosine.

A new pyrimidine nucleoside, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil, previously has been shown to be active against the herpes group of viruses in vitro and in vivo. It is also active against mouse and human leukemic cells in culture and against mouse leukemias L1210, P388, and P815 in vivo. In contrast to other 1-beta-D-arabinofuranosylcytosine (ara-C) derivatives, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil, when given either i.p. or p.o., is highly active against lines of leukemias P815 and L1210 made resistant to ara-C. Against P815/ara-C and L1210/araC, it is more effective than is 5-azacytidine, a drug which has shown definite effectiveness in patients with acute leukemia whose disease has become resistant to ara-C. For these reasons, 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil would seem to merit clinical trial in patients with acute nonlymphocytic leukemia whose disease has become resistant to ara-C.

Anti-B4-blocked ricin immunotoxin shows therapeutic efficacy in four different SCID mouse tumor models.

Anti-CD19 monoclonal antibody anti-B4 (IgG1) conjugated to the novel toxin-blocked ricin forms a potent immunotoxin, anti-B4-blocked ricin, that kills greater than 4.5 logs of CD19-positive cells in vitro after a 24-h exposure to a conjugate concentration of 5 x 10(-9) M (1.11 micrograms/ml). The efficacy of anti-B4-blocked ricin in vivo was assessed in survival models of SCID mice bearing either a human B-cell lymphoma (Namalwa), a human non-T and non-B acute lymphoblastic leukemia (Nalm-6), or a murine B-cell lymphoma transfected with the human CD19 gene (300B4). In one model, 5 x 10(7) tumor cells were injected i.p., and 1 h later the mice were treated with i.v. bolus injections of anti-B4-blocked ricin at 100 micrograms/kg/day for 5 days. Controls included similar treatment with anti-B4 antibody (72 micrograms/kg/day or 2 mg/kg/day for 5 days) alone or with the isotype-matched nonspecific immunotoxin, N901-blocked ricin (100 micrograms/kg/day). In a second model, 4 x 10(6) tumor cells were injected i.v., and 7 days later mice were treated i.v. as above. Anti-B4-blocked ricin showed efficacy by killing in vivo up to 3 logs of tumor cells, which was manifested in significant prolongation of the life of the treated animals. Only very limited or no effects were observed in animals treated with either anti-B4 antibody alone or N901-blocked ricin control conjugate. The concentration of anti-B4-blocked ricin in the blood of animals was 150 ng/ml after the first i.v. injection and about 800 ng/ml following the fifth injection of conjugate. This increase may be due to damage to the reticuloendothelial system by anti-B4-blocked ricin, since the rate of clearance of carbon from blood also decreased 5-fold after five injections as compared to the rate after only one injection. These studies indicate that anti-B4-blocked ricin has the potential to increase survival times of hosts with malignant disease.

An improved method for analyzing survival data from combination chemotherapy experiments.

In this paper, we present a new method for analyzing survival data from combination chemotherapy experiments. The analysis consists of relating survival to the dosage level of each drug in the combination and using response surface techniques to determine the importance of drug interactions and to estimate optimal doses. A combination experiment using cyclophosphamide, mechlorethamine, and mitomycin C in early L1210 leukemia, advanced L1210 leukemia, and advanced P388 leukemia is used to illustrate the analyses. A therapeutic synergism has been shown. As a result of the various drug interactions, the predicted optimal dose of mitomycin C is found to be zero. This result was duplicated in each tumor system studied.

Biological effects and structure-activity relationships of 1,2-dihydropyrido[3,4-b]pyrazines.

The effects of a number of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines) upon the proliferation and the mitotic index of cultured L1210 cells and upon the survival of mice bearing P388 leukemia were determined. The 1,2-dihydrostructure and amino groups or masked amino groups at positions 5 and 7 were necessary for activity, and various substituents at positions 2 and 3 had considerable influence upon the activity. A number of these pyrazines had significant activity against i.p. P388 leukemia in mice, and several pyrazines were more active than the corresponding oxazines or thiazines in both the in vitro and the in vivo systems. The effects of the pyrazines upon the cultured cells were reversible, and the rate and degree of reversibility were influenced by the substituents at positions 2 and 3. Tests performed with two of the pyrazines yielded results that indicate that these compounds, like the known agent nocodazole, might compete with colchicine for binding to tubulin. Synergistic killing of cultured L1210 cells was obtained with combinations of one of the pyrazines and vincristine.

Deregulated FADD expression and phosphorylation in T-cell lymphoblastic lymphoma.

In the present work, we show that T-cell lymphoblastic lymphoma cells exhibit a reduction of FADD availability in the cytoplasm, which may contribute to impaired apoptosis. In addition, we observe a reduction of FADD phosphorylation that inversely correlates with the proliferation capacity and tumor aggressiveness. The resultant balance between FADD-dependent apoptotic and non-apoptotic abilities may define the outcome of the tumor. Thus, we propose that FADD expression and phosphorylation can be reliable biomarkers with prognostic value for T-LBL stratification.

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

Antileukemic effect of interleukin-7-transduced bone marrow stromal cells in mice following allogeneic T-cell-depleted bone marrow transplantation.

Impaired immune reconstitution following allogeneic bone marrow transplantation (BMT) remains a major obstacle to its clinical application. In this study, interleukin (IL)-7-transduced bone marrow stromal cells (MSC-IL7, 1 x 10(6)/mouse) were transfused into lethally irradiated C57BL/6 recipient mice. By day 40 after transplantation, the recipient mice were challenged with the lymphoma cell line EL4. MSC-IL7 co-transplantation protected recipient mice from leukemic mortality (MST >120 days after BMT vs mean survival time (MST) 70 days in the PBS group) It enhance the PFC count and DTH responses of recipients after transplantation. In conclusion, MSC mediated IL-7 gene therapy and may be a more feasible strategy to restore immune function following allo-TCD-BMT.