Efficient selection of genetically modified human T cells using methotrexate-resistant human dihydrofolate reductase.

Genetic modification of human T cells to express transgene-encoded polypeptides, such as tumor targeting chimeric antigen receptors, is an emerging therapeutic modality showing promise in clinical trials. The development of simple and efficient techniques for purifying transgene(+) T cells is needed to facilitate the derivation of cell products with uniform potency and purity. Unlike selection platforms that utilize physical methods (immunomagnetic or sorting) that are technically cumbersome and limited by the expense and availability of clinical-grade components, we focused on designing a selection system on the basis of the pharmaceutical drug methotrexate (MTX), a potent allosteric inhibitor of human dihydrofolate reductase (DHFR). Here, we describe the development of self inactivating (SIN) lentiviral vectors that direct the coordinated expression of a CD19-specific chimeric antigen receptor (CAR), the human EGFRt tracking/suicide construct, and a methotrexate-resistant human DHFR mutein (huDHFR(FS); L22F, F31S). Our results demonstrate that huDHFR(FS) expression renders lentivirally transduced primary human CD45RO(+)CD62L(+) central memory T cells resistant to lymphotoxic concentrations of MTX up to 0.1 µM. Our modular complementary DNA (cDNA) design insures that selected MTX-resistant T cells co-express functionally relevant levels of the CD19-specific CAR and EGFRt. This selection system on the basis of huDHFR(FS) and MTX has considerable potential utility in the manufacturing of clinical-grade T cell products.

Atomic-scale structure and stability of the square root(31) x square root(31)R9 degrees surface of Al2O3(0001).

Through the interplay of noncontact atomic force microscopy studies and density functional theory calculations, an atomistic model for the Al2O3(0001)-square root(31) x square root(31)R9 degrees surface reconstruction is revealed. The surface is found to consist of an Al adlayer on the Al2O3 substrate, and the driving force for the formation of the reconstruction is related to a detailed balance between strain in the adlayer and the preference for Al atoms to be located on distinct substrate sites.

Transgene-enforced co-stimulation of CD4+ T cells leads to enhanced and sustained anti-tumor effector functioning.

Background The role of co-stimulation in CD4+ T cell activation by professional APC is well established, while less is known of the role co-stimulation plays when CD4+ T cells interact directly with tumor cells. Methods Through genetic engineering of human CD4+ T cells, we tested the hypothesis that integration of co-stimulatory signaling domains within a tumor-targeting chimeric Ag receptor (CAR), the IL-13Ralpha2-specific IL-13-zetakine (IL13zeta), would enhance CD4+ T cell mediated responses against tumors that fail to express ligands for co-stimulatory receptors. Results Compared with CD3zeta-mediated activation alone, CD4+ effector T cells expressing the IL13-CD28-41BBzeta CAR exhibited augmented/sustained MAPK and AKT activity, up-regulated Th1 cytokine production, and enhanced cytolytic potency against tumor targets. Moreover, upon recursive stimulation with tumor, the IL13-CD28-41BBzeta+ cells retained/recycled their lytic function, whereas IL-13zeta+ CD4+ cells became anergic/exhausted. These in vitro observations correlated with enhanced in vivo control of established orthotopic CNS glioma xenografts in immunodeficient mice mediated by adoptively transferred ex vivo-expanded CD4+ T cells expressing the co-stimulatory CAR. Discussion Together these studies demonstrate the importance of integrating co-stimulation with CD3zeta signaling events to activate fully CD4+ anti-tumor effector cells for sustained function in the tumor microenvironment.

Lentiviral vector conferring resistance to mycophenolate mofetil and sensitivity to ganciclovir for in vivo T-cell selection.

Several clinical studies of gene-modified T cells have shown limited in vivo function of the cells, immunogenicity of the transgene, and lack of a selective advantage for gene-modified T cells. To address these problems, we developed a lentiviral vector (LV) that provides a selectable, proliferative advantage and potentially decreases immunogenicity for transduced T cells. The bicistronic vector expressed two genes linked with an internal ribosomal entry site. One gene is a variant of the inosine monophosphate dehydrogenase 2, inosine monophosphate dehydrogenase (IMPDH(IY)), conferring resistance to the immunosuppressive drug mycophenolate mofetil (MMF). The other is a suicide gene, herpes simplex virus thymidine kinase (HSV-TK), rendering proliferating cells sensitive to ablation with ganciclovir, fused to the selectable transmembrane marker DeltaCD34 (DeltaCD34/TK). Cells transduced with LV-DeltaCD34/TK.IMPDH(IY) were efficiently enriched by immunomagnetic selection for CD34, proliferated in 0.5-5 microM MMF, and were killed by 0.5-25 microg ml(-1) ganciclovir. We demonstrate efficient selection and killing of gene-modified cells and suggest LV-DeltaCD34/TK.IMPDH(IY)-transduced T cells could be used to facilitate allogeneic hematopoietic cell engraftment. The expression of IMPDH(IY) would allow in vivo selection with MMF, and DeltaCD34/TK expression would allow rapid and safe elimination of transduced T cells if graft-versus-host disease developed.

Conversion of a tumor-binding peptide identified by phage display to a functional chimeric T cell antigen receptor.

Adoptive transfer of ex vivo expanded tumor-specific T cells is a promising therapeutic modality for promoting or augmenting antitumor immunity. Several groups, including ours, are developing antigen receptor gene transfer strategies as a means of generating effector cells for adoptive therapy. Chimeric antigen receptors (CARs) have been described that use single-chain antibodies or cytokine ligands as tumor targeting domains. Here, we describe the capacity of a tumor-binding peptide identified by phage display combinatorial library screening to serve as a CAR targeting domain. A phage library-selected high-affinity 12-mer peptide (Bpep) specific for alpha(v) beta(6) integrin (alpha v beta6) was chosen for these studies. Primary human T cells were genetically modified to express the Bpep-CAR consisting of an alpha v beta6-specific peptide and human IgG4 hinge-Fc extracellular domain fused to the cytoplasmic tail of CD3-zeta. T cell expression of the Bpep-CAR was assessed by Western blot analysis, and trafficking of the Bpep-CAR to the cell surface was demonstrated by flow cytometry. Functionally, Bpep-CAR redirected cytotoxic T lymphocytes specifically kill integrin alpha v beta6+ ovarian tumor targets, and are activated for interferon gamma secretion. Our data suggest that large new repertoires of tumor-specific T cell antigen receptor transgenes might be available through merging combinatorial peptide libraries with CAR construct design.

Manufacturing of gene-modified cytotoxic T lymphocytes for autologous cellular therapy for lymphoma.

BACKGROUND: The production of therapeutic T-cell populations for adoptive immunotherapy of cancer requires extensive ex vivo cell processing, including the isolation or creation of Ag-specific T cells and their subsequent propagation to clinically relevant numbers. These procedures must be performed according to the principles of current good manufacturing practices (cGMP) for phase I clinical trials to ensure the identity, purity potency and safety of the cellular product. In this report we describe our approach to manufacturing and characterizing bulk populations of gene-modified autologous T cells for use in treating follicular lymphoma. METHODS: PBMC from healthy donors, obtained after informed consent, were stimulated in vitro with Ab to CD3epsilon (OKT3) and recombinant human IL-2 and then electroporated with plasmid DNA containing a human CD19-specific chimeric Ag receptor (CAR) gene and HSV-1 thymidine kinase (TK) gene. Stably transfected cells were selected in cytocidal concentrations of hygromycin B over multiple 14-day stimulation culture cycles and then cryopreserved. Vials of cryopreserved/selected T cells were used to initiate T-cell expansion cultures to produce cell products for clinical infusion. These cultures were characterized for phenotype, function and suitability for use in adoptive immunotherapy studies. RESULTS: Our results demonstrate that bulk populations of gene-modified T cells derived from peripheral blood of healthy donors express CD19+ chimeric Ag receptor at low levels and can specifically lyse CD19+ target cells in vitro. These cells display a differentiated T-effector phenotype, are sensitive to ganciclovir-mediated killing and display a non-transformed phenotype. TCR Vbeta usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5 x 10(9) and 1 x 10(10) cells/run. One of three transductions resulted in a population of cells that was not suitable for infusion but was identified during release testing. No populations displayed any evidence of bacterial, fungal or mycoplasma contamination. DISCUSSION: We have established a manufacturing strategy that is being used to produce T cells for a phase I clinical trial for follicular lymphoma. Genetically modified T cells have been characterized by cell-surface marker phenotype, functional activity against CD19+ targets and requisite safety testing. These pre-clinical data confirm the feasibility of this approach to manufacturing T-cell products.

Engineered CD20-specific primary human cytotoxic T lymphocytes for targeting B-cell malignancy.

BACKGROUND: Immunotherapy for B-cell lymphomas has evolved significantly with the advent of CD20-targeted Ab-based therapeutics. Strategies to invoke or augment cellular anti-lymphoma immune responses may also have considerable therapeutic potential and serve to further augment the clinical efficacy of MAbs. METHODS: We report here the aquisition by priming human cytotoxic T lymphocyte (CTL) effectors of re-directed CD20 specificity by their genetic modification to express a chimeric immunoreceptor consisting of an anti-CD20 single chain Ab extracellular domain molecularly fused to the T-cell receptor complex CD3-zeta cytoplasmic tail (scFvFczeta). Peripheral blood-derived human T-cells were transduced with naked DNA plasmid vector by electoporation then selected for G418 resistance. RESULTS: Following cloning in limiting dilution and ex vivo expansion to large numbers scFvFczeta+ TCRalpha/beta+ CD4- CD8+ CTL display re-directed HLA-unresricted CD20-specific lymphoma cell cytolysis proportional to the cell-surface density of the chimeric immunoreceptor. Engineered CTL clones are also activated through the chimeric immunoreceptor to produce Tc1 cytokines (IFN-gamma) upon co-culture with CD20+ lymphoma stimulator cells. Additionally, CD20-specific CTL proliferate in the presence of lymphoma stimulators and IL-2 (5 U/mL). DISCUSSION: These studies provide the rationale for exploring the clinical utility of adoptive therapy with CD20-specific CTL as a component of immunotherapeutic targeting of CD20+ malignancy.

Human T lymphocyte genetic modification with naked DNA.

Endowing T lymphocytes with novel functional attributes by genetic modification is under development for a broad range of clinical cellular immunotherapy applications. To circumvent many of the limitations associated with viral vector systems, a plasmid-based electroporation system that reliably generates G418-resistant primary human T lymphocyte clones was developed. TCR alpha/beta+ CD4+CD8-, and CD4-CD8+ T lymphocyte clones can be routinely isolated from OKT3-stimulated peripheral blood mononuclear cells electroporated with linear plasmid DNA in a limiting dilution drug selection format. Fluorescence in situ hybridization (FISH) studies performed on T cell metaphase spreads using a probe specific for plasmid sequence demonstrated a single FISH signal doublet that varied in chromosomal location from clone to clone. Southern blot analysis using a Neo-specific probe verified chromosomal integration of plasmid vector at a single site. Band intensity quantitation of blots developed with a zeta-specific probe capable of annealing to both endogenous TCR-zeta and the introduced chimeric zeta sequence demonstrated that integrated plasmid was present at a single copy number. Expression levels of the CD20-specific chimeric immunoreceptor construct from a CMV immediate/early promoter present in the plasmid vector varied widely from clone to clone but remained stable during ex vivo expansion to cell numbers in excess of 10(10). This T lymphocyte genetic modification strategy is currently being piloted in a FDA-sanctioned adoptive therapy trial for recurrent lymphoma.

Interobserver and intraobserver variability in interpretation of lumbar disc abnormalities. A comparison of two nomenclatures.

STUDY DESIGN: A double-blind prospective study was used to measure interobserver and intraobserver variability when interpreting lumbar spine magnetic resonance imaging studies of disc abnormalities. OBJECTIVES: To evaluate reader consistency when interpreting disc extension beyond the interspace, and assess the effect of two distinct nomenclatures on reader consistency. SUMMARY OF BACKGROUND DATA: Interobserver and intraobserver variability in interpretation of lumbar disc abnormalities is an important consideration in analyzing the technical efficacy of an imaging modality. However, this has not been well measured (particularly for standardized nomenclature). METHODS: Magnetic resonance imaging studies of the lumbar spine performed prospectively in 98 asymptomatic volunteers, and an additional 27 selected studies from symptomatic patients, were read blindly by two experienced neuroradiologists, using two separate nomenclatures. Only the discs were evaluated (625 interspaces). Nomenclature I was normal, bulge, herniation. Nomenclature II was normal, bulge, protrusion, extrusion. Intraobserver and interobserver variation were measured with Kappa statistic analysis. RESULTS: Interobserver agreement was 80% for both nomenclatures with a Kappa statistic of 0.58. Intraobserver agreement was 86% for each reader, with a Kappa statistic of 0.71 and 0.69, respectively. The most common disagreement was for normal versus bulge. The next most common disagreement (5-6%) was for bulge versus herniation (or protrusion in Nomenclature II). Herniation was read in 23% of the asymptomatic subjects. Using Nomenclature II, protrusion was seen in 27% of these subjects. Extrusion was read in only two asymptomatic subjects. CONCLUSIONS: Experienced readers using standardized nomenclature showed moderate to substantial agreement with interpreting disc extension beyond the interspace on magnetic resonance imaging.

MRI of degenerative disease of the lumbar spine.

Low back pain (LBP) is one of the most common reasons that patients seek medical attention. Although acute LBP is generally a self-limiting condition, the estimated cost for this health care problem exceeds $8 billion annually. MR accurately depicts both the morphologic as well as biochemical sequelae of disc degeneration. Additionally, MR is superior in its ability to depict disease processes that can present in an indistinguishable fashion. Although multiple mechanisms have been proposed for the possible etiology of disc degeneration, it remains incompletely understood at this time. In addition to the unknown etiology of disc degeneration, the relationship between degenerative disc disease and LBP has not been firmly established. Substantial percentages of people without a history of LBP or sciatica have been shown to have abnormal imaging examinations. Mechanical compression of neural elements by disc herniation, as well as direct biochemical and inflammatory effects of the contents of the nucleus pulposus upon neural structures, have been proposed as possible sources of LBP. Due to the above, caution is urged before attributing a particular anatomic finding as the patient's source of low back pain.

Magnetic resonance imaging of the lumbar spine in people without back pain.

BACKGROUND: The relation between abnormalities in the lumbar spine and low back pain is controversial. We examined the prevalence of abnormal findings on magnetic resonance imaging (MRI) scans of the lumbar spine in people without back pain. METHODS: We performed MRI examinations on 98 asymptomatic people. The scans were read independently by two neuroradiologists who did not know the clinical status of the subjects. To reduce the possibility of bias in interpreting the studies, abnormal MRI scans from 27 people with back pain were mixed randomly with the scans from the asymptomatic people. We used the following standardized terms to classify the five intervertebral disks in the lumbosacral spine: normal, bulge (circumferential symmetric extension of the disk beyond the interspace), protrusion (focal or asymmetric extension of the disk beyond the interspace), and extrusion (more extreme extension of the disk beyond the interspace). Nonintervertebral disk abnormalities, such as facet arthropathy, were also documented. RESULTS: Thirty-six percent of the 98 asymptomatic subjects had normal disks at all levels. With the results of the two readings averaged, 52 percent of the subjects had a bulge at at least one level, 27 percent had a protrusion, and 1 percent had an extrusion. Thirty-eight percent had an abnormality of more than one intervertebral disk. The prevalence of bulges, but not of protrusions, increased with age. The most common nonintervertebral disk abnormalities were Schmorl's nodes (herniation of the disk into the vertebral-body end plate), found in 19 percent of the subjects; annular defects (disruption of the outer fibrous ring of the disk), in 14 percent; and facet arthropathy (degenerative disease of the posterior articular processes of the vertebrae), in 8 percent. The findings were similar in men and women. CONCLUSIONS: On MRI examination of the lumbar spine, many people without back pain have disk bulges or protrusions but not extrusions. Given the high prevalence of these findings and of back pain, the discovery by MRI of bulges or protrusions in people with low back pain may frequently be coincidental.

Pictorial review of the usual and unusual roentgen manifestations of childhood tuberculosis.

Despite advances in management of infectious diseases, tuberculosis remains a significant cause of morbidity and mortality in urban populations. In the pediatric age group, 65% of patients presenting with tuberculosis are asymptomatic. Recognition of the usual and unusual roentgen manifestations of this infectious disease often provides the only clue to the diagnosis.

MR imaging of the brain in patients with AIDS: value of routine use of i.v. gadopentetate dimeglumine.

OBJECTIVE: A prospective study was conducted to explore the value of routine administration of IV gadopentetate dimeglumine for MR imaging of the brain in patients with AIDS. SUBJECTS AND METHODS: Over a 19-month period, MR images of the brain in 51 consecutive AIDS patients were obtained routinely both with and without IV gadopentetate dimeglumine. Unenhanced and contrast-enhanced images from the resulting 63 studies were viewed together. Findings were classified into one or more of three categories: normal, mass or focal lesions, or white matter disease. The number of focal or mass lesions was recorded. Lesion conspicuity on the unenhanced and enhanced images was compared. Ventricular enlargement was also graded. Available medical records and laboratory data of the patients were reviewed. RESULTS: Of the 63 MR studies reviewed, 39 (62%) were abnormal. In no case was a normal unenhanced MR study rendered abnormal after the administration of gadopentetate dimeglumine. Overall, T2-weighted images showed twice as many focal or mass lesions than contrast-enhanced T1-weighted images did. Most lesions detected on the T2-weighted images did not show enhancement with contrast material. White matter disease was the most common abnormality detected. The group of patients with white matter disease also had the highest occurrence of ventriculomegaly. CONCLUSION: Our study does not support the routine use of gadopentetate dimeglumine for MR imaging of the brain in patients with AIDS. Our experience emphasizes the importance of a normal T2-weighted image.

Whole body nitrogen kinetics and their relationship to growth in short children treated with recombinant human growth hormone.

We studied the effects of growth hormone on retention of 15N-labeled amino acids in 34 short, prepubertal, growth hormone-sufficient children and three growth hormone-deficient subjects. All 34 non-growth hormone-deficient children had apparently normal circulating growth hormone molecules and no mutations were detected in the growth hormone or IGF-I genes of any subjects. Fibroblasts from 34 children responded normally when challenged with recombinant human IGF-I. During the last 72 h of a 4-d challenge with recombinant human growth hormone (16 micrograms/kg body wt), retention of a mixed 15N-amino acid dose varied between 5.7 and 50.5%. Whole body protein synthesis, breakdown, and net anabolism calculated from the 15N kinetics were all increased by the acute growth hormone challenge. However, no routine clinical feature or laboratory determination correlated with the nitrogen retention response. After subsequent treatment (75 micrograms/kg three times a week) with recombinant human growth hormone for 1 y, there was a significant increase in height velocity, but this increase was not related significantly to pretreatment variables other than inversely to pretreatment height velocity. There was a significant (p = 0.03) correlation between the change in height velocity Z score and the degree of nitrogen retention to acute challenge with growth hormone, but this correlation was too weak (r = 0.37) to be of practical value in predicting the treatment growth response in an individual child.

Color Doppler sonography in acute epididymitis and orchitis.

Clinical diagnosis of patients with acute scrotal pain is frequently imperfect. Imaging, using nuclear medicine scintigraphy and hand-held continuous-wave Doppler ultrasound devices, has been used in these patients. We retrospectively analyzed 28 consecutive patients referred for scrotal sonography, all of whom had been imaged using color Doppler sonography. Of 22 patients with confirmed diagnoses, 11 had acute epididymitis/orchitis and 11 had another diagnosis. Ten of 11 patients with acute epididymitis/orchitis had increased epididymal flow. Eight also had increased testicular flow. None of the 11 patients without acute epididymitis/orchitis had increased flow. Our data suggest that color Doppler sonography may be useful in establishing the diagnosis of acute epididymitis/orchitis. This might decrease the need for scrotal exploration. No distinction could be made among scrotal lesions in the nonacute epididymitis/orchitis group. Sensitivity was inadequate to reliably detect flow in normal testicles, a prerequisite to accurately diagnose torsion. Newly improved sensitivity may enhance the utility of color Doppler sonography in assessing patients with acute scrotal pathology.

CEO incentives-its not how much you pay, but how.

The arrival of spring means yet another round in the national debate over executive compensation. But the critics have it wrong. The relentless attention on how much CEOs are paid diverts public attention from the real problem-how CEOs are paid. The authors present an in-depth statistical analysis of executive compensation. The study incorporates data on thousands of CEOs spanning five decades. Their surprising conclusions are at odds with the prevailing wisdom on CEO pay: Despite the headlines, top executives are not receiving record salaries and bonuses. Cash compensation has increased over the past 15 years, but CEO pay levels are just now catching up to where they were 50 years ago. Annual changes in executive compensation do not reflect changes in corporate performance. For the median CEO in the 250 largest public companies, a $1,000 change in shareholder value corresponds to a change of just 6.7 cents in salary and bonus over a two-year period. With respect to pay for performance, CEO compensation is getting worse rather than better. CEO stock ownership-the best link between shareholder wealth and executive well-being-was ten times greater in the 1930s than in the 1980s. Compensation policy is one of the most important factors in an organization's success. Not only does it shape how top executives behave but it also helps determine what kind of executives an organization attracts. That's why it's so urgent that boards of directors reform their compensation practices and adopt systems that reward outstanding performance and penalize poor performance.