Jeffrey J. Molldrem

Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia

Although the immune system has long been implicated in the control of cancer, evidence for specific and efficacious immune responses in human cancer has been lacking. In the case of chronic myelogenous leukemia (CML), either allogeneic bone marrow transplant (BMT) or interferon-α2b (IFN-α2b) therapy can result in complete remission, but the mechanism for prolonged disease control is unknown and may involve immune anti-leukemic responses. We previously demonstrated that PR1, a peptide derived from proteinase 3, is a potential target for CML-specific T cells. Here we studied 38 CML patients treated with allogeneic BMT, IFN- α2b or chemotherapy to look for PR1-specific T cells using PR1/HLA-A*0201 tetrameric complexes. There was a strong correlation between the presence of PR1-specific T cells and clinical responses after IFN-α and allogeneic BMT. This provides for the first time direct evidence of a role for T-cell immunity in clearing malignant cells.

Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.

Immunocompromised individuals are at high risk for life-threatening diseases, especially those caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus. Conventional therapeutics are primarily active only against CMV, and resistance is frequent. Adoptive transfer of polyclonal cytotoxic T lymphocytes (CTLs) specific for CMV or EBV seems promising, but it is unclear whether this strategy can be extended to adenovirus, which comprises many serotypes. In addition, the preparation of a specific CTL line for each virus in every eligible individual would be impractical. Here we describe genetic modification of antigen-presenting cell lines to facilitate the production of CD4+ and CD8+ T lymphocytes specific for CMV, EBV and several serotypes of adenovirus from a single cell culture. When administered to immunocompromised individuals, the single T lymphocyte line expands into multiple discrete virus-specific populations that supply clinically measurable antiviral activity. Monoculture-derived multispecific CTL infusion could provide a safe and efficient means to restore virus-specific immunity in the immunocompromised host.

Cord-blood engraftment with ex vivo mesenchymal-cell coculture.

BACKGROUND Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. METHODS We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. RESULTS Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram--doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). CONCLUSIONS Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316.).

Antithymocyte globulin for patients with myelodysplastic syndrome

Twenty‐five transfusion‐dependent myelodysplastic syndrome (MDS) patients (with < 20% blasts) were treated in a phase II study with antithymocyte globulin (ATG) at 40 mg/kg/d for four doses and then followed with blood counts every 2 weeks and clinic visits every 3 months, for a median of 14 months (range 1–38 months). 11 (44%) patients responded and became transfusion‐independent after ATG, including three complete responses, six partial responses, and two minimal responses. Responses were observed in 9/14 patients (64%) with refractory anaemia (RA) and 2/6 patients (33%) with refractory anaemia with excess blasts (RAEB). Median response duration was 10 months (range 3–38 months). The Kaplan‐Meier estimate of overall survival was 84% at 38 months, with one early death due to pneumonia and two deaths from disease progression to leukaemia. Side‐effects consisted mainly of mild serum sickness in all patients. A single course of ATG restored haemopoiesis in some patients with MDS and was well tolerated.

PD-L1 Expression in Triple-Negative Breast Cancer

Using tissue microarrays containing 105 triple-negative breast cancer (TNBC) specimens, Mittendorf and colleagues show that 20% of the TNBC specimens express PD-L1, half have lost PTEN, and inhibitors of PI3K pathway decrease PD-L1 expression, providing a rationale for therapeutic targeting of PD-L1 for TNBC. Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (n = 120) compared with non-TNBC (n = 716; P < 0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1+ tumors had greater CD8+ T-cell infiltrate than PD-L1− tumors (688 cells/mm vs. 263 cells/mm; P < 0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA (shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses. Cancer Immunol Res; 2(4); 361–70. ©2014 AACR.

Display technologies: Application for the discovery of drug and gene delivery agents☆

Abstract Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed.

Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells

We have shown that cytotoxic T lymphocytes specific for PR1, an HLA-A2-restricted nonopeptide derived from proteinase 3, kill leukemia cells and may contribute to the elimination of chronic myelogenous leukemia (CML) after treatment with IFN or allogeneic bone marrow transplant. Some patients with persistent disease also have circulating PR1-specific T cells, however, suggesting the likelihood of immune tolerance. Here we show that both high- and low-avidity PR1-specific T cells from the peripheral blood of healthy donors can be identified and selectively expanded in vitro. Although high-avidity PR1-specific T cells killed CML more effectively than low-avidity T cells, only high-avidity T cells underwent apoptosis when stimulated with high PR1 peptide concentration or when exposed to leukemia that overexpressed proteinase 3. No high-avidity PR1-specific T cells could be identified or expanded from newly diagnosed leukemia patients, whereas low-avidity T cells were readily expanded. Circulating high-avidity PR1-specific T cells were identified in IFN-sensitive patients in cytogenetic remission, however. These results provide evidence that CML shapes the host immune response and that leukemia outgrowth may result in part from leukemia-induced selective deletion of high-avidity PR1-specific T cells.

Antithymocyte Globulin for Treatment of the Bone Marrow Failure Associated with Myelodysplastic Syndromes

Context Myelodysplastic syndromes are bone marrow disorders that are characterized by ineffective hematopoiesis and leukemic transformation. Almost half of the deaths caused by these syndromes are from cytopenia. Standard supportive care (red blood cell and platelet transfusions and hematopoietic growth factors) often fails. Contribution This prospective case series found that one third of 61 patients with red blood cell transfusion-dependent myelodysplastic syndrome became transfusion independent within 8 months of a 4-day course of intravenous antithymocyte globulin. Cautions Before instituting treatment changes on the basis of these exciting preliminary findings, physicians should watch for controlled studies that compare transfusion and survival outcomes in patients treated with antithymocyte globulin and patients given usual care (or other therapies). The Editors Myelodysplastic syndromes are clonal bone marrow disorders that usually affect older adults. An estimated 20 000 new cases are documented annually in the United States (1, 2). The incidence seems to be rising, partly because older persons represent an increasing proportion of the population and partly because of improved diagnostic ability (3). Myelodysplastic syndromes are characterized by ineffective hematopoiesis that leads to varying degrees and combinations of anemia, neutropenia, and thrombocytopenia and by a tendency to evolve to acute leukemia that is refractory to treatment. Treatment of the bone marrow failure that accompanies myelodysplastic syndromes is unsatisfactory. It relies mainly on supportive care with red blood cell and platelet transfusions, antibiotics, and combinations of hematopoietic growth factors, which may partially improve blood cell counts. Although death from myelodysplastic syndromes can occur from leukemic progression, almost half of all deaths are due to cytopenia occurring without leukemic transformation (4). Therefore, improved treatments to relieve bone marrow failure associated with myelodysplastic syndromes should be greatly beneficial in terms of survival and quality of life. Laboratory data suggest that immune mechanisms may contribute to the cytopenia of myelodysplastic syndromes (5-7). Case reports and small studies have shown that in some patients with the hypoplastic myelodysplastic syndrome, cytopenia responds to immunosuppressive treatment with antithymocyte globulin (ATG) or cyclosporine (8-10). In a previous report, we documented the remission of dependence on red blood cell transfusions and correction of neutropenia and thrombocytopenia in 12 of 25 patients with myelodysplastic syndromes who were treated with ATG (11). In this study, we attempted to establish the frequency of red blood cell, neutrophil, and platelet response after ATG treatment in 61 patients (including the 25 reported in our earlier study [11]) with myelodysplastic syndromes. We also wanted to determine the survival and risk for leukemia progression in patients with myelodysplastic syndromes and to identify independent risk factors for treatment response, survival, and leukemic progression. Methods Patients Between January 1994 and June 1998, patients were entered into a phase II trial of ATG treatment for myelodysplastic syndromes (study 95-H-0189). Of the 71 patients who were evaluated for the study, 61 met the eligibility criteria. All patients gave written informed consent, and the Institutional Review Board of the U.S. National Heart, Lung, and Blood Institute approved the study. Patients 18 years of age or older with a diagnosis of myelodysplastic syndrome and Eastern Cooperative Oncology Group (ECOG) performance status score of 2 or less were eligible for the study. Protocol entry was restricted to patients who were dependent on red blood cell transfusions, with or without concurrent neutropenia or thrombocytopenia. Transfusion dependence was defined as having had at least three separate transfusions of two or more units of red blood cells at intervals of 2 to 4 weeks to maintain the hemoglobin level (after exclusion of blood loss as a cause of anemia). To minimize the effect of previous treatments on ATG responses, patients were required to discontinue all other treatments capable of stimulating bone marrow (that is, growth factors, cyclosporine, steroids, and androgens) at least 1 month before entering the study. Myelodysplastic syndromes were confirmed by bone marrow aspirate and biopsy and were classified according to the FrenchAmericanBritish criteria as refractory anemia, refractory anemia with ringed sideroblasts, or refractory anemia with excess of blasts (12). Patients with chronic myelomonocytic leukemia and those with refractory anemia and 20% or more blasts in the bone marrow in transformation to leukemia (refractory anemia with excess blast cells in transformation) were excluded. Dysplasia affecting a minimum of two hematopoietic lineages was required for diagnosis. Marrow cellularity was assessed by surveying a 1- to 2-cm core biopsy specimen taken from the posterior iliac crest. Before treatment, bone marrow cytogenetic analysis was performed for all patients. These data were used to assign a score to each patient by using the International Prognostic Scoring System (IPSS) (1). The most common reasons for excluding patients from the study were dysplasia affecting only a single lineage or a bone marrow biopsy that was inadequate to render a diagnosis. Study Protocol Before treatment, allergy to ATG was determined by intradermal injection of 0.1 mL of ATG at a concentration of 5 mg/mL. Antithymocyte globulin treatment was contraindicated if erythema larger than 5 mm in diameter, compared with the saline control, developed. Eligible patients were given ATG (Pharmacia, Peapack, New Jersey), 40 mg/kg of body weight daily for 4 days. Antithymocyte globulin was administered intravenously over 4 to 8 hours with oral prednisone, 1 mg/kg daily [minimum of 40 mg/d], for 10 days and then tapered by day 17. Clinic visits were scheduled at 3, 6, and 12 months and then annually. Bone marrow aspirate, biopsy specimens, and bone marrow samples for cytogenetic analysis were obtained at 6 and 12 months and then annually. Excluding transfusions and antibiotics, patients received no other treatment for myelodysplastic syndromes for at least 6 months. The main criterion for response was independence from red blood cell transfusion. This criterion was met if 1) the patient was independent from transfusion for a minimum of 6 weeks with a sustained increase in hemoglobin level, or 2) the patient maintained a stable hemoglobin level within 8 months of ATG treatment, as measured from the time the patient last received a transfusion until reinstitution of red blood cell transfusions. Standard criteria for red blood cell transfusion were followed. Transfusions were given for hemoglobin nadirs between 7 and 10 g/dL, depending on subjective tolerance of anemia in individual patients. Platelet and neutrophil counts that were the highest at 3 and 6 months of follow-up were used as the peak response. Thrombocytopenia was defined as a platelet count less than 150 109cells/L, and neutropenia was defined as a neutrophil count less than 1.0 109 cells/L. In patients with severe thrombocytopenia with a platelet count less than 20 109 cells/L, a response was defined as a sustained increase to at least 25 109 cells/L. In patients with severe neutropenia with a neutrophil count of 0.5 109 cells/L, a response was defined as an increase to 1.0 109 cells/L or greater. Morphologic characteristics of bone marrow, cellularity, and karyotype were compared before treatment, after 6 months, and then annually. Statistical Analyses Results were analyzed on an intention-to-treat basis. Therefore, we evaluated all patients enrolled in the study and receiving ATG, regardless of whether they completed the course of ATG. Survival was censored in December 2001, with the exception of three bone marrow transplant recipients who were censored at the time of transplantation. Survival and time to progression for the entire cohort and response duration for the responders were evaluated by using the KaplanMeier method (13), with pointwise confidence intervals based on the variance formula of Greenwood (14). Within-patient differences before and after ATG treatment were evaluated by using the paired t-test for continuous variables and permutation tests for categorized variables. Prognostic factors for response were assessed by using logistic regression (15). The Fisher exact test was used to compute univariate P values for categorical predictors, and the likelihood ratio chi-square test with one degree of freedom was used for continuous predictors. Cox proportional-hazards regression was used to ascertain the effects of study entry characteristics and response on survival and disease progression (16). The effect of response on survival and disease progression was analyzed by modeling response as a time-varying covariate. With this approach, when a patient is in response, the model assumes that the relative risk for death or disease progression differs by a fixed amount compared with a similar patient who is not in response. Significance in Cox analyses was determined according to the score statistic. All P values are two sided; a P value less than 0.05 was considered statistically significant. Confidence intervals, determined by using Wald methods, were used for odds ratios. Stepwise regression methods using a modified step-up algorithm were used. At each step, a score statistic criterion (P = 0.25) was used for a variable to be entered into the model and a Wald statistic criterion (P = 0.30) was used for a variable to remain in the model. Role of the Funding Source These studies were conducted at the U.S. National Institutes of Health and were funded by the U.S. government. No outside agency influenced our analysis or interpretation of the data or the decision to submit th

Functionally active virus-specific T cells that target CMV, adenovirus, and EBV can be expanded from naive T-cell populations in cord blood and will target a range of viral epitopes

The naive phenotype of cord blood (CB) T cells may reduce graft-versus-host disease after umbilical cord blood transplantation, but this naivety and their low absolute numbers also delays immune reconstitution, producing higher infection-related mortality that is predominantly related to CMV, adenovirus (Adv), and EBV. Adoptive immunotherapy with peripheral blood-derived virus-specific cytotoxic T lymphocytes (CTLs) can effectively prevent viral disease after conventional stem cell transplantation, and we now describe the generation of single cultures of CTLs from CB that are specific for multiple viruses. Using EBV-infected B cells transduced with a clinical-grade Ad5f35CMVpp65 adenoviral vector as sources of EBV, Adv, and CMV antigens, we expanded virus-specific T cells even from CB T cells with a naive phenotype. After expansion, each CTL culture contained both CD8(+) and CD4(+) T-cell subsets, predominantly of effector memory phenotype. Each CTL culture also had HLA-restricted virus-specific cytotoxic effector function against EBV, CMV, and Adv targets. The CB CTLs recognized multiple viral epitopes, including CD4-restricted Adv-hexon epitopes and immunosubdominant CD4- and CD8-restricted CMVpp65 epitopes. Notwithstanding their naive phenotype, it is therefore possible to generate trivirus-specific CTLs in a single culture of CB, which may be of value to prevent or treat viral disease in CB transplant recipients. This study is registered at www.clinicaltrials.gov as NCT00078533.

Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte‐mediated inhibition of CFU‐GM and alterations in T‐cell receptor Vβ profiles

We have demonstrated that 44% of myelodysplastic syndrome (MDS) patients with cytopenia have a haematological response to antithymocyte globulin (ATG). Three ATG responders and two non‐responders with refractory anaemia were further studied for lymphocyte‐mediated inhibition of bone marrow using a standard CFU‐GM assay. In responders, peripheral blood lymphocytes (PBL) added at a 5:1 ratio suppressed CFU‐GM by 54 ± 9% (P = 0.04) and was reversed by ATG treatment. Pre‐treatment marrow depleted of CD3 lymphocytes, increased CFU‐GM by 32% (P = 0.02) in an ATG responder, but not in a non‐responder. CD3 lymphocytes from 6‐month post‐treatment marrow did not inhibit pre‐treatment CFU‐GM, indicating ATG had affected the T cells. Pre‐treatment marrow depleted of CD8 lymphocytes, increased CFU‐GM by 60% (P = 0.01) and 49% (P = 0.03) in two ATG responders, but not in a non‐responder. Inhibition required cell–cell interaction through MHC I. TCR Vβ families, analysed by SSCP, changed from clonal to polyclonal in one ATG responder after 6 months, but clones persisted in a non‐responder. These results indicate patients with refractory anaemia who respond to ATG have CD8 T‐cell clones that mediate MHC‐I‐restricted suppression of CFU‐GM which are replaced by polyclonal T cells that do not suppress CFU‐GM after ATG treatment.