ALBERT

Description: CTLA-4 and PD-1 are receptors that negatively regulate T cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28 mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4 mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28 mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/Atk activation was dependent upon the ITSM located in its cytoplasmic tail. Lastly, PD-1 ligation is more effective in suppressing CD3/28 induced changes in the T cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T cell activation through distinct and potentially synergistic mechanisms.

Description: T-bodies that combine the antigen recognition capability of single chain antibody molecules (scFv) with the signal transduction domains of activating receptors can be used to generate T cells with novel, engineered specificities independent of their endogenous TCR. We have developed a series of T-bodies with specificity towards CD19, a molecule with expression that appears restricted to normal and malignant B cells. These T-bodies contain the activating signals from the costimulatory T cell receptors, CD28 and/or 4-1BB, in tandem with the TCR-ζ signal transduction domain. Using lentiviral gene transfer and T cells expansion via bead-immobilized anti-CD3 and anti-CD28 antibodies, we can generate T cells 95% of which express the T-bodies. Lentiviral transduction combined with this efficient T cell expansion system has the potential to produce 〉1011 antigen-specific T cells without the need for a cell selection step. Interestingly, we have noted significant constitutive signaling effects of T-body overexpression on transduced T cells that may been overlooked previously due to lower transduction efficiency. Despite the observed constitutive effects, we show that lentiviral-engineered T cells expressing anti-CD19 T bodies efficiently kill CD19 expressing cell lines and primary B-ALL cells in an antigen-specific manner. T-body engineered T cells also produce cytokines in response to antigen triggering with different patterns of cytokine production by T cells engineered with different T-bodies. In particular, a T-body receptor containing the 4-1BB signal transduction domain combined with the TCR-ζ domain stimulates a 〉6-fold increase in IL-6 production compared with other receptor combinations in CD4+ T cells. In contrast, a CD28 and TCR-ζ domain containing T body uniquely enhances IL-2 (〉3-fold) and TNF-α (〉 7-fold) secretion by CD8+ T cells. These results indicate that the functional characteristics of our tested T-body signaling constructs are distinct and depend upon the particular receptor and T cell subset combination. We are also comparing the anti-tumor response generated by adoptively transferred T-body engineered T cells in leukemia bearing immunodeficient mice as a pre-clinical, therapeutic model. In addition to elucidating important functional differences of our lentiviral constructs, these studies indicate that an important variable in clinical trials will be optimizing the cytosolic signaling domain for the desired T cell subset. The present results suggest that distinct combinatorial signaling domains may be required for optimal function in CD4 and CD8 engineered T cells.

Description: Previously, we showed that human umbilical cord blood (UCB) regulatory T cells (Tregs) could be expanded approximately 100-fold using anti-CD3/28 monoclonal antibody (mAb)–coated beads to provide T-cell receptor and costimulatory signals. Because Treg numbers from a single UCB unit are limited, we explored the use of cell-based artificial antigen-presenting cells (aAPCs) preloaded with anti-CD3/28 mAbs to achieve higher levels of Treg expansion. Compared with beads, aAPCs had similar expansion properties while significantly increasing transforming growth factor β (TGF-β) secretion and the potency of Treg suppressor function. aAPCs modified to coexpress OX40L or 4-1BBL expanded UCB Tregs to a significantly greater extent than bead- or nonmodified aAPC cultures, reaching mean expansion levels exceeding 1250-fold. Despite the high expansion and in contrast to studies using other Treg sources, neither OX40 nor 4-1BB signaling of UCB Tregs reduced in vitro suppression. UCB Tregs expanded with 4-1BBL expressing aAPCs had decreased levels of proapoptotic bim. UCB Tregs expanded with nonmodified or modified aAPCs versus beads resulted in higher survival associated with increased Treg persistence in a xeno-geneic graft-versus-host disease lethality model. These data offer a novel approach for UCB Treg expansion using aAPCs, including those coexpressing OX40L or 4-1BBL.

Description: Abstract 513 UCB transplantation (UCBT) is associated with greater risk of graft rejection and, in particular in double UCBT, with graft vs. host disease (GVHD). Our group has shown in mouse models that ex vivo activated and expanded Tregs improve engraftment and reduce and treat GVHD. We here report on the first clinical trial to study the safety of the infusion of UCB Tregs in patients receiving a nonmyeloablative double UCB transplant. We enrolled 19 patients (pts) (age 25-65 yrs; weight 52-133; male 9; CMV seropositive 6) with high risk or advanced leukemia (n=12) and lymphoma/CLL (n=7). All patients received the same nonmyeloablative conditioning consisting of cyclophosphamide 50mg/kg/×1day, fludarabine 40 mg/m2/×5d, and total body irradiation 200 cGy/×1d; immunosuppression was cyclosporine A/mycophenolate mofetil (MMF) in the first 17 and sirolimus (Siro)/MMF in the last 2 pts. Tregs were obtained from a 3rd UCB unit and after CD25+ selection (Miltenyi CliniMACS) were activated and expanded in the presence of anti-CD3/CD28 mAb coated beads and IL-2 for 18±1 days. Treg dose escalation levels were 1, 3, 10, 30 × 105/kg on day +1, and 30 × 105/kg on days +1 and +15 after UCBT. After CD25+ selection the median % CD4+/CD25+ was 62% (range , 11-87%) and following culture was 86% (r, 62-97%). Median fold expansion was 198 (r, 13-1169); median mixed lymphocyte reaction suppression at a 1:4 ratio post-culture was 86% (39-95%). All products achieved lot release. Pts were monitored for 48h post-infusion and no dose limiting or serious adverse event was observed. After infusion an increase in the proportion of peripheral blood comprised of CD4+FoxP3+CD127- cells was observed. Donor Tregs were clearly detected in all pts receiving Tregs that were HLA disparate with the pt and graft donor units (Figure 1). Mixed day 21 donor graft UCB chimerism was observed in 59% (10/19) of pts vs. 35% (38/107) in historical controls. The cumulative incidence of grades II-IV acute GVHD was 47% (95%CI, 24-71) and III-IV 16% (95%CI, 0-33). In pts receiving ≥ 30 × 105/kg (n=14), grades II-IV acute GVHD was 43% (95%CI, 16-70) and III-IV 7% (95%CI, 0-21). Compared to historical controls receiving the same conditioning regimen, the median time to the development of acute GVHD was longer (Figures 2), although not statistically significant. Neutrophil recovery was 95% (95%CI, 79-100) at median of 8.5 days (r, 3-36), platelet recovery 〉50,000/mL was achieved in 74% (95%CI, 53-95) at a median 43 days (r, 27-57), cumulative incidence of opportunistic infections (fungal + viral) at 100 days was 32% (95%CI, 11-53), treatment related mortality at 100 days was 11% (95%CI, 0-25), and disease free-survival was 40% (95%CI, 15-65%). None differed from historical controls. Based on our results, the co-infusion of ex vivo expanded and activated UCB-derived Treg to recipients of nonmyeloablative UCBT 1) is safe at the tested dose levels, 2) leads to detectable increased in Treg-donor unit derived circulating CD4+FoxP3+CD127- cells, and 3) results in an increased the proportion of mixed chimerism at day +21. Identification of the Treg MTD in the context of Siro/MMF immunosuppression will set the stage for the planned definitive studies that will establish the true impact of Tregs on engraftment and GVHD and for future studies in the treatment of autoimmune disease. Disclosures: No relevant conflicts of interest to declare.

Description: A hallmark of various human malignancies is the expression of immunoinhibitory factors within the tumor microenvironment. There is indirect evidence based on in vitro experiments that tumor-infiltrating T cells in human malignancies are suppressed by such factors. Still, direct evidence of the influence of individual inhibitory factors on immune cells in human cancer in vivo is lacking. To address this question, we used Hodgkin lymphoma (HL) as a model because histopathological characteristics of HL are thought to be due mostly to the effects of a wide variety of cytokines, including TGFβ or membrane-bound receptors such as PD-1 that are suspected to contribute to immune evasion of tumor cells. Using a genome-wide transcriptional approach, we established specific RNA fingerprints of TGFβ and PD-1 signaling in human T cells in vitro. Applying these specific fingerprints, we directly demonstrate that CD4+ T cells in HL—but not in follicular lymphoma (FL)—are under the inhibitory influence of both TGFβ and PD-1 in vivo. This approach can be easily generalized to provide direct evidence of the impact of any given soluble or cell-bound factor on any cell type within diseased tissue.

Description: Abstract 3580 Poster Board III-517 Immune cell expression of programmed death ligand-1 (PD-L1) represents a particularly important molecular mechanism responsible for control of auto- and allo-immunity mediated by effector memory T cells expressing PD1 receptor. As such, we have reasoned that an immuno-gene therapy approach that enables T cell expression of PD-L1 will represent a novel method of immune regulation. Advantageous features of this proposed therapy include a capacity to: (1) enforce long-term, stable expression of PD-L1; (2) build-in an independent surface marker to allow specific transduced cell enrichment; (3) utilize cellular delivery vehicles comprised of highly functional T cells that persist in vivo after adoptive transfer; and (4) incorporate an enhanced cell fate control or ‘suicide’ gene to permit in vivo control of the immuno-gene therapy. Given these considerations, we developed a recombinant lentiviral vector (LV) incorporating an EF1-α promoter that first encodes the cDNA for a fusion protein consisting of human CD19 (truncated, non-signaling) combined with mutated human TMPK that efficiently activates AZT as a pro-drug (Sato et al; Mol Therapy, 2007); then, after an IRES element, the vector encodes full-length human PD-L1. LV was made after transfection of 293T cells and then concentrated and titered. Initial experiments used Jurkat cells to optimize virus infection and to confirm co-expression of CD19 and PD-L1 by flow cytometry. In previous work, we have demonstrated that ex vivo T cell expansion in rapamycin induces an anti-apoptotic phenotype that permits enhanced in vivo T cell persistence in murine models and human-into-mouse xenogeneic transplant models. As such, we established the goal of infecting primary human CD4+ T cells manufactured using ex vivo co-stimulation (anti-CD3, anti-CD28), Th1-type polarization (inclusion of IFN-α), and exposure to high-dose rapamycin (1 μM); using a 6-day culture system and subsequent anti-CD19 column purification, 〉90% of resultant transduced T cells expressed PD-L1. Next, we utilized a xenogeneic transplantation model (Rag2−/−γc−/− hosts) to assess in vivo persistence of the gene-modified T cells and transgene expression (10,000 T cells transferred i.v. into each host). In vivo experiment #1 demonstrated that recipients of gene-modified T cells had increased numbers of human T cells in the spleen that co-expressed CD19 and PD-L1 relative to recipients of non-transduced but identically expanded human T cells (harvested at day 5 after adoptive transfer; 38,000 cells/spleen vs. 1000 cells/spleen, p=0.02). Such in vivo harvested T cells were secondarily co-stimulated ex vivo and propagated for an additional 5 days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells harvested from the gene-modified T cell cohort, and T cell numbers were maintained ex vivo (yield of CD19+PD-L1+ cells, 28,600 vs. 1500; p=0.0001). In vivo experiment #2 confirmed and extended these results. At day 21 after adoptive transfer, recipients of gene-modified T cells had increased numbers of human T cells that co-expressed CD19 and PD-L1 relative to recipients of non-transduced but identically expanded human T cells in both the spleen (2800 cells/spleen vs. 390 cells/spleen, p=0.01; n=10 per cohort) and bone marrow (71,600 cells/marrow vs. 6500 cells/marrow, p=0.0001; n=10 per cohort). Such in vivo harvested T cells at day 21 after adoptive transfer were secondarily co-stimulated ex vivo and propagated for an additional 6 days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells harvested from the gene-modified T cell cohort, and T cell numbers were maintained ex vivo (yield of CD19+PD-L1+ cells harvested from spleen, 71,200 vs. 1800, p=0.0008; yield of CD19+PD-L1+ cells harvested from marrow, 226,000 vs. 1400, p=0.0001). Because the rapamycin-resistant T cell vehicle utilized in these experiments manifests an anti-apoptotic phenotype that confers long-term engraftment potential, it is likely that the demonstrated durability in transgene expression relates both to the efficiency of the LV method utilized and to a T cell pro-survival function. In conclusion, the LV-mediated transfer of this novel combination of CD19/TMPK fusion protein and PD-L1 results in stable transgene expression in primary human T cells in vitro and in vivo, thereby opening an avenue to assess PD-L1 mediated immuno-gene therapy under cell fate control. Disclosures: No relevant conflicts of interest to declare.

Description: The CD28 family of receptors (CD28, cytotoxic T-lymphocyte–associated antigen 4 [CTLA-4], inducible costimulator [ICOS], program death-1 [PD-1], and B- and T-lymphocyte attenuator [BTLA]) plays a critical role in controlling the adaptive arm of the immune response. While considerable information is available regarding CD28 and CTLA-4, the function of the more recently discovered members of the CD28 family is less well understood. This review will highlight recent findings regarding the CD28 family with special emphasis on effects the CD28 family has on immunopathology, the discovery of costimulatory antibodies with superagonist function, and the status of clinical trials using various strategies to augment or block T-cell costimulation.