Purpose: A phase I study was conducted to assess the safety of adoptive immunotherapy using gene-modified autologousTcells for the treatment of metastatic ovarian cancer. Experimental Design: T cells with reactivity against the ovarian cancer^associated antigen a-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor g chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1received a dose escalation of Tcells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specificTcells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells.

Purpose Adoptive transfer of genetically modified T cells is being explored as a treatment for patients with metastatic cancer. Most current strategies use genes that encode major histocompatibility complex (MHC) class I-restricted T-cell receptors (TCRs) or chimeric antigen receptors to genetically modify CD8 T cells or bulk T cells for treatment. Here, we evaluated the safety and efficacy of an adoptive CD4 T-cell therapy using an MHC class II-restricted, HLA-DPB1*0401-restricted TCR that recognized the cancer germline antigen, MAGE-A3 (melanoma-associated antigen-A3). Patients and Methods Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4 T cells, retrovirally transduced with MAGE-A3 TCR plus systemic high-dose IL-2. A cell dose escalation was conducted, starting at 10 total cells and escalating at half-log increments to approximately 10 cells. Nine patients were treated at the highest dose level (0.78 to 1.23 × 10 cells). Results Seventeen patients were treated. During the cell dose-escalation phase, an objective complete response was observed in a patient with metastatic cervical cancer who received 2.7 × 10 cells (ongoing at ≥ 29 months). Among nine patients who were treated at the highest dose level, objective partial responses were observed in a patient with esophageal cancer (duration, 4 months), a patient with urothelial cancer (ongoing at ≥ 19 months), and a patient with osteosarcoma (duration, 4 months). Most patients experienced transient fevers and the expected hematologic toxicities from lymphodepletion pretreatment. Two patients experienced transient grade 3 and 4 transaminase elevations. There were no treatment-related deaths. Conclusion These results demonstrate the safety and efficacy of administering autologous CD4 T cells that are genetically engineered to express an MHC class II-restricted antitumor TCR that targets MAGE-A3. This clinical trial extends the reach of TCR gene therapy for patients with metastatic cancer.

Between 1989 and 1993, 255 tumor biopsies representing 4 tumor histologies (melanoma, breast cancer, colon cancer and renal cell cancer) were received by the Surgery Branch of the National Cancer Institute. Tumor-infiltrating lymphocytes (TIL) were grown from single-cell suspensions of tumor biopsies over the course of 30-45 days. The TIL were grown in medium containing IL-2. To obtain numbers suitable for therapy (>10(11)), TIL were expanded using a large-scale system of cell culture and harvesting. While the largest number of biopsies was obtained from melanoma patients, TIL were successfully grown from 160 of 255 tumor biopsies representing all 4 histologies. Under the culture conditions employed, several characteristics of TIL expansion were observed. The cell surface phenotype of TIL which grew out from the tumor biopsies was generally a mix of CD3+/CD4+ or CD3+/CD8+ lymphocytes. Only TIL from melanoma biopsies were found to be consistently cytolytic and, in many cases, lysed autologous tumor cells preferentially. Interestingly, TIL derived from extra-nodal sites of metastatic melanoma biopsies (subcutaneous, lung, bowel; 36 of 67, 54%) were more likely to have these cytolytic characteristics than TIL derived from tumor-involved lymph node biopsies (7 of 39, 18%). The present study summarizes 5 years of laboratory effort and validates the technologies developed for the large-scale growth and harvesting of TIL. In addition, it summarizes the laboratory effort supporting previously published clinical reports on TIL from our group.

Increasing attention has been devoted to elucidating the mechanism of lost or decreased expression of MHC or melanoma-associated antigens (MAAs), which may lead to tumor escape from immune recognition. Loss of expression of HLA class I or MAA has, as an undisputed consequence, loss of recognition by HLA class I-restricted cytotoxic T cells (CTLs). However, the relevance of downregulation remains in question in terms of frequency of occurrence. Moreover the functional significance of epitope down-regulation, defining the relationship between MHC/epitope density and CTL interactions, is a matter of controversy, particularly with regard to whether the noted variability of expression of MHC/epitope occurs within a range likely to affect target recognition by CTLs. In this study, bulk metastatic melanoma cell lines originated from 25 HLA-A*0201 patients were analyzed for expression of HLA-A2 and MAAs. HLA-A2 expression was heterogeneous and correlated with lysis by CTLs. Sensitivity to lysis was also independently affected by the amount of ligand available for binding at concentrations of 0.001 to 1 mM. Natural expression of MAA was variable, independent from the expression of HLA-A*0201, and a significant co-factor determining recognition of melanoma targets. Thus, the naturally occurring variation in the expression of MAA and/or HLA documented by our in vitro results modulates recognition of melanoma targets and may (i) partially explain CTL-target interactions in vitro and (ii) elucidate potential mechanisms for progressive escape of tumor cells from immune recognition in vivo.

Suboptimal N nutrition increased the water potential for stomatal closure in water stressed cotton (Gossypium hirsutum L.) leaves. This increased sensitivity to water stress had two components, increased accumulation of abscisic acid (ABA) and increased apparent stomatal sensitivity to ABA. Low N increased the threshold water potentials for stomatal closure and ABA accumulation by about 4 bars and 2 bars, respectively. Low N also greatly increased stomatal response to low concentrations of exogenous ABA applied to excised leaves through the transpiration stream. In low N leaves, kinetin decreased stomatal response to ABA to the level observed with high N leaves. Kinetin by itself had little effect on stomata, nor did it alter stomatal response to ABA in high N leaves. The results suggest a cytokinin-ABA balance which is altered by suboptimal N nutrition to favor stomatal closure during stress.Ambient temperature and N nutrition interacted to alter stomatal response to water stress. Stress-induced ABA accumulation and apparent stomatal sensitivity to ABA were independently affected. The effects of each treatment, and their interaction, could be explained as the net result of changes in both accumulation and apparent sensitivity. Although the results document environmental control of stomatal response to ABA, either altered partitioning of ABA between active and inactive pools, or altered sensitivity of the guard cells, could account for the data.Suboptimal N nutrition sensitizes stomata to water stress, causing stomatal closure at a higher water potential than normal (10,12). This early closure is not based upon a difference in leaf turgor (13). Some evidence indicates that low N promotes ABA accumulation during stress in parallel with stomatal closure (12). Closure in both low N and high N plants is accompanied by increased stomatal sensitivity to CO2 (12), a characteristic considered diagnostic for a role of ABA (16,17). These results strongly suggest that N effects on stomatal closure during drought are mediated by ABA. High temperature also affects stomatal closure (15). In cotton, N nutrition and ambient temperature interact to control stomatal responses to water stress (14). However, no data are available to show whether temperature effects on stomata are also mediated by ABA. Answering this question was one goal of the present study.Trewavas (22) recently suggested that stomatal sensitivity to ABA, rather than the concentration of ABA at the guard cells, may control stomatal closure in water stressed leaves. This proposal is consistent with stomatal sensitivity to CO2 (12), because it accommodates a role of ABA in stomatal closure (albeit a noncontrolling role). Control of sensitivity to ABA is a concept which has received little attention. Davies (3) and Ackerson (1) reported that water stress increased stomatal sensitivity to applied ABA, but Raschke and Zeevaart (18) found no effect of leaf age on stomatal sensitivity to ABA in immature Xanthium leaves. Therefore, our second goal was to det...

Cotton plants (Gossypium hinaum L.) grown on deficient levels of N exhibited many of the characteristics associated with dronght resistance. In N-deficient plants, leaf areas and leaf epidermal cells were smaller than at the same nodes in highN plants. N-deficient Drought resistance of plants, and the water deficit-induced development of characters associated with drought resistance (adaptation) have received much attention in recent years (1-3, 8, 9, 19). Characters which could have adaptive value include decreased leaf size and shoot: root ratio, often with increased root dry weight (2); decreased succulence (% water) (3, 5); decreased loss of water per unit change in '1 (1, 2, 19); and osmotic adjustment (8, 9).Many of these responses are the same as or similar to responses to N deficiency (7,12). Thus, the question may be posed whether N nutrition can affect drought adaptation. Because NO3 assimilation is' quite sensitive to water deficit (21), any effects of N deficiency also need to be assessed in terms of their possible role in adaptation induced by water deficit. In this paper we compare the water relations of leaves from N-deficient plants to those of leaves from normal plants. In Relative Water Content and Water Potential. When low-N plants reached the stage of full expansion of the fifth leaf, watering was stopped. At various times after initiation of drying, leaves were removed from the plants and small interveinal segments were excised, weighed, and floated on distilled H20 in Petri dishes.After 24 h at 30 C in a lighted growth cabinet, the leaf pieces were blotted, reweighed, then dried for 24 h in a forced-draft oven at 70 C for dry weight determination. RWC was calculated according to Slatyer (23).Immediately after removal of the small segments, leaves were transferred to a pressure chamber (Soilmoisture Equipment Co., Santa Barbara, Calif.) and I determined by standard techniques (10). All measurements were made in early afternoon, at the time of minimum I. Segment excision had no apparent effect on determination of I.Pressure-induced Exudation. Small interveinal segments were excised from leaf blades of intact well-watered plants, and the RWCs were determined as above. Leaves were then cut from the plant and placed in the pressure chamber. A thin tapered glass tube (made from a Pasteur pipette tip bent to an acute angle) was placed over the cut end of the petiole and sealed to it with silicone stopcock lubricant. The pressure was raised to the threshold for exudation, then slowly increased further in increments of 2 to 4 bars. After each step, gas escaping through the cut end of the petiole forced droplets of exuding sap through the tube. The sap was collected in a preweighed glass vial and the amount collected was followed as increase in vial weight.

Adoptive immunotherapy with genetically modified T lymphocytes is being utilized in clinical trials for the treatment of a broad range of diseases including cancer and HIV infection. To improve on these treatments, and to better understand their mechanisms of action, it is necessary to develop techniques to generate large numbers of cells and characterize the functional heterogeneity of the cells produced. In this study, patient peripheral blood lymphocytes were transduced with a chimeric antigen receptor (MOv-gamma) derived from a mouse monoclonal antibody against folate-binding protein, which is overexpressed on many ovarian cancers. Thus, irrespective of their original specificity, normal human T lymphocytes were redirected to react against ovarian cancer cells. Lymphocytes from five patients were transduced and grown to large numbers, with a median expansion of more than 7000-fold. When proliferation was inadequate, the cells were expanded by stimulation utilizing anti-CD3, IL-2, and irradiated allogeneic PBMCs. The cells maintained their functional ability to recognize ovarian cancer over several months. Cloning of transduced cells was undertaken to determine the level of gene expression and function of individual cells making up the bulk population. Transduced CD4(+) and CD8(+) cell clones were isolated from the bulk and demonstrated antitumor activity. These clones had a diverse repertoire with respect to secretion of cytokines, and individual clones maintained their cytokine profile on subsequent expansion. These studies establish the feasibility of consistently generating large numbers of gene-modified tumor-reactive lymphocytes, with a stable and diverse cytokine repertoire, that could be utilized for patient treatment.

SummaryThe ability to selectively enrich or deplete T lymphocytes of specific phenotype and function holds significant promise for application in adoptive immunotherapy protocols. Although CD4 + T cells can have an impact on CD8 + T-cell effector function, memory, and maintenance, a subset of CD4 + T cells, CD25 + regulatory T cells (T reg ), can regulate peripheral self tolerance and possess the ability to suppress antitumor responses. The authors report the ability to selectively deplete CD25 + T reg cells from patient leukapheresis samples using a clinical-grade, large-scale immunomagnetic system. Using leukapheresis samples containing up to 1.3 × 10 10 white blood cells, efficient depletion of T reg cells was measured by flow cytometric analysis of CD25 expression and FOXP3 expression on post-depletion products. Remnant CD25 + cells could not be detected in CD25-depleted products after short-term culture in IL-2 or enriched following secondary immunomagnetic selection for CD25 + cells, confirming that efficient depletion had occurred. In parallel to efficient enrichment of CD25 − cells, immunomagnetic selection resulted in the recovery of T reg cells, since CD25 + lymphocytes removed during depletion were primarily composed of CD4 + T cells that expressed high levels of FOXP3 and possessed suppressive activity against autologous TCR-stimulated CD4 + CD25 − T cells in vitro. These results show that selective separation of functional CD25 + T reg cells from large-scale samples can be performed in large scale under clinical-grade conditions with sufficient selection, recovery, viability, ability to expand, and function for potential use in adoptive immunotherapy. Keywordshuman; CD25; regulatory T cells; clinical; depletion Naturally occurring CD4 + CD25 + T regulatory (T reg ) cells are involved in the maintenance of homeostatic peripheral self tolerance by suppressing autoreactive T cells. 1 In mice and humans, T reg cells develop in the thymus and represent about 5% to 10% of the peripheral CD4 + T-cell compartment. These regulatory cells are characterized by their constitutive expression of CD25, CTLA-4, glucocorticoid-induced TNFR (GITR), and the transcription factor Forkhead box P3 (Foxp3). 1 Compared with CD25 − CD4 + T cells, T reg cells exhibit a hypoproliferative capacity and possess the ability to suppress CD8 + and CD25 − CD4 + T cell activation in vitro. The mechanism of suppression is cell contact-dependent, but how regulatory T cells induce and maintain self tolerance is unknown.The importance of T reg cells as key mediators of self tolerance is witnessed in individuals genetically predisposed to the immune dysregulation, polyendocrinopathy, enteropathy, Xlinked syndrome (IPEX), a recessive and often fatal disorder of early childhood caused by loss Reprints: