Chromium Release Assay Research Articles

Abstract 5597: NK cells and CD8+ T cells in orthotopic pancreatic cancer model: Mediation of anti-cancer immune response by 5-fluorouracil and interferon-α

Abstract Pancreatic cancer has the poorest prognosis of all cancers urging the necessity of new therapeutic approaches. Recent clinical data show encouraging results for combining Interferon-α (IFN-α) immunotherapy with 5-Fluorouacil (5-FU) chemotherapy. However, the role of IFN-α in this immunochemotherapy scheme is still elusive. Here we investigate in vivo the relevance of different immune subpopulations in the anti-cancer immune response mediated by IFN-α in combination with 5-FU. Luciferase-transfected Panc02 cells were implanted in the pancreas of C57BL/6 mice. Five days later, mice were treated with 5-FU alone, 5-FU + IFN-α or with a vehicle control. In parallel, CD4+ T cells, CD8+ T cells, and NK cells were depleted by neutralizing antibodies. Depletion of CD11c+ dendritic cells (DCs) was performed using transgenic CD11c. DTR mice. On day 21, mice were sacrificed and tumors were measured and frozen for further immunohistochemical analysis to evaluate the immune cells infiltration. NK cells were isolated and enriched from spleens of mice and their lytic activity was tested against Panc02 cells using a standard chromium release assay. Our data show that treatment with IFN-α + 5-FU has significantly decreased tumor volume in comparison with control or 5-FU treatments. On the other hand, this decrease in tumor volume was remarkably abolished by depleting CD8+ T cells or NK cells. Furthermore, NK cells isolated from spleens of IFN-α + 5-FU treated mice showed enhanced cytotoxicity towards pancreatic cancer cells (Panc02). Meanwhile, studying the role of CD4+ T cells and DCs besides immunohistochemical analysis is still ongoing. To conclude, this study gives more insight about the mechanism of action of INF-α in combination with 5-FU and introduces CD8+ T cells and NK cells as main players in the observed anti-cancer immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5597.

Abstract 4409: A bispecific T-cell engager effectively eradicates EGFRvIII-expressing glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor and remains uniformly lethal despite the progress of conventional therapies, which lack specificity and thus result in high toxicity to normal cells. Immunotherapy promises to induce robust tumor-specific immune responses that eliminate neoplastic cells with unparalleled specificity and with no additional toxicity to multimodality therapy. Recently, an approach using recombinant bispecific T-cell engagers (BiTEs), which consist of a tumor-targeting, single-chain antibody conjugated to a single-chain antibody directed against a T-cell activation ligand such as CD3, has arisen as a promising means for treating tumors. Human trials using a CD19XaCD3 BiTE confirmed the potency of these constructs by producing tumor regression in 7/7 patients with non-Hodgkin's lymphoma. The most significant limitation of these promising constructs, however, is the lack of tumor-specific targets that are frequently and homogeneously expressed. The epidermal growth factor receptor mutation EGFRvIII is a consistent 801-base-pair in-frame deletion in the extracellular domain of EGFR, not expressed in normal tissues but widely expressed in GBM and other common neoplasms. EGFRvIII also produces a constitutively active tyrosine kinase that enhances neoplastic cell growth and migration and confers radiation and chemotherapeutic resistance. Thus EGFRvIII appears to be a perfect anti-tumor target for these novel constructs. Here, we hypothesize that by targeting EGFRvIII through the T-cell engager MR1-1XaCD3, GBM will be efficiently eradicated. To test our hypothesis, we constructed the molecule MR1-1XaCD3, which consists of MR1-1, the murine anti-human EGFRvIII single-chain Fv, and aCD3, the murine anti-human CD3 single-chain Fv. MR1-1XaCD3 was expressed in and purified from bacteria BL21 (DE3), and the activity of this double function molecule was confirmed by FACS showing its specific binding to EGFRvIII-expressing cell lines, as well as human T cells. The cytotoxicity of MR1-1XaCD3 on EGFRvIII-expressing GBM D54MG. EGFRvIII cell lines was measured in vitro by standard chromium release assay. Preliminary results show that the MR1-1 construct is highly cytotoxic and antigen-specific, with an 8-fold increase in specific lysis for D54MG. EGFRvIII over the wild-type control. Currently, the efficacy of MR1-1XaCD3 is being evaluated in NOD/SCID gamma mice where human EGFRvIII-expressing xenografts have been implanted. In summary, our experiments showed that a human EGFRvIII-specific T-cell engager, MR1-1XaCD3, is effective on EGFRvIII-expressing tumor cells and provide new insights into GBM treatments. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4409.

The DNA demethylating agent 5-aza-2′-deoxycytidine induces expression of NY-ESO-1 and other cancer/testis antigens in myeloid leukemia cells

Azanucleoside DNA-hypomethylating agents have remarkable clinical activity in myelodysplastic syndromes and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs) encoding immunogenic proteins are not expressed in almost all normal tissues and many tumor types, but are consistently derepressed by epigenetically active agents in various cancer cell lines. Since the expression of CTA genes is usually very low or absent in myeloid leukemias, we treated various AML cell lines with 5-aza-2′-deoxycytidine (DAC) and quantified mRNA expression of the CTAs NY-ESO-1, MAGEA1, MAGEA3 and MAGEB2. Consistent time- and dose-dependent reactivation of all 4 CTA genes was observed, with maximum mRNA levels 72–144 h after treatment start. As determined by RNA microarray analyses, numerous other CTA genes (all located on the X-chromosome) were also derepressed in a time-dependent fashion by DAC. NY-ESO-1 derepression was confirmed at the protein level. By Elispot and chromium release assays we showed that the de novo expressed NY-ESO-1 protein was naturally processed and presented in a time- and dose-dependent fashion up to 8 days after the start of DAC treatment, and converted the cell lines susceptible to antigen-specific recognition by CD8+ T-cell clones. In conclusion, NY-ESO-1 and numerous other CTAs localized on the X-chromosome are readily and transiently derepressed in AML cell lines treated with DAC. The susceptibility of DAC-treated AML cell lines to antigen-specific T-cell recognition has clear implications for future clinical trials combining DAC and specific immunotherapy in AML.

Selective targeting of tumor vasculature by cytotoxic T lymphocytes specific for tumor endothelial marker-8 (131.36)

Abstract Tumor endothelial marker 8 (TEM8) is uniquely expressed on tumor vasculature but is absent on normal adult vasculature or somatic tissues, making it an attractive target for immunotherapy. The aim of the current study was to induce human cytotoxic T lymphocytes (CTL) with specificity for TEM8 with the end objective of starving tumors by sustained immunological disruption of their vascular supply. Two HLA-A0201 binding peptide epitopes, TEM8207-215 and TEM8298-306 were identified based on predictive algorithms in silico and T2 binding assays in vitro. Individual CTL cultures were established by stimulation of peripheral blood mononuclear cells with peptides in HLA-A2+ healthy donors. CTL induced with each of these peptides mediated lysis of T2 cells pulsed with the respective TEM8 peptide, but not control HIV-RT476-484 peptide, in chromium release assays. However, only CTL induced with TEM8298-306 peptide exhibited specific lysis of an HLA-A2+ TEM8+ HepG2 tumor cells, indicating that TEM8298-306 is a naturally processed peptide epitope. Further characterization of this CTL demonstrated specific secretion of INF-γ and TNF-α per multiplex cytokine arrays. TEM8298-306 peptide does not share sequence homology with peptides derived from other members of the vWF type-A domain family, notably capillary morphogenesis protein-2 which is widely expressed on normal tissues. These results suggest that TEM8298-306 peptide can be safely used in vaccine formulations for tumor immunotherapy.

Combination of CTLA-4 blockade with anti-CD40/IL-2 immunotherapy augments the CD8+ T cell response in resting and tumor-bearing mice (131.17)

Abstract Immunotherapy is a promising new option for the treatment of disseminated cancers. We have demonstrated that combined interleukin-2 (IL-2) and anti-CD40 (αCD40) therapy results in synergistic anti-tumor effects. While this strategy has shown success, it does not work well on less immunogenic tumors and can be extremely toxic. We sought to determine if removal of negative regulators such as cytotoxic T lymphocyte antigen-4 (CTLA-4) could result in augmentation of CD8+ T cell effects. We assessed total CD8+ T cell numbers and lytic ability after combination therapy in naïve and tumor-bearing mice. Here we present evidence that when the therapies are combined, there are increases in CD8+ T cells both systemically and intratumorally as well as a prolonged CD8+ memory expansion. We also saw increases in the lytic ability of CD8+ T cells against tumor cells as measured by chromium-51 release assay after combination with CTLA-4 blockade. We hypothesize that the amplification of T cell responses due to CTLA-4 blockade will enhance the anti-tumor effects of immunostimulatory therapies. It may also be possible to augment anti-tumor responses such that less immunotherapy will be needed to achieve equivalent anti-tumor responses.

Abstract B15: Immunochemotherapy with 5-fluorouracil and interferon-enhances innate and adoptive immune responses against pancreatic cancer in vivo

Abstract Pancreatic cancer has the poorest prognosis of all cancers urging the necessity of new therapeutic approaches. Recent clinical data show encouraging results for combining Interferon-α (IFN-α) immunotherapy with 5-Fluorouacil (5-FU) chemotherapy. However, the role of IFN-α in this immunochemotherapy scheme is still elusive. Here we investigate in vivo the relevance of different immune subpopulations in the anti-cancer immune response mediated by IFN-α in combination with 5-FU. Luciferase-transfected Panc02 cells were implanted in the pancreas of C57BL/6 mice. Five days later, mice were treated with 5-FU alone, 5-FU + IFN-α or with a vehicle control. In parallel, CD4+ T cells, CD8+ T cells, and NK cells were depleted by neutralizing antibodies. Depletion of CD11c+ dendritic cells (DCs) was performed using transgenic CD11c.DTR mice. On day 21, tumor volume was measured and NK cells were isolated and enriched from spleens of mice then used as effectors against Panc02 cells in a standard chromium release assay. Tumors were frozen for further immunohistochemistry analysis to evaluate the immune cells infiltration. In parallel, we performed flow cytometry analysis to evaluate the effects of different treatments on the expression of MHC-I and NKG2D ligands (MULT-1 and Rae-1) on Panc02 cells. Our data show that treatment with IFN-α + 5-FU has significantly decreased tumor volume in comparison with control or 5-FU treatments. This decrease in tumor volume was remarkably abolished by depleting CD8+ T cells or NK cells. Furthermore, NK cells isolated from spleens of IFN-α + 5-FU treated mice showed enhanced cytotoxicity towards Panc02 cells. Moreover, IFN-α + 5-FU treatment enhances the expression of MHC-I and NKG2D-ligand MULT-1 on Panc02 cells which could be the possible key for the enhanced recognition and per se killing by CD8+ T cells and NK cells respectively. To conclude, this study gives more insight about the mechanism of action of INF-α in combination with 5-FU and introduces CD8+ T cells and NK cells as main players in the observed anti-cancer immune response. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B15

Pluripotent stem cells are highly susceptible targets for syngeneic, allogeneic, and xenogeneic natural killer cells

Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.

CD94/NKG2C is a killer effector molecule in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are severe, bullous cutaneous diseases with uncertain pathogenesis, although cytotoxic T cells seem to be involved. Natural killer (NK)-like activity has been found in blister infiltrates. Cytotoxic T lymphocytes (CTLs) with NK-like activity (NK-CTLs) have been shown to express T-cell receptors restricted by the HLA-Ib molecule HLA-E. Alternatively, the HLA-E-specific activating receptor CD94/NKG2C can trigger T-cell receptor-independent cytotoxicity in CTLs. Our aim was to test whether HLA-E expression sensitizes keratinocytes to killing by CTLs with NK-like activity and to explore the expression of activating receptors specific for HLA-E in blister cytotoxic lymphocytes. We used flow cytometry and immunohistochemistry to analyze HLA-E expression in keratinocytes from affected skin in patients with SJS, TEN, and other less severe drug-induced exanthemas. The expression of CD94/NKG2C was analyzed by means of flow cytometry in PBMCs and blister cells from patients. PBMCs and blister cells were analyzed for their ability to kill HLA-E-expressing cells. Involvement of CD94/NKG2C in triggering degranulation of cytolytic cells was explored by means of CD107a mobilization assays and standard cytotoxicity chromium release assays. We found that keratinocytes from affected skin expressed HLA-E and that cell-surface HLA-E sensitizes keratinocytes to killing by CD94/NKG2C(+) CTLs. Frequencies of CD94/NKG2C(+) peripheral blood T and NK cells were increased in patients with SJS and TEN during the acute phase. Moreover, activated blister T and NK lymphocytes expressed CD94/NKG2C and were able to degranulate in response to HLA-E(+) cells in an NKG2C-dependent manner. CD94/NKG2C might be involved in triggering cytotoxic lymphocytes in patients with SJS and TEN.

Nonintegrating Lentiviral Vector-Based Vaccine Efficiently Induces Functional and Persistent CD8+ T Cell Responses in Mice

CD8+ T cells are an essential component of an effective host immune response to tumors and viral infections. Genetic immunization is particularly suitable for inducing CTL responses, because the encoded proteins enter the MHC class I processing pathway through either transgene expression or cross-presentation. In order to compare the efficiency and persistence of immune response induced by genetic vaccines, BALB/c mice were immunized either twice intramuscularly with DNA plasmid expressing a codon-optimized HIV-1 gp120 Envelope sequence together with murine GM-CSF sequence or with a single immunization using an integrase defective lentiviral vector (IDLV) expressing the same proteins. Results strongly indicated that the schedule based on IDLV vaccine was more efficient in inducing specific immune response, as evaluated three months after the last immunization by IFNγ ELISPOT in both splenocytes and bone marrow- (BM-) derived cells, chromium release assay in splenocytes, and antibody detection in sera. In addition, IDLV immunization induced high frequency of polyfunctional CD8+ T cells able to simultaneously produce IFNγ, TNFα, and IL2.

Allo-Reactive Potential of Multi-Virus Specific Cytotoxic T Cell Lines Recognizing Adenovirus Hexon, Cytomegalovirus pp65 and Epstein Barr Virus.

Abstract 5121 IntroductionCytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are major pathogens after allogeneic hematopoietic stem cell transplant. The current antiviral therapy is associated with significant myelosuppression, impaired hematopoietic reconstitution and consequently, higher rates of other infections. Adoptive immunotherapy utilizing allogeneic donor-derived virus specific cytotoxic T cell lines (CTL) has been shown to effectively treat refractory viral infections. Although such lines carry the theoretical risk of allo-reactivity and the resultant graft versus host disease, this complication has not been widely reported in recipients of single virus specific cell lines. To improve the efficiency of this therapeutic modality we have developed an approach to generate a single cell line that recognizes all three of these clinically relevant viruses while also having greatly reduced allo-reactive potential. MethodsPeripheral blood mononuclear cells (PBMC) from 3 healthy CMV seropositive donors were primed 3 times with autologous monocyte-derived dendritic cells pulsed with overlapping pools of pentadecapeptides spanning the antigen coding region of highly conserved regions of the Ad hexon protein and/or pools spanning the entire CMV pp65 protein. The lines were expanded for minimally one additional week using peptide loaded EBV transformed B cells (BLCL) as antigen presenting cells (APC). CMV, Ad and EBV anti-viral activity was monitored weekly by chromium release assay and the specificity of the lines was controlled by varying the peptide pools used for subsequent priming. The IFN-gamma ELISPOT assay was used to identify single peptides and to determine HLA restriction. The frequency and subset of single and pool peptide-reactive cells was determined using IFN-gamma intracellular cytokine production by flow cytometry. Fresh PBMC and established lines were additionally primed with fully allogeneic stimulators and response measured using intracellular cytokine production. ResultsEach of the 3 lines had robust proliferation and CTL activity against CMV and Ad peptide-pulsed targets. The lines consisted of 44.5-82.4% CD3+CD8+cells and 18.1-56.7% CD3+CD4+cells. CMV induced IFN-gamma response in 4.3-76.6% of CD3+CD8+cells and 0.4-47.5% of CD3+CD4+cells. Ad induced 1.4-43.9% of CD3+CD8+cells and 6.9-45.7% of CD3+CD4+cells to produce IFN-gamma. The 3 lines recognized 2-4 single CMV peptides and 2-6 single Ad peptides. The reactivity against these viral peptides was HLA restricted. At the time of testing none of the lines showed a strong EBV response. However, EBV reactivity could be detected with 1-2 additional primings, indicating that these precursors were present. Compared to fresh PBMC from the same donors, there was a 34.0±5.6 fold decrease in the frequency of alloreactive CD3+CD8+cells demonstrated in the intracellular cytokine assay. ConclusionsGeneration of multi-viral specific CTL in a single culture is feasible so long as antigen stimulation is controlled and this approach will greatly reduce the time, cost and number of cells required to restore viral immunity for patients receiving allogeneic stem cell transplant. The risk of allo-reactivity in these lines is greatly reduced compared to fresh PBMC. This makes adoptive cell therapy more practical, easier to implement and safer. DisclosuresNo relevant conflicts of interest to declare.

Potent Alloreactive Effector T Cells Cause Limited Damage to Non-Hematopoietic Tissues Under Non-Inflammatory Conditions, Despite Proper Expression of the Relevant Target Antigens.

Abstract 2454Poster Board II-431Donor T cells specific for minor histocompatibility antigens (mHags) contribute to graft-versus-leukemia (GvL) reactivity and graft versus host disease (GvHD) following HLA-matched stem cell transplantation. Hematopoiesis-restricted mHags may give rise to a more specific GvL effect without GvHD, whereas ubiquitously expressed mHags can be targets for both GvHD and GvL. Interestingly, high frequencies of circulating T cells specific for ubiquitously expressed mHags (e.g. male-specific epitopes, LB-ADIR-1F) have been detected in patients showing strong GvL with no or limited GvHD. Although both hematopoietic and non-hematopoietic cells express the proteins encoding these mHags, the immune effector response in these patients appears to be skewed towards hematopoietic cells. Therefore, we hypothesized that non-hematopoietic tissues are relatively unsusceptible to the cytotoxic effect of these mHag-specific T cells. To test this hypothesis, we developed an in vitro system in which HLA-A2+, ADIR+ primary human fibroblasts were exposed to mHag LB-ADIR-1F or allo-A2 specific CD8+ T cells. ADIR+ EBV-LCL were used as a control for hematopoietic cells. CTL-mediated cytotoxicity was measured using 4, 8 and 20 hours chromium release assays. Whereas all EBV-LCL were killed within 4 hours, 10% lysis of fibroblasts was found after 4 hours, only gradually increasing to 70% lysis after 20 hours in the continuous presence of the CTLs at 10/1 effector/target (E/T) ratios. To analyze whether this inefficient killing was due to low antigen expression by the human primary fibroblasts, we examined cytotoxicity against targets loaded with increasing concentrations of ADIR-peptide. Exogenous loading of fibroblasts with saturating concentrations (10-6M) of ADIR-peptide did not increase the sensitivity to T cell mediated cell death. To exclude that the observed inferior cell killing was due to the number of T cells available for engaging the fibroblasts, E/T ratios were increased to >100/1, but this did not change the poor susceptibility of the fibroblasts to CTL-mediated killing. Since apparently inefficient elimination of non-hematopoietic targets was found despite proper expression of the relevant antigen and the presence of functional T cells, we investigated whether insufficient engagement of T cells during contact with the fibroblasts was the cause of the impaired killing. To analyze this, kinetics of T cell degranulation upon target encounter was measured by CD107a, perforin and granzyme B expression. Although the onset of T cell activation was already seen after 2 hours, stimulation with fibroblasts resulted in degranulation in only a proportion of the T cells (50-60%) after 4hrs, resulting in full activation only after prolonged co-culture. In contrast, stimulation with EBV-LCL resulted in rapid, complete, and profound degranulation (100% of T cells within 2hrs). In concordance with this, T cell activation, as measured by the production of interferon gamma (IFNg) after stimulation with the fibroblasts, was suboptimal as compared to stimulation with EBV-LCL. These data suggest formation of an initial low avidity interaction with fibroblasts under steady state conditions, possibly only leading to efficient T cell engagement and fibroblast killing after a prolonged period of co-culture, resulting in the formation of a local pro-inflammatory environment. To confirm whether susceptibility to mHag-specific T cells was increased under pro-inflammatory conditions, we pre-treated fibroblasts with IFNg. Analysis of the expression of a panel of known adhesion and co-stimulatory molecules revealed that IFNg treatment resulted in a 2-5 fold upregulation of expression of HLA class I, HLA class II and ICAM-1, leading to a more uniform and profound T cell activation. This increased T cell activation resulted in more efficient direct fibroblast killing. In conclusion, despite proper expression of the relevant target antigens, low-avidity interaction between effector T cells and non-hematopoietic tissues results in minimal damage under non-inflammatory conditions. Disclosures:No relevant conflicts of interest to declare.

Adoptively Transferred Expanded Natural Killer Cells Inhibit Myeloma Tumor Growth In Vivo.

Abstract 953Recently developed culture conditions for expanding and activating natural killer (NK) cells may improve immunotherapeutic options for patients with multiple myeloma. We have previously shown that co-incubation of NK cells from multiple myeloma patients and healthy donors with K562 cells genetically modified to express membrane-bound interleukin 15 and the co-stimulatory molecule 41BBL (K562-mb15-41BBL) leads to a dramatic increase in both NK cell number and anti-myeloma activity in vitro. In this study, we tested the anti-myeloma activity of these expanded NK cells in vivo using a murine model, which supports the growth of myeloma cells in a microenvironment that reproduces that of the human bone marrow. We first implanted human fetal bones subcutaneously into NOD/SCID/IL2Rγ null mice and allowed them to engraft. Luciferase transfected OPM2 myeloma cells were then injected into the human bone fragment. Tumor burden was followed by bio-imaging and ELISA for human Ig. NK cells were expanded from healthy donor PBMC by co-culture with irradiated K562-mb15-41BBL cells in the presence of 300U/ml IL2 for 10-12 days. Myeloma tumor bearing mice were dosed 6-7 days after OPM2 injection (bio-imaging confirmed myeloma engraftment) with expanded NK cells via tail vein injection followed by subcutaneous IL2 to prolong NK cell survival. Flow cytometry was used to track the human NK cells in blood. Histology was performed by H&E staining of formalin-fixed, paraffin embedded tissues harvested at the end of the study. Sufficient NK cells (3×109) for dosing mice were obtained from one unit of healthy donor blood and their ability to kill OPM2 myeloma was confirmed in vitro by chromium release assays. In experiment 1, OPM2-bearing mice received two tail vein injections, 48h apart, comprising PBS, 4 ×107, or 16 ×107 (total dose) expanded NK cells with 100U IL2 given twice per week. We observed significant myeloma growth inhibition in the cohort given 16 ×107 NK cells (p<0.04) and circulating human NK cells could be detected up to day 21 post-administration. Measurement of tumor burden by bio-imaging and ELISA were highly concordant (correlation coefficient = 0.995). Histologic analyses confirmed a dramatic reduction in tumor burden in the mice treated with expanded NK cells and revealed that bone loss was more pronounced in the implanted fetal bones of the control cohort not receiving NK cells. We observed in a subsequent experiment that increasing the IL2 dose to 1000U daily led to in vivo expansion of CSFE-labeled NK cells of up to 10 generations by day 6 post-infusion, which was associated with an enhanced anti-tumor effect in the 16 ×107 dose cohort. Furthermore, we found that multiple injections of NK cells (four injections of 4 ×107 NK cells versus two injections of 8 ×107) were better tolerated in the highest dose cohort (3/3 surviving versus 6/12 surviving). In conclusion, we observed a significant anti-myeloma effect against the aggressive plasma cell leukemia cell line OPM2 with expanded NK cells and low dose IL2. Adjusting the IL2 dose to 1000U daily led to in vivo expansion of NK cells, longer term NK cell persistence, and an increased anti-myeloma effect. Importantly, this IL2 dose is comparable to the 3×106 U daily dose well tolerated in a previous trial with non-expanded NK cells in humans. Single doses of NK cells >8×107 led to high morbidity in this model while multiple doses of 4×107 were well tolerated. Experiments are in progress testing the in vivo effect of expanded NK cells on primary myeloma cell tumors in this model. Our findings support the planned clinical application of ex vivo expanded NK cells. Disclosures:No relevant conflicts of interest to declare.

Drug-Induced, NKG2D-Dependent Sensitization of ALL Cell Lines to NK Recognition.

Abstract 2024Poster Board II-1The combination of tumor-sensitizing drugs with NK cell infusion is beginning to emerge as a novel anti-tumor therapy. A growing body of in vitro studies show that drugs such as proteosome inhibitors, histone deacetylase inhibitors and thiazolidinediones are able to sensitize tumor cells but not their healthy counterparts to NK-mediated lysis. Drug induced NK-sensitization has shown promise in acute myeloid leukemias but no studies have yet proven this principle in acute lymphoblastic leukemia (ALL); a tumor phenotype reported to be relatively NK-resistant. The mechanisms underlying sensitization have not been fully identified but up regulation of ligands for TRAIL and the NK activating receptor NKG2D: MICA MICB and the UL16-binding proteins, may have a role. We set out to explore ALL susceptibility to NK cytotoxicity and whether this could be modulated by drug treatment. In contrast to published data, untreated ALL cell lines were positive for surface expression of MICB and ULBP2. Median fluorescence intensity ratios (mean ± SD; n = 6) for MICB detection on the cell lines 697, NALM-6, BV173 and SEM were: 3.2 ± 0.9; 3.8 ± 1.3; 4.0 ± 0.5; 2.5 ± 0.9, respectively and for ULBP-2: 2.3 ± 0.4; 55 ± 4.9; 2.9 ± 0.2; 1.8 ± 0.4, respectively. NALM-6 was also positive for ULBP1 (3.3 ± 0.6) while all were negative for MICA and ULBP3. Susceptibility of untreated ALL lines to NK mediated killing was assessed by chromium release assay using an IL-2 stimulated primary NK cell line. At effector to target ratio 40:1, specific release was 2.3% with cell line 697, 12% with NALM-6, 36% with BV173 and 63% with SEM. These results correlated with CD107a exposure in a degranulation assay using IL-2 stimulated peripheral blood lymphocytes: specific degranulation (% CD107a+ target with effector minus %CD107a+ effector alone) was 0.68% (697), 7.1% (NALM-6), 10% (BV173) and 17% (SEM). There was no correlation between baseline expression of NKG2DL and susceptibility to NK killing. Bortezomib, sodium valproate and troglitazone were added to cell cultures at sub-IC50 doses for 48 hours and compared with equimolar vehicle controls. Surface NKG2DL expression was measured by flow cytometry. On NALM-6 troglitazone treatment increased ULBP1 MFI by 2.0 ± 0.33 fold compared with vehicle control and increased percentage of ULBP1 positive cells by 39.6% (paired t-test: p=0.063). Sodium valproate increased MICA expression by 2.91 ± 1.18 fold and percentage of MICA positive cells by 12.3% (p=0.0382). On BV173, sodium valproate treatment increased ULBP2 MFI by 1.55 ± 0.07 fold and percentage of ULBP2 positive cells by 8.6% (p=0.04). There were no significant ligand changes after drug treatment on cell line 697. No NKG2DL changes were seen after Bortezomib treatment on any cell line. The functional significance of NKG2DL changes was assessed by CD107a degranulation assay. NALM-6 treated for 48 hours with drugs yielded the following fold increases in specific degranulation of NK cells compared to NALM-6 vehicle controls: 5.02 ± 5.98 for Bortezomib (mean ± SD), 2.4 ± 0.67 for Troglitazone and 1.44 ± 0.13 for Valproate. Levels of NK degranulation with 697 were very low (<5%) and drug treatment had no effect. Finally, we demonstrated that sensitization of NALM-6 was at least partly dependent on NKG2DL recognition, since blocking antibody to NKG2D reduced CD107a exposure by all three drugs. Compared with controls, blocking reduced CD107a expression by 59 ± 12% for Bortezomib, 47 ± 1.1% for Troglitazone and 48 ±11% for valproate-treated cells. This result was unexpected for Bortezomib as no changes in surface NKG2DL expression were detected after drug treatment. However, we are investigating the possibility that Bortezomib may down-regulate HLA class I expression, thus reducing inhibitory signaling upon NALM-6/NK interaction and unmasking an activation pathway that signals through NKG2D. In conclusion, we found basal levels of expression of NKG2DL on ALL cell lines. There was no correlation between NKG2DL expression and susceptibility to NK lysis, although this was to be expected given that a wealth of other activating and inhibitory receptors that contribute to NK activation. Bortezimib, valproate and troglitazone induced NKG2DL expression and sensitization to NK recognition in a cell line-specific manner. These drugs may therefore be useful to augment conventional chemotherapy or immunotherapeutic approaches to ALL. Disclosures:No relevant conflicts of interest to declare.

Characterization of Early Natural Killer Cell Reconstitution Following Autologous Transplantation in Multiple Myeloma.

Abstract 4641 BackgroundDespite the advent of novel therapies, multiple myeloma (MM) remains incurable and high-dose chemotherapy (HDC) with autologous stem cell transplantation (SCT) remains an important therapy for this incurable malignancy. Prior research has suggested that early lymphocyte immune reconstitution following HDC/SCT may be associated with improved outcomes; however, little is known regarding the biologic mechanisms underlying this phenomenon. A potent natural killer (NK) cell versus MM effect has also been demonstrated by our group and others previously, and NK cells are the first lymphocyte subset to recover following HDC/SCT. Herein, we characterize the nature of this recovery to suggest that the early post-transplant period may be an especially favorable environment in which to augment the NK cell versus MM effect to improve outcomes for patients with MM undergoing HDC/SCT. MethodsWith informed consent and under institutional review board approval, peripheral blood samples from patients with multiple myeloma undergoing HDC/SCT were collected on the day of first peripheral white cell count > 1500/uL post- HDC/SCT. All patients received intravenous melphalan (200mg/m2) as the preparative regimen. Mononuclear cells were isolated and, by flow cytometry, CD56(+),CD3(-) NK cells were comprehensively immunophenotyped for expression of inhibitory and activating ligands. NK cell activation and cytotoxicity were assessed by ELISPOT and chromium release assays. Clinical outcome was assessed as a function of early NK cell recovery. Results11 patients (median age 57, 8 male, 3 female) with a median of 1 prior regimen to transplant (range 1-9) have been assessed to date. 1 patient was mobilized with AMD-3100, 3 had cyclophosphamide mobilizations and 7 were mobilized with neupogen alone. The average CD34(+) cell dose received was 5.22 (+/- SD 2.39). The median day of peripheral white cell recovery was day +12 (range 11-18). The median absolute lymphocyte count on sample procurement day was 410 (range 340 – 820), of which an average of 37% (range 13 – 70) were NK cells. Although total white count on procurement day correlated with CD34(+) cell dose (p = 0.02), neither absolute lymphocyte count or NK cell count correlated significantly with CD34(+) cell dose and mobilization strategy did not affect NK cell count. NK cell reconstitution correlated with best response to HDC/SCT (p=0.02), the average NK cell count for patients ultimately achieving CR was 389 (+/- 131) as compared to those achieving PR or less (171 +/- 101). Interestingly, nearly half of reconstituting NK cells (46% +/- SD 20) expressed CD117 (c-kit receptor), a marker shown previously to be associated with earlier stages of NK cell development. Inhibitory KIR (CD158a on 36% +/-17 and CD158b on 36% +/- 24) and NKG2A (76% +/- 10) expression also seemed restricted to the CD117(+) NK cells. This population of NK cells also displayed NKG2D (25% +/- 22) and CD16 (73% +/-8). Reconstituting NK cells were capable of interferon gamma production against MM tumor targets similar to NK cells isolated from healthy donors (E:T = 20:1, 956 +/- 98 spots/well versus 896 +/-85, p = n/s). Granzyme B production against MM tumor targets also compared to normal controls (E:T = 20:1, 547 +/- 66 versus 573 +/- 50, p= n/s). Moreover, reconstituting NK cells showed enhanced cytotoxicity against MM tumor targets when pre-treated with an inhibitory KIR blocking antibody (IPH2101/1-7F9) similar to healthy donor NK cells. DiscussionTaken together, we provide quantitative and qualitative data to suggest that early NK cell recovery post-SCT may contribute to outcome in patients with MM. This may be an optimal period to implement immunomodulatory strategies to augment NK cell function to improve outcomes for patients with MM undergoing HDC/SCT. Reconstituting NK cells, although exhibiting a relatively unique immunophenotype compared to mature NK cells in healthy, normal donors, appear to retain properties of activation and cytotoxicity. Given the favorable effector:target ratio in vivo following SCT, these results support further research into immunotherapeutic manipulation of the reconstituting NK cell clone to improve efficacy of HDC/SCT in patients with MM. Disclosures:Andre:Innate Pharma: Employment. Squiban:Innate Pharma: Employment. Romagne:Innate Pharma: Employment.

Co-Existing Serological Immune Responses against RHAMM Might Be a Prerequisite for Strong Cellular Immune Responses of CD8-Positive T Cells in RHAMM-R3 Peptide Vaccination for Patients with Different Hematological Malignancies.

Abstract 3671Poster Board III-607For effective elimination of malignant cells by specific T cells a co-activation of CD4- and CD8-positive T cells might be important. We performed two RHAMM-R3 peptide vaccination trials using 300μg and 1000μg for patients with AML, MDS and multiple myeloma overexpressing RHAMM. Similar mild toxicity of both cohorts was found, only mild drug-related adverse events were observed such as erythema and induration of the skin. In the 300μg cohort we detected in 7/10 (70 %) patients specific immune responses and also positive clinical effects in 5/10 (50 %) patients. In the high dose peptide vaccination trial (1000μg peptide) 4/9 (44 %) patients showed positive immune responses. These patients showed an increase of CD8+RHAMM-R3 tetramer+/CD45RA+/CCR7-/CD27-/CD28- effector T cells and an increase of R3-specific CD8+ T cells. In the higher peptide dose cohort three patients showed positive clinical effects. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial and might induce immune tolerance.In this work, we investigated the co-existence of serological immune responses against RHAMM detected by a RHAMM-specific ELISA of patients with AML, MDS and multiple myeloma treated in these two peptide vaccination trials. We correlated these results to specific T cell responses of CD8-positive T cells measured by ELISpot assays for interferon gamma and Granzyme B, tetramer staining and chromium release assays. Moreover, these results were compared to the frequency of regulatory T cells. 4/19 patients have a positive serological immune response in ELISA assay, all of these patients developed also strong specific CD8-positive T cell responses during peptide vaccination detected by ELISpot assays and tetramer staining. As expected, peptide vaccination did not result in the induction of humoral immune responses. In further ELISA assays we measured IL-2 and IL-10 levels in the sera of the patients before and three weeks after four vaccinations. While IL-10 levels remained at a rather low level over the time of vaccination, we detected an increase of IL-2 up to the five-fold of the initial levels in four of ten patients.Moreover, we performed a proteome array to detect cytokine and chemokine regulation in sera of patients vaccinated in these two trials during and after RHAMM-R3 peptide vaccination. 36 cytokines, chemokines and acute phase proteins were measured and both cohorts vaccinated with different peptide doses were compared.Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Co-existence of immune responses of CD4-positive T cells against the target RHAMM seems to be important for an induction of strong immune responses of CD8-positive T cells. Disclosures:No relevant conflicts of interest to declare.

Identification of RHAMM-Derived CD8+ Restricted, Heteroclitical, Cryptic Epitope R9Y as a Promising Target for Immunotherapy of Chronic Lymphocytic Leukemia.

Abstract 3034Poster Board II-1010The receptor for hyaluronic acid mediated motility (RHAMM) is leukemia associated antigen (LAA) strongly correlated with proliferation and bearing negative prognosis for chronic lymphocytic leukemia (CLL). Recently, we completed a peptide vaccination clinical trial with dominant RHAMM-derived epitope (R3) for patients with CLL. To our surprise, R3 vaccination induced regulatory T cells (Treg) in 66% vaccinated patients which might limit the clinical efficacy of peptide immunotherapy. Obtained results also pointed to a pre-existing tolerance to LAA-derived peptides. While the tolerance is limited to dominant epitopes we aimed to identify a crypitic epitopes that might circumvent tolerance mechanisms and in order to increase affinity modified them heteroclitically.We screened in silico 50 peptides with different heteroclitical modifications using SYFPEITHI algorithm and calculate affinity to MHC-I complex. Further, we choose 10 peptide pairs (cryptic – modified) which affinity increased to the highest extent and screened them in vitro assay for the affinity as well as the peptide-MHC-I complex stability in T2 binding test. In functional studies we screened 5 peptide pairs by ELISPOT analysis using CD8+ T cells isolated from peripheral blood (PB) of CLL patients and healthy donors. We found that CD8+ cells from CLL patients presensitized with peptides: R9Y (YLQLDAFEV); R13YL (YLVQSLEDV); R15Y (YLKQTLDEL); R17YL (YLQEQLNKI) as well as R19YL (YLIKHVVKL) specifically recognized T2 cells pulsed with respective peptides with the highest frequency for R9Y (90%). In chromium-51 release assays, R9Y-primed CD8+ T cells from CLL patients were able to effectively lyse R9Y-peptide pulsed T2 cells as well as CLL leukemic cells presenting RHAMM in a HLA-A2 restricted manner; in contrast, K562 cells and T2 cells lacking either RHAMM or HLA-A2 expression were not lysed above background levels. In addition, mAb-based MHC I blockade demonstrated that the observed peptide-specific lysis was MHC I-restricted.In conclusion, we defined a novel RHAMM-derived CD8+ restricted, heteroclitical, cryptic epitope R9Y which might represent an interesting target for immunotherapy of CLL. In future clinical administration the R9Y peptide might circumvent tolerance to dominant epitopes and thereby increase efficacy of peptide vaccination. DisclosuresStilgenbauer:GSK: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Generation of CMV Specific T Cells From CMV Seronegative Donors by T Cell Receptor RNA Transfer.

Abstract 3577Poster Board III-514Reactivation of latent human cytomegalovirus (CMV) infection is a frequent complication in CMV seropositive patients after allogeneic hematopoietic stem cell transplantation (HSCT). Although antiviral drug therapy is successfully used to reduce the risk of CMV disease, long-term virus control requires the re-establishment of protective antiviral T cell immunity in the host. The latter is challenging, particularly if the donor is CMV seronegative and thus, no CMV specific T cells are being transferred from donor to recipient during HSCT. Grafting nonreactive T cells of CMV seronegative donors by virus-antigen specific T cell receptors (TCR) may be an efficient means to transfer CMV specific T cell function into allogeneic HSCT recipients. In this study, we intended to reprogram T cells of CMV seronegative donors with human TCR recognizing the immunodominant HLA-A*0201 binding peptide epitope NLVPMVATV (495-503) derived from the CMV pp65 protein. A common approach for TCR gene transfer into T cells is retroviral transduction bearing the risk of insertional mutagenesis which hampers clinical translation. In addition, heterologous recombination between introduced and naturally expressed TCR chains might lead to the induction of harmful autoimmunity. Herein we used in vitro transcribed RNA encoding the CMV pp65/HLA-A*0201-specific TCR for electroporation of anti-CD3 stimulated T cells in peripheral blood mononuclear cells (PBMC) of CMV seronegative donors. This procedure resulted in transient surface expression of the introduced TCR chains up to 5 days as shown by flow cytometry. Maximum expression level was observed at 4 to 24 h after electroporation, with up to 70% of total CD8+ and CD4+ T cells staining positive for the vβ13.1 subfamily domain of the TCRβ chain. After introduction of TCR RNA, the intensity of CMV pp65/HLA-A*0201 tetramer staining was 60% and 50% of total CD8+ and CD4+ cells, respectively. In IFN-γ ELISPOT and 51Chromium-release assays, TCR RNA transfected T cells recognized HLA-A*0201 expressing T2 cells pulsed with titrated amounts of CMV pp65 (495-503) peptide. Minimal peptide concentration triggering specific lysis was 0.1 nM to 1 nM at a CD8+ to target (CD8+:T) ratio of 2:1. The EC50 value (0.2 nM) was in the same range of avidity compared to that of a retrovirally transduced counterpart construct of this TCR. Most importantly, TCR recipient CD8+ T cells gained the ability to lyse HLA-A*0201 positive human fibroblasts upon infection with CMV. Specific lysis between 20% and 100% was observed at a CD8+:T ratio of 1:1 or higher. We next sorted CD8+ T cells from PBMC of CMV seronegative donors into naïve and memory cells according to expression of the differentiation markers CD45RA and CD45RO. Although 90% of naïve CD8+ T cells stained positive for the CMV pp65/HLA-A*0201 tetramer after electroporation of TCR RNA, they mediated only marginal lysis toward CMV infected fibroblasts. In contrast, TCR RNA transfected memory CD8+ T cells showed strong lysis against CMV infected fibroblasts at a CD8+:T ratio of 0.7:1 or higher. Specific lysis was detected for at least 3 days after electroporation. In summary, our data demonstrate that nonreactive human T cells can be successfully redirected with CMV pp65 TCR RNA. The expression level of the introduced TCR is sufficient to trigger IFN-γ production and cytolytic activity toward CMV infected human fibroblasts. Electroporation of TCR RNA is comparably easy and eliminates the risk of retroviral transduction. We therefore believe that CMV pp65 TCR RNA has the potential to be further developed as a therapeutic “off-the-shelf” reagent for patients who undergo drug-resistant CMV reactivation after HSCT. Disclosures:No relevant conflicts of interest to declare.

In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Abstract 4079Poster Board III-1014MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures:No relevant conflicts of interest to declare.

Immune-Based Therapies Targeting Mage-A4 for Relapsed/Refractory Hodgkin's Lymphoma After Stem Cell Transplant.

Abstract 4089Poster Board III-1024Stem cell transplantation (SCT) from autologous and, recently, allogeneic (unrelated adult donor and umbilical cord blood) sources is the treatment of choice for patients with relapsed/refractory Hodgkin's Lymphoma (HL). However, HL patients who relapse after SCT have limited options for long-term cure – necessitating the development of novel therapies for the treatment of this disease. We have previously shown that infusion of cytotoxic T cells (CTLs) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV+ HL patients. Unfortunately, approximately 70% of relapsed HL tumors are EBV-negative. Therefore, we investigated another promising T cell target, the cancer/testis antigen (CTA) MAGE-A4 as a potential target for CTL therapy. We hypothesize that adoptive transfer of MAGE-A4-specific T cells may be a feasible therapeutic strategy for patients with relapsed HL after SCT. MAGE-A4 is expressed by >30% of EBV antigen-negative HL tumors and expression can be enhanced in MAGE-A4-negative tumors by epigenetic modification using HDAC inhibitors/demethylating agents that are currently in clinical use. We therefore determined whether MAGE-A4-specific cytotoxic T lymphocytes (CTL) could be expanded from healthy adult donor peripheral blood (PB) (n=9), cord blood (CB) (n=2) and HL patient blood (n=3). Dendritic cells (DCs) pulsed with overlapping peptides spanning MAGE-A4 were used as antigen presenting cells to stimulate peripheral blood mononuclear cells in the presence of IL-7, IL-12, and IL-15. After 9-12 days in culture, cells were stimulated weekly (with at least one additional stimulation) with MAGE-A4 peptide-pulsed DCs in the presence of IL7 and IL2. Analysis of the stimulated T cells using interferon gamma (IFN-γ) ELISPOT assays showed high specificity against MAGE-A4 with mean 96.6 (range 11-716) IFNγ-secreting spot forming cells (SFC)/10e5 cells in response to MAGE-A4 peptides compared to a mean of 7.8 (range 0-46.5) SFC/10e5 cells in response to irrelevant peptides. In addition, specific killing was measured in chromium (Cr51) release assays following co-culture with MAGE-A4 positive targets, showing that we successfully generated cytotoxic T cells. All evaluable T cell lines generated from healthy adult donors were predominantly CD8+, and 4 of the 8 showed specificity for a single, previously uncharacterized MAGE A4 epitope (aa266-285, NPARYEFLWGPRALAETSYV). In contrast, 6/8 T-cell lines derived from HL patients and CB were predominantly CD4+, and showed a range of MAGE-A4 epitope specificities. Mechanisms for these differences are now being investigated. These early results suggest that strategies using healthy donor cord blood and patient T cells for adoptive immunotherapy may be a novel and feasible approach for the treatment of relapsed HL post SCT. Disclosures:No relevant conflicts of interest to declare.

In vitro activity of pertuzumab in combination with trastuzumab in uterine serous papillary adenocarcinoma

Background:Uterine serous papillary adenocarcinoma (USPC) is a rare but highly aggressive variant of endometrial cancer. Pertuzumab is a new humanised monoclonal antibody (mAb) targeting the epidermal growth factor type II receptor (HER2/neu). We evaluated pertuzumab activity separately or in combination with trastuzumab against primary USPC cell lines expressing different levels of HER2/neu.Methods:Six USPC cell lines were assessed by immunohistochemistry (IHC), flow cytometry, and real-time PCR for HER2/neu expression. c-erbB2 gene amplification was evaluated using fluorescent in situ hybridisation (FISH). Sensitivity to pertuzumab and trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) was evaluated in 5 h chromium release assays. Pertuzumab cytostatic activity was evaluated using proliferation-based assays.Results:Three USPC cell lines stained heavily for HER2/neu by IHC and showed amplification of the c-erbB2 gene by FISH. The remaining FISH-negative USPCs expressed HER2/neu at 0/1+ levels. In cytotoxicity experiments against USPC with a high HER2/neu expression, pertuzumab and trastuzumab were similarly effective in inducing strong ADCC. The addition of complement-containing plasma and interleukin-2 increased the cytotoxic effect induced by both mAbs. In low HER2/neu USPC expressors, trastuzumab was more potent than pertuzumab in inducing ADCC. Importantly, in this setting, the combination of pertuzumab with trastuzumab significantly increased the ADCC effect induced by trastuzumab alone (P=0.02). Finally, pertuzumab induced a significant inhibition in the proliferation of all USPC cell lines tested, regardless of their HER-2/neu expression.Conclusion:Pertuzumab and trastuzumab induce equally strong ADCC and CDC in FISH-positive USPC cell lines. Pertuzumab significantly increases tratuzumab-induced ADCC against USPC with a low HER2/neu expression and may represent a new therapeutic agent in patients harbouring advanced/recurrent and/or refractory USPC.