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Biomedical subjects

M K Brenner

Publications and source records attributed to M K Brenner.

At least 19 recordsLinked to original sources
PubMed2006

Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells.

T cells can be engineered to target tumor cells by transduction of tumor-specific chimeric receptors, consisting of an extracellular antigen-binding domain and an intracellular signaling domain. However, the peripheral blood of cancer patients frequently contains an increased number of T regulatory cells, which appear to inhibit immune reactivity. We have investigated the effects of T regulatory cells on chimeric T cells specific for the B-cell antigen CD19, as B-cell malignancies are attractive targets for chimeric T-cell therapy. When a CD19 single-chain Fv antibody was coupled to the CD3 zeta (zeta) chain, there was sharply reduced activity on exposure to T regulatory cells, measured by CD19+ target-induced proliferation and cytotoxicity. By contrast, expression in T cells of a chimeric receptor consisting of the intracellular portion of the CD28 molecule fused to the zeta-chain and CD19 single-chain Fv not only produced a higher proliferative response and an increased nuclear factor kappaB activation but also sustained these activities in the presence of T regulatory cells. These effects are seen whether the chimeric T cells are derived from normal donors or from patients with B-cell chronic lymphocytic leukemia, indicating the potential for clinical application in B cell malignancies.

Antigens, CD19↗
PubMed2006

Outcome of reduced-intensity allogeneic hematopoietic stem cell transplantation (RISCT) using antilymphocyte antibodies in patients with high-risk acute myeloid leukemia (AML).

Hemopoietic stem cell transplantation (SCT) with fully ablative conditioning is associated with an age-related increase in treatment-related mortality. It is therefore particularly unsuited to older individuals, who are most at risk of developing acute myeloid leukemia (AML). Reduced-intensity SCT (RISCT) may be of value in this group. We report 17 consecutive patients with high-risk AML whose median age was 58 years and who received stem cells from HLA-matched siblings (n=5), or alternative donors (n=12). We used lymphodepleting antibodies as a part of the reduced-intensity conditioning regimen to limit the risk of graft rejection and graft-versus-host disease (GVHD). All patients engrafted. One patient developed severe fatal GVHD, and two patients died of infection. At a median follow-up of 861 days (372-1957 days), seven patients are alive in remission, which includes two patients treated in relapse and five patients who lacked an MHC identical sibling donor. Both progression-free survival and overall survival are 40% (95% CI, 17-64%). Hence, RISCT using lymphodepleting antibodies may be of value for older patients with AML, even in those with active or high-risk disease, and even if they lack an MHC-identical sibling donor.

Acute Disease↗
PubMed2005

A distinct "side population" of cells in human tumor cells: implications for tumor biology and therapy.

Stem cells have an extensive capacity to proliferate, differentiate and self-renew. In many mammals, including humans, an adult stem cell subpopulation termed the "side population" (SP) has been identified. SP cells can rapidly efflux lipophilic fluorescent dyes to produce a characteristic profile based on fluorescence-activated flow cytometric analysis. Previous studies have demonstrated SP cells in bone marrow obtained from patients with acute myeloid leukemia, suggesting that these cells might be candidate leukemic stem cells, and recent studies have found a SP of tumor progenitor cells in human solid tumors. These new data indicate that the ability of malignant SP cells to expel anticancer drugs may directly improve their survival and sustain their clonogenicity during exposure to cytostatic drugs, allowing disease recurrence when therapy is withdrawn. Identification of a tumor progenitor population with intrinsic mechanisms for cytostatic drug resistance might also provide clues for improved therapeutic intervention.

ATP-Binding Cassette Transporters↗
PubMed2005

CD52 and CD45 monoclonal antibodies for reduced intensity hemopoietic stem cell transplantation from HLA matched and one antigen mismatched unrelated donors.

Allogeneic hemopoietic stem cell transplantation (HSCT) is the only curative option for many patients with hematological malignancies. Since many of these patients lack HLA-identical sibling donors and are older or have comorbidity, a fully ablative HSCT is not feasible and an alternative approach is required. We studied 22 consecutive patients who could not have myeloablative conditioning because of comorbidity or age - 21/22 being over the age of 50 years (median 58 years range 20-70 years). A conditioning regimen consisting of fludarabine, total body radiation 450 cGy and alemtuzumab (CD52 mAb) was used for 15 patients. A second group of seven patients received CD45 monoclonal antibodies in addition. Unrelated donor stem cells were HLA matched (15 patients - 68%) or one locus mismatched (seven patients - 32%). In all, 16 patients had high-risk disease, including 12 with active malignancy at the time of transplant. With a median follow-up of 715 (216-1470) days, nonrelapse mortality, actuarial event-free and overall survival is 27, 45 and 45%, respectively. Hence the outcome of reduced intensity HSCT with lymphodepleting antibodies in older patients with intermediate/high-risk hematological malignancies appears comparable to that obtained with fully ablative transplantation in younger patients, even when these older recipients lack HLA-identical sibling donors.

Adult↗
PubMed2005

Cytomegalovirus (CMV) infections and CMV-specific cellular immune reconstitution following reduced intensity conditioning allogeneic stem cell transplantation with Alemtuzumab.

We studied the incidence and recurrence of Cytomegalovirus (CMV) infection and reactivation in 38 recipients of Alemtuzumab reduced intensity conditioning-stem cell transplantation, and used CMV-HLA tetramer studies to discover if these events correlated with recovery of circulating CMV-specific CD8+ T cells (cytotoxic T lymphocyte (CTLs)). The cumulative incidence of CMV infection was 60% at 1 year (95% CI, 45-78%) with a median reactivation time of 24 days (range 5-95 days). All patients with CMV reactivation received Ganciclovir or Foscarnet, and only one developed CMV disease. More strikingly, only 8/21 patients had relapse of CMV antigenemia. Tetramer analysis in 13 patients showed that 11 reconstituted CMV CTLs (7/11 by day 30 and 10/11 by day 90). The development of CMV infection was accompanied by a >5-fold rise of CMV CTLs. Recurrence of CMV infection occurred only in the patients who failed to generate a CTL response to the virus. Hence, recipients of SCT using Alemtuzumab-RIC are initially profoundly immunosuppressed and have a high incidence of early CMV reactivation. However, in the majority of patients, infection is transient, and antiviral T cell reconstitution is rapid. Monitoring with CMV-specific CTLs may help identify the subset of patients at risk from recurrent infection or disease.

Adult↗
PubMed2005

Add-back of allodepleted donor T cells to improve immune reconstitution after haplo-identical stem cell transplantation.

Poor immune reconstitution after haplo-identical stem cell transplantation results in high mortality from viral infections and relapse. One approach to overcome this problem is to deplete alloreactive cells selectively by deleting T cells activated by recipient stimulators, using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient PBMC, and this can result in GvHD. We have shown that using recipient EBV-transformed LCL as stimulators to activate donor alloreactive T cells results in more consistent depletion of in vitro alloreactivity while preserving T-cell responses to viral and potential myeloid tumor Ag. Based on these data, we have embarked on a phase I clinical dose escalation study of add-back of allo-LCL-depleted donor T cells in the haplo-identical setting, to determine if the allodepletion we achieve to allow infusion of sufficient T cells to restore useful antiviral/anti-leukemic responses without causing GvHD. Fifteen patients have so far been treated. The incidence of significant acute or chronic GvHD has been low (2/15), as has mortality from infection (1/15). Preliminary data show accelerated immune reconstitution in dose level 2 patients. Infused allodepleted donor T cells appear able to expand significantly in the face of viral reactivations, and doses as low as 3 x 10(5)/kg may be sufficient to confer useful antiviral immunity in this setting. At a median follow-up of 19.5 months, nine of 15 patients are alive and disease-free. Five patients have relapsed, all of whom have died.

Clinical Trials, Phase I as Topic↗
PubMed2004

A distinct "side population" of cells with high drug efflux capacity in human tumor cells.

A subset of stem cells, termed the "side population" (SP), has been identified in several tissues in mammalian species. These cells maintain a high efflux capability for antimitotic drugs. We have investigated whether functionally equivalent stem cells also may be detected in human cancers. We initially examined primary tumor cells from 23 patients with neuroblastoma and cell lines derived from a range of other tumors. A distinct SP was found in neuroblastoma cells from 15 of 23 patients (65%). The SP was capable of sustained expansion ex vivo and showed evidence for asymmetric division, generating both SP and non-SP progeny. These cells also expressed high levels of ABCG2 and ABCA3 transporter genes and had a greater capacity to expel cytotoxic drugs, such as mitoxantrone, resulting in better survival. A SP also was detected in breast cancer, lung cancer, and glioblastoma cell lines, suggesting that this phenotype defines a class of cancer stem cells with inherently high resistance to chemotherapeutic agents that should be targeted during the treatment of malignant disease.

ATP-Binding Cassette Transporters↗
PubMed2004

Liposomal enhancement of the antitumor activity of conditionally replication-competent adenoviral plasmids.

Many human tumors have a functional deficiency in p53. Numerous studies have taken advantage of this phenomenon to use a conditionally replication-competent adenovirus (Ad dl1520) that will grow in and lyse tumor cells while sparing normal tissues. However, success has been limited, in part due to difficulties in reaching a sufficiently high proportion of tumor cells. Preexisting or developing immune responses directed toward viral proteins further decrease the efficacy of the approach. We have developed a liposome-encapsulated conditionally replication-competent plasmid based on the dl1520 virus. Like the parent virus, this plasmid generates infectious particles following transfection of p53-defective, but not p53-wild-type tumor cells, but unlike the parent virus it is able to infect CAR-negative tumor cells. The antitumor efficacy of this infectious plasmid was demonstrated in mice with xenografted human tumors, in which it was active after both local and intravenous administration for subcutaneous tumors and following intravenous administration for disseminated malignancy. Activity was retained systemically, even in the presence of neutralizing antibody. Such liposomally encapsulated conditionally replication-competent plasmids may complement the use of conventional viral particles, particularly in settings in which liver uptake of adenoviral vector is undesirable or there are problematic inhibitory effects from humoral immune responses.

Adenoviridae↗
PubMed2004

Comparison of the efficiency of transduction of leukemic cells by fiber-modified adenoviruses.

Efficient gene transfer with adenoviral type 5 (Ad5) vectors depends on the initial attachment of their fiber, which binds the coxsackie-adenovirus receptor (CAR), and their subsequent internalization, mediated by the interaction of viral penton base with target cell alphav integrins. We previously demonstrated that human leukemic cells lack these receptors and are therefore resistant to Ad5 transduction, limiting efforts to genetically modify these cells. Human leukemic blasts are, however, susceptible to transduction with an adenovector made CAR independent by substitution of a chimeric Ad5/35 fiber [Yotnda et al. (2001). Gene Ther. 8, 930-937]. Other receptors can also be targeted with recombinant ligand moieties incorporated into adenovirus fiber. We have determined which of these fiber-modified adenovectors is most effective at modifying human primary leukemia cells, and lines. We used a replication-incompetent Ad5-beta-gal vector, in which the Ad5 fiber was replaced with fiber from various adenovirus serotypes (Ad35 and Ad11), or modified either with variable length polylysine (K4, K7, K21) or RGD-4C peptide. All the modified fiber vectors transduced primary leukemia cells and cell lines more efficiently than Ad5. Polylysine-substituted Ad5F/K21 and peptide-modified Ad5F/RGD vectors were most effective overall (up to 100% efficiency), whereas Ad5F/RGD was the most effective at transducing B cell acute lymphoblastic leukemia cells (90% efficiency). Ad5F/K21 and Ad5F/RGD should be of value for the genetic modification of human primary leukemia cells.

Adenoviridae↗
PubMed2003

Expansion of EBV latent membrane protein 2a specific cytotoxic T cells for the adoptive immunotherapy of EBV latency type 2 malignancies: influence of recombinant IL12 and IL15.

BACKGROUND: EBV-associated malignancies with a Type II latency gene expression pattern, such as EBV-positive HD, or nasopharyngeal carcinoma, frequently express the EBV latency Ag LMP2a. Hence, they provide a potential target for adoptive immunotherapy using in vitro-generated LMP2a-specific cytotoxic T lymphocytes (CTL). In this study, LMP2a-specific CTL were specifically amplified and the influence of rIL12 and rIL15 on the culture outcome was tested. METHODS: PBMC from donors were stimulated twice with autologous DC transduced with an adenovirus vector expressing LMP2a. This led to a significant expansion of LMP2a-tetramer-specific CTL, which were subsequently further expanded with autologous EBV-transformed B-lymphoblastoid cells (LCL). The addition of rIL12 and rIL15 to the standard IL2-containing culture medium enhanced the proliferation of LMP2a-specific CTL. RESULTS: While rIL15 did not change the pattern of cytokines secreted by LMP2a-CTL, rIL12 enhanced the production of Th1/Tc1 cytokines, such as IFN-n, while suppressing the production of the Th2/Tc2 cytokine IL5. DISCUSSION: Stimulation of CTL cultures with rIL12 or rIL15 will generate CTL more rapidly, facilitating the application of this approach for patients with these EBV-associated disorders.

Adenoviridae↗
PubMed2002

Immunotherapy for Hodgkin's disease.

EBV proteins present in the malignant Hodgkin Reed-Sternberg (HR-S) cells of about 40% of patients with Hodgkin's Disease (HD) provide targets for immunotherapy with virus-specific cytotoxic T lymphocytes (CTL). However, Hodgkin tumors use multiple strategies to avoid CTL, including down-regulation of immunodominant EBV antigens, and secretion of cytokines and chemokines such as TGF-beta, that inhibit the activation of CTL and professional antigen-presenting cells (APC). To be effective against this tumor, CTL must resist some or all of these strategies. Thirteen patients with multiply-relapsed HD received EBV-specific CTL, generated ex vivo using the autologous EBV-transformed B cells (LCL) as stimulator cells. After CTL infusion, EBV-specific immunity increased, virus load decreased, CTL homed to sites of malignancy and persisted for up to ten months. Clinically, CTL produced resolution of B symptoms and mixed tumor responses including one complete remission of residual disease remaining after autologous bone marrow transplant. However, no complete remission of bulky disease was achieved. Although LMP2-specific CTL activity could be detected in some of the infused CTL lines, they were present in low frequency. In pre-clinical studies, LMP1 and LMP2-specific CTL could be produced by stimulating PBMC from patients and normal donors with autologous dendritic cells expressing LMP1 or LMP2 from adenoviral vectors. Further, CTL could be rendered resistant to the devastating effects of TGF-beta by transduction with a retrovirus vector expressing a dominant-negative TGF-beta receptor, while transgenic IL-12 could increase the expression of Th1 and decrease that of Th2 cytokines. Future clinical studies will test the efficacy of CTL with improved antigen-specificity and resistance to Hodgkin immune evasion strategies.

Antigens, Viral↗
PubMed2002

Targeting CD19 with genetically modified EBV-specific human T lymphocytes.

Human Epstein-Barr virus-specific T cells were genetically modified to express chimeric receptors specific for human CD19, which is expressed on the cell surface of most B cell malignancies. The receptor-modified EBV-specific T cells can be expanded and maintained long term in the presence of EBV-infected B cells. They recognize autologous EBV-infected targets through their conventional T cell receptor, and allogeneic EBV-infected targets and tumor targets through their chimeric receptor. They efficiently lyse both EBV and CD19-positive tumor targets in the absence of background cytotoxicity against CD19-negative targets. Donor-derived EBV-specific T cells expressing chimeric anti-tumor receptors may represent a source of effector cells that could be safely administered to leukemia patients to eradicate minimal residual disease after allogeneic bone marrow transplantation.

Antigens, CD19↗
PubMed2001

Targeting of G(D2)-positive tumor cells by human T lymphocytes engineered to express chimeric T-cell receptor genes.

Genetic engineering of human T lymphocytes to express tumor antigen-specific chimeric immune receptors is an attractive means for providing large numbers of effector cells for adoptive immunotherapy while bypassing major mechanisms of tumor escape from immune recognition. We have applied this strategy to the targeting of a G(D2)-positive tumor, neuroblastoma, which is the commonest extracranial solid tumor of childhood. Chimeric immune receptors were generated by joining an extracellular antigen-binding domain derived from either of the 2 ganglioside G(D2)-specific antibodies sc7A4 and sc14.G2a to a cytoplasmic signaling domain. The variable domains of hybridoma antibody 14.G2a were cloned and selected using a phage display approach. Upon coincubation with G(D2)-expressing tumor cell targets, human T lymphocytes transduced with recombinant retroviruses encoding chimeric receptors based on sc14.G2a, but not sc7A4, secreted significant levels of cytokines in a pattern comparable to the cytokine response obtained by engagement of the CD3 receptor. T cells transduced with the sc14.G2a-based chimeric T-cell receptors also displayed specific lysis of G(D2)-positive neuroblastoma cells, which was blocked in the presence of monoclonal antibody 14.G2a. In the absence of nonspecific stimulation of transduced cells, their functionality declined over time and antigenic stimulation of the chimeric receptor alone did not induce commitment to proliferation. These results support the feasibility of redirecting human T lymphocytes to a tumor-associated ganglioside epitope but emphasize that successful chimeric receptor-mediated adoptive immunotherapy will require additional strategies that overcome functional inactivation of gene-modified primary T lymphocytes.

Cytokines↗
PubMed2001

Generation of autologous Epstein-Barr virus-specific cytotoxic T cells for adoptive immunotherapy in solid organ transplant recipients.

BACKGROUND: Epstein-Barr virus (EBV)-driven posttransplant lymphoproliferative disorders (PTLD) affect 2%-27% of solid organ transplant (SOT) recipients. Adoptive immunotherapy may have therapeutic potential in this setting, but there is little experience in generating autologous EBV-specific cytotoxic T-cell lymphocytes (EBV-CTLs) from SOT recipients, and their efficacy and persistence in an immunosuppressed environment is unknown. METHODS: EBV-CTLs were generated from eight SOT recipients, using weekly stimulations with autologous lymphoblastoid cell lines (LCLs) and interleukin-2. CTL phenotype and function were evaluated in the presence of therapeutic concentration of cyclosporin A or FK506. RESULTS: In all cases, CTLs expanded with normal kinetics. The majority was CD3+CD8+ (mean, 76%), with less than 3% of natural killer cells. All ex vivo-generated CTLs produced significantly higher killing of autologous LCLs than of HLA-mismatched LCLs (mean, 56% vs. 14% at 20:1 ratio). No lysis of autologous or allogeneic PHA blasts was observed. The CTL expansion rate was reduced in a concentration-dependent manner in the presence of immunosuppressive drugs; however, neither lytic activity nor phenotype was affected. CONCLUSIONS: Using methods that are approved for clinical application, EBV-CTLs can be generated from SOT recipients, even those with frank lymphoma, or who are receiving immunosuppressive drugs. These CTLs retain their function in the presence of immunosuppressive agents. Although in vivo efficacy, safety, and persistence can be assessed only in clinical trials, our results suggest that CTLs can be effective for the treatment of PTLD, even when immunosuppression cannot be reduced because of the high risk of graft rejection.

Antibody Specificity↗