ALBERT

Description: Introduction: Patients receiving an allogeneic stem cell graft from a cytomegalovirus (CMV) seronegative donor are prone to CMV reactivation with high risk of disease and mortality. Therefore we initiated a clinical phase I trial with a novel vaccine designed by our group: a CMV phosphoprotein 65 (CMVpp65)-derived peptide in water-in-oil emulsion (Montanide™) plus administration of granulocyte-macrophage colony stimulating factor. Material and Methods: 10 patients after allogeneic stem cell transplantation received four vaccines s.c. at a biweekly interval. Patients were monitored for clinical course and CMVpp65 antigenemia. CMV-specific CD8+ and gamma/delta T cells were analyzed by multi-color flow cytometry. Neutralizing anti-CMV antibody assays were established and correlated to clinical parameters. Results: Peptide vaccination was well tolerated, no drug-related serious adverse events were detected. 7 of 9 patients with CMVpp65 antigenemia cleared the CMV with enduring response 〉 1.5 years after 4 vaccinations and are still free from antigenemia until present. 2 patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An up to 6-fold increase in frequency of both CMV-specific CD8+ T cells or Vdelta2- gamma/delta T cells with a maximum of 1.32% CMV-specific CD8+ T cells ex vivo without priming and 13.7% Vdelta2- gamma/delta T cells of all CD8+ T cells was detected in five patients. Also titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. Humoral and cellular immune responses correlated with clearance of the CMV load. Conclusion: Administration of CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing. Disclosures Kuball: Gadeta B.V.: Membership on an entity's Board of Directors or advisory committees. Wuchter:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Gilead: Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy. Greiner:BMS: Research Funding.

Description: Background: After transplantation of solid organs like allogeneic kidneys, the administration of immunosuppressive drugs such as cyclosporine A (CSA) and steroids is mandatory. This regimen exerts toxicity to the graft and makes transplant recipients prone to opportunistic infections. Replacement of the immunosuppressive drugs by a transfusion of tolerogenic cells might overcome these noxious side effects. Mitomycin-induced cells (MICs) are donor-derived monocytes that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C and have a myeloid-derived suppressor cell (MDSC) character. Materials and methods: Peripheral blood mononuclear cells (PBMCs) were harvested from living kidney donors by leukapheresis and MIC cells were manufactured under Good Manufacturing Practice (GMP) conditions in the clean room of our University Hospital. Kidney transplant recipients received either 1.5x10E6 MIC cells per kg body weight on day -2 (N=3, group A) or 1.5x10E8 MIC cells per kg body weight on day -2 (N=3, group B) or on day -7 (N=4, group C) before living donor kidney transplantation. Patients received immunosuppressive therapy with cyclosporine a (CSA), enteric coated mycophenolate sodium (EC-MPS) and corticosteroids. The primary outcome was measured by the frequency of adverse events (AEs) on post-transplant day 30 with a follow-up until post-transplant day 360 for all patients. Results: Clinically, all kidney transplant recipients showed a median serum creatinine of 1.4 mg/dL at day 30 and remained stable with a median creatinine of 1.48 mg/dL at day 180 without significant proteinuria (median 10 g/mol creatinine at day 180) and without rejection episode. In total 72 AEs were observed including three severe AEs which were not associated with the MIC cell transfusion. Besides two infectious complications, no positive cross match results, no de novo donor-specific antibodies or rejection episodes were recorded. In group C, a reduction of immunosuppressive therapy was effective in the observational phase with low-dose CSA and low-dose EC-MPS. Immunologically, CD19+ B cells increased up to a median of 300/µL until day 30, followed by a decrease to a median of 35/µL at day 180 in group C. Notably, CD19+CD24highCD38high regulatory B cells were significantly increased from a median of 2% on day 30 to a median of 20% on day 180. The plasma IL-10/TNF-α ratio increased from a median of 0.05 before cell therapy to a median of 0.11 at day 180. Moreover, recipient lymphocytes showed no or only minimal reactivity against irradiated donor PBMCs, while reactivity against 3rd party healthy donor PBMCs in vitro was not impaired. Additionally, the quality assessment demonstrated that MIC cells have the capability to induce tolerogenic dendritic cells (tDCs) by down-regulating the costimulatory molecules CD80 and CD86, and the maturation molecule CD83, while up-regulating the immunosuppressive molecule CD103. MIC-induced tDCs showed the capacity to inhibit donor specific allo-reactive CD4 and CD8 T cell proliferation. Conclusion: A stable function was observed in all transplant recipients receiving the MIC cells product without any allograft injury or rejection episodes even under reduction of conventional therapy with immunosuppressive drugs. MIC cells constitute a novel tool for immunotherapy with a high potential in transplantation medicine. Disclosures No relevant conflicts of interest to declare.

Description: Adoptive TCR transfer against rapidly mutating targets, such as HIV-1 or cancer, must counteract corresponding immune escape. Hence, we generated T cells expressing two additional receptors (TETARs) specific for HIV-1 by TCR mRNA electroporation. An HLA-A2–restricted gag-specific TCR and an HLA-B13–restricted nef-specific TCR were chosen. When both TCRs were transfected simultaneously, strong competitive effects occurred that were overcome by replacing the human constant domains of one TCR with murine counterparts and adapting the amounts of TCR-RNA used for transfection. The resulting TETAR responded to both epitopes with cytokine secretion and cytotoxic function. Cell sorting revealed that one individual cell indeed recognized both epitopes. The T cells diminished their reactivity to each epitope after stimulation but sequentially killed targets that presented the gag epitope and then targets that presented the nef epitope, or vice versa. Taken together, TETARs represent a sophisticated tool to study TCR functionality and might be a useful strategy in immunotherapy.

Description: Introduction The advent of T cells expressing chimeric antigen receptors (CART) for the treatment of cancer patients has been a milestone in the field of immunotherapy. For clinical application, reproducibility and safe generation of CART must be guaranteed. We investigated how different culture conditions of human peripheral blood mononuclear cells (PBMCs) influence the phenotype, i.e. naïve (N), central / effector memory (CM/EM), effector (E) cells, and the cellular composition of the resulting CART preparation, among others: the percentage of CD56+, γ/δ TCR+ and CD4+CD8+ double positive cells. Eventually, freezing/thawing of CART might also have an impact on their number, phenotype and function. Methods CART were generated by transducing human PBMCs from healthy donors with a CD19.CAR-CD28-CD137zeta vector (kindly provided by the Center for Cell and Gene Therapy, Houston) under two different stimulating culture conditions - anti-CD3/anti-CD28 antibodies with the addition of either IL-7/IL-15 or IL-2. CART generation lasted for 15 days. Cytotoxic ability of the generated CART was assessed by a standard chromium (Cr-51) release assay. CD19+ (Daudi) and CD19- (K562) cells were labeled for 2h with Cr-51 and coincubated with CART for 4h. Cr-51 release was measured in a liquid scintillation counter. In addition, multi-parametric flow cytometry was performed using a FACS LSR device. Results Overall, the transduction rate for both cytokine cocktails was around 50% of all CD3+ T cells. Freezing/thawing of CART reduced their cytotoxic activity: Lysis of the CD19+ target cell line (Daudi) reached a maximum of 80% for fresh cells and 50% for freshly thawed cells, both stimulated with IL-7/IL-15. FACS analysis revealed that freshly prepared cells showed an increased percentage of activated lymphocytes compared to cryopreserved cells. The majority of these activated lymphocytes were CD4+CD8+ double positive. Using freshly thawed cells, CART stimulated with IL-7/IL-15 showed higher cytotoxic activity than CART stimulated with IL-2: 50% vs. 38% (Daudi) and 7% vs. 5% (K562). Stimulation with IL-7/IL-15 led to an increase in the CD56+ CAR NK T cell population (20%) compared to IL-2 (8%). γ/δ TCR+ CART differed as well (3% for IL-7/IL-15, 8% for IL-2). The percentage of CD8+ and CD4+ CART was similar for both cytokine cocktails with around 60% and 30%, respectively. However, stimulation with IL-7/IL-15 showed a trend towards generation of a lower percentage of CD4+CD8+ double positive CART than stimulation with IL-2 (2% vs. 6%). The analysis of the different CART phenotypes showed significant differences: N 24% vs. 3%, CM 21% vs. 13%, EM 24% vs. 77%, E 31% vs. 7%, for IL-7/IL-15 vs. IL-2, respectively. A general phenomenon observed for both cytokine cocktails, was the difference in phenotype for CD4+ vs. CD8+ CART: CD4+ CART showed a more distinct memory phenotype, i.e. central and effector memory cells. Correspondingly, the CD8+ CART shifted more towards the naïve and effector phenotype. The percentage of highly effective stem cell memory CART (TSCM: naïve CD27+CD95+) was assessed. Using IL-7/IL-15, CAR TSCM cells accounted for 4.8%, whereas for IL-2, 2.7% had a TSCM phenotype. 75% of the CART were HLA-DR positive (activation marker) and 100% were positive for CD95 (FAS receptor, inhibition marker). Using PBMCs from healthy donor samples, the generated CART products did not contain any contamination of either B (CD19+) or stem cells (CD34+). Conclusions We have established a combination of immunophenotyping and cytotoxicity assays for CART as a preclinical potency assay. Stimulation with IL-7/IL-15 led to a balanced phenotype expression, with a similar number of N, CM, EM and E CART, whereas the majority of CART under IL-2 stimulation adopted an EM phenotype. Correlation of these data with the clinical outcome of patients receiving the corresponding cell products will allow optimization and standardization of CART therapy. Disclosures Wuchter: Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Gilead: Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy.

Description: Introduction: Graft-versus-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), results in post-transplant morbidity and mortality. Steroids with strong immunosuppressive and anti-inflammatory effects remain the gold standard for initial management of GvHD. However, high doses of steroids are usually accompanied by an increased risk of infections and secondary malignancies. ECP, due to its good clinical response, is recommended as a second-line treatment for GvHD. Notably, no clinical data showed that ECP therapy results in relapse and infection. However, the mechanism of action behind that is barely known. Therefore, we conducted this study to figure out how ECP preserves the anti-viral and anti-leukemia effects. Materials and Methods: 34 patients with steroid-refractory/resistant aGvHD ≥ °II and moderate to severe cGvHD received ECP therapy at the University Hospitals Heidelberg, Greifswald and Tel Hashomer. ECP therapy was performed according to the current European guidelines. For clinical staging of aGvHD under ECP treatment patients were evaluated according to Glucksberg criteria, for quality of life according to Karnofsky and for clinical staging of cGvHD according to NIH criteria. Phenotypical analysis of different cellular subsets was evaluated by multicolor flow cytometry. The quantity and quality of virus-specific CD8+ T cells were evaluated by Tetramer staining and IFN-γ-ELISPOT. NK activity in terms of killing function and cytokine release function was monitored by chromium-51 release assay and intracellular cytokine staining. Results: ECP seems to be favorable in the treatment of GvHD. Clinically, an overall response of 75% for aGvHD and 78% for cGvHD patients was achieved. A steroid-sparing effect in addition to the resolution of GvHD manifestations was observed in responders. Our patients showed no increased susceptibility to infections. On the cellular level, the frequency of cytotoxic CD8+ T cells, the most important mediators of GvL activity, as well as CD4+CD8+ T cells, γδ T cells, NKT cells and CD56dimCD57+NKG2C+ NK cells as other well-established protective cell subsets remained constant under ECP therapy. Moreover, neither frequency nor IFN-γ release of CMV specific CD8+ T cells was hampered by ECP. 51Cr-release assay revealed stable functional cytotoxicity of NK cells. Additionally, ECP therapy did not affect the capacity of cytokine release by NK cells, including quality, quantity and multifunctional release. Furthermore, no significant influence of ECP therapy on antigen recall function of NK cells, CD4+ and CD8+ T cells could be observed. Conclusion: ECP therapy represents an attractive strategy to treat GvHD. Moreover, our results reflect that ECP therapy preserves immunity against infections as well as the GvL effect via maintaining the quality and quantity of effector cells. Disclosures Hilgendorf: Novartis: Other: Travel support, Research Funding; Medac: Other: Travel support, Research Funding.