BACKGROUND: Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to ...depletion of normal CD4+ T-cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells (ACTC) expanded ex vivo may enhance immune reconstitution and improve disease control.
METHODS: We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1–3 and cyclophosphamide (250 mg/m2) days 1–3. Four weeks after the last cycle, responding patients (CR/CRu, PR) receive escalating doses of ACTC prepared ex vivo from autologous T-cellss collected prior to chemotherapy and depleted of regulatory CD4+/CD25+ cells, then expanded and activated using anti-CD3 and anti-CD28.
RESULTS: Eleven pts have been enrolled to date. Median age is 49 y (range: 33–64y). Median number of prior therapies is 2 (range: 1 – 3). Two pts were withdrawn from the study due to hematologic toxicity related to chemotherapy, one patient was withdrawn for progressive disease during chemotherapy, and one patient has not yet completed chemotherapy. Of the 7 pts completing chemotherapy and proceeding to T-cell infusion, 5 pts achieved a CR/CRu and 2 pts achieved a PR; 5 pts received 1 – 5 x 109 CD3+ cells and 2 patients received 5 – 10 x 109 CD3+ cells. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 19 mos (range: 3 – 26 mos). CD4+ counts increased in all patients by 1 month after T-cell infusion, with a median increase of 3.2 fold (range: 1.5 – 70, p= .028)(see figure). For patients at dose level 1, the median increase was 2.2 fold (n=4; range: 1.5 – 3.3); at dose level 2 it was 37 fold (n=2; range: 3.8 – 70). CD8+ counts also increased, with a median increase of 8.1 (range: 1.0 – 30, p= .046)(see figure). All 7 pts receiving ACTC were anergic to candida antigen by delayed type hypersensitivity (DTH) skin testing before chemotherapy. Four pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. For patients receiving ACTC, median follow-up is 24 mos (range: 7 – 31 mos) with a median progression-free survival of 18 months which is significantly longer than the time to progression from last therapy (p= .024).
CONCLUSIONS: ACTC infusion results in significant CD4+ and CD8+ numerical and functional lymphocyte recovery after cyclophosphamide-fludarabine chemotherapy in pts with low-grade FL. This compares very favorably to historical controls treated with fludarabine-based regimens. T-cell dose escalation is ongoing.
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Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of ...normal CD4 T-cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) may enhance immune reconstitution and improve disease control. We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1–3 and cyclophosphamide (250 mg/m2) days 1–3. Four weeks after last chemotherapy, responding patients (CR, CRu, PR) receive escalating doses of ACTC prepared ex vivo from autologous T-cells collected prior to chemotherapy and depleted of regulatory CD4+/CD25+ cells, then expanded and activated using anti-CD3 and anti-CD28. Eight pts have been enrolled to date. Median age is 40.5 y (range: 32–64). Median number of prior therapies is 2 (range: 1–3). Two pts were withdrawn from the study due to hematologic toxicity related to the chemotherapy; one patient has not completed chemotherapy. Of the 5 pts completing chemotherapy, 3 pts achieved a CR and 2 pts achieved a PR; 4 pts received 5 x 109 and 1 patient received 1 x 1010 CD3+ ACTC. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 9 months (range: 2–15 months). Median time to CD4 count >200 /uL was 29 days following T-cell infusion (range 28–127 days). CD4 counts increased in all patients by 1 month after T-cell infusion, with a median increase of 126% from baseline (range 50 to 484%). CD8 counts also increased, with a median increase of 82% (range −4 to 976%). All 5 pts receiving ACTC were anergic to candida antigen by Delayed Type Hypersensitivity (DTH) skin testing before chemotherapy. Three pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. From the start of therapy for patients receiving T-cells, median follow-up is 14 months (range 7–20 months) with median progression-free survival not reached; 4 pts remain in remission and 1 patient had progression of disease. ACTC results in significant CD4+ lymphocyte recovery in previously treated pts receiving cyclophosphamide-fludarabine chemotherapy and compares very favorably with historical controls. T-cell dose escalation is ongoing.
Although immunizations against microbial infections are recommended following autologous stem cell transplantation (ASCT), the antibody responses are often suboptimal. To assess improved immunization ...strategies, we tested the ability of autologous T cells that were co-stimulated ex vivo with anti-CD3 and -CD28 coated beads as part of their therapy for MM, as well as pre-ASCT immunization to enhance the antibody response to PCV post-ASCT. Subjects were randomized to receive PCV (groups 1 and 2) or no immunization (groups 3 and 4) before T cell harvest, and subsequent mobilization chemotherapy. All subjects received PCV at days 30 and 90 post-ASCT; however, groups 1 and 3 were given T cells before (day 12) post-ASCT PCV immunization, while groups 2 and 4 received T cells after PCV immunization (day 100). Patients receiving adoptive T cell therapy for MM without PCV immunization (group 5) served as controls. The results of the adoptive transfer of T cells on MM therapy are reported elsewhere. Anti-pneumococcal (Pn) antibody levels for serotypes 6B, 14, 23F, 19F were measured in sequential samples for up to 180 days for each patient by ELISA (response defined as ≥ 2-fold increase over baseline and ≥ 0.5 μg/ml) and opsonophagocytosis assay (OPA) (response defined as ≥ 1-log bacterial kill or ≥ 70% kill when ≤ 30% kill at baseline).
Groups12345No. PCV33220T given atD12D100D12D100D12≥3 types6/7a3/83/93/61/44 types6/7*1/81/93/60/4OPA8/9b**5/81/83/51/4a Numbers of evaluable subjects per group responding by ELISA to 3 or more of 4 pneumococcal serotypes tested. bNumber of evaluable subjects with OPA response to 19F only. T=T cells; D=day;* P=0.02, and** P=0.05 v. group 5 by Fisher's Test.
Responses in group 1 were of longer duration and greater magnitude than in other groups. We conclude that (1) MM patients immunized with PCV pre-ASCT respond better to PCV post-ASCT; (2) T cell administration before PCV immunization enhances protective anti-Pn antibody levels and for a longer duration. Our vaccine schedule that leads to improved PCV responses may also be used to improve the responses of ASCT patients to other microbial and perhaps cancer vaccines. (This study was supported by NIH contract N01-AI-85342 and LSA SCOR #7000-002).
Ex-vivo co-stimulation of autologous T-cells with anti-CD3/anti-CD28-conjugated magnetic beads followed by adoptive transfer may augment T-cell responses toward tumor antigens or infectious agents. ...54 patients (pts) were treated with ex-vivo co-stimulated autologous T-cells after autotransplantation for myeloma. The median age was 56 (range 38–71), 67% were male, 20% were African-American, 22% had IgA paraproteins, 11% had del 13 or complex karyotypes and the median β2m level at diagnosis was 3.31 mg/L (range 1.09–73.7). After lymphocyte collections, pts received cyclophosphamide (4.5 g/m2) + G-CSF for stem cell mobilization, and high dose melphalan (200 mg/m2 or 140 mg/m2 for pts ≥ 70) for conditioning. T-cells were cultured for ~12 days with anti-CD3/anti-CD28-immobilized immunomagnetic beads + IL-2 supplementation (100 units/ml). During a run-in phase, 12 pts received co-stimulated T-cells post-transplant ( day +12) alone. In a second phase, 42 pts who participated in a 2 x 2 randomization, received T-cells either early (day +12) or late (day + 100) after transplant. These pts also received either 2 immunizations with the pneumococcal conjugate vaccine (PCV, Prevnar) at days +30, +90 or 3 immunizations (prior to T-cell collection, at days + 30, + 90) to test immune responses to a well-defined antigen. 42 pts received PCV immunizations with no grade 3/4 adverse events. Anti-pneumococcal antibody responses developed in 51% of 31 pts tested thus far. Details of these studies will be presented separately. 52 pts received a mean dose of 8.04 x 109 costimulated T-cells (range 1.6–11). Infusion-related adverse effects included grade I–II rigors/chills (40%), grade I–II facial/upper body rashes (12%) at a median of 13 days after T-cells, grade I cardiovascular events (10%), grade I–III hypoxia (5%), grade II fever (5%), and 1 episode of DVT. At T-cell harvesting, the mean % of CD3 + cells in culture was 94.8%, the mean T-cell doubling level was 4.81 (28-fold expansion). Among the 42 randomized pts, at day + 42 post-transplant ( 30 days after T-cell infusion for the early groups), the median CD4/CD3 count was 462/mcl (range 202–1439) for the early T-cell recipients vs 230/mcl (range 50–915) for the late T-cell recipients (T-cells not yet infused) P=0.004. The median CD8/CD3 counts were 1399/mcl (range 465–2810) vs 1084/mcl (range 103–3422) for the early and late T-cell recipients respectively at day +42 P=0.04. For clinical responses there were 11 CRs, 22 VGPRs ( 90% reduction in paraprotein levels), 18 PRs (50–90% reductions), 2 pts had no response and 1 pt was unevaluable. 2 pts had delayed reductions in M-protein levels of ~ 50% between day 42 and day 180 or 270. For the entire cohort, the probability of overall survival at 1.5 years was 78% 95%CI 64%–92%. Infusions of ex-vivo expanded autologous T-cells are well-tolerated and associated with accelerated T-cell recovery early after autotransplantation. This study may provide a platform for combining costimulated T-cells and tumor vaccines in the autograft setting.
To study the safety and feasibility of T-cell reconstitution in HIV-infected individuals, we adoptively transferred activated autologous CD4 super(+) T cells. Polyclonal peripheral blood CD4 super(+) ...cells were costimulated ex vivo and subjects were given infusions of up to 3 x 10 super(10) activated CD4 super(+) cells. Dose-dependent increases in CD4 super(+) cell counts and in the CD4:CD8 ratio were observed. Sustained increases in the fraction of cytokine-secreting T cells and decreases in the percentage of CD4 super(+)CCR5 super(+) cells were noted in vivo, suggesting enhanced function and resistance to HIV infection. The frequency of CD4 super(+)Ki-67 super(+) cells increased whereas CD4 super(+) T cells containing T cell-receptor rearrangement excision circles (TRECs) decreased. These findings indicate that expansion of the peripheral T-cell pool mediated the increase in CD4 counts and suggest that approaches to reconstitute CD4 helper cell activity and decrease CCR5 expression may augment natural immunity to HIV infection.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells. This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 ...coated beads (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtained by density gradient centrifugation of an apheresis product. Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG. The cells were then magnetically depleted of B cells and CD8+ cells with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads. The remaining cells were >80% CD4+ and were transferred to gas-permeable bags containing CD3/CD28 beads and cultured in a closed system. After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+. Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents. Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow. The cells were then concentrated to 300 ml in an automated centrifuge. This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors.
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