ALBERT

Description: Transfer of tumor antigen–specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRα/β chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRα constant α (Cα) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Cα domain preceded by a TCRα signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Cα were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector ζ-chain–associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Cα transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Description: Reactivation of latent human cytomegalovirus (HCMV) infection is a frequent complication in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Preclinical research in murine models as well as clinical phase I/II trials have shown that the adoptive transfer of virus-specific CD8+ T cells is a therapeutic option for preventing HCMV disease. However, the feasibility of HCMV-specific immunotherapy is currently limited in clinical routine due to technical restrictions. It has also limitations, if the donor is HCMV-seronegative or carries only low numbers of HCMV-specific memory T cells. In this situation, grafting non-reactive T cells by virus-antigen specific T-cell receptors (TCR) may be an alternative means to transfer HCMV-specific T-cell function into HSCT recipients. Nevertheless, improvement of clinical protocols is needed before HCMV-specific cell therapy can be implemented in general clinical practice. Here we describe a novel preclinical mouse model that will help to evaluate new HCMV-immunotherapy options. Due to the strict species specificity of CMV, we constructed a recombinant murine CMV (mCMV) coding for the HLA-A2 restricted pp65495-503 (NLV) peptide epitope of HCMV. This peptide is expressed with flanking amino acids within the mCMV-IE2-protein in the context of experimental infection with mCMV-IE2/NLV. The NLV peptide was found to be presented by infected murine fibroblasts from NOD/SCID/IL-2Rγc-/--HLA-A2-transgenic (NSG-A2) mice as shown by T cell specific recognition in ELISPOT and cytotoxicity assays. Interestingly, this presentation proved to be susceptible to the inhibitory function of mCMV immune evasion proteins. NSG-A2 mice were then used to evaluate antiviral control by HCMV-NLV-specific TCR (TCRNLV)-engineered human T cells. After infection with mCMV-IE2-NLV, the NSG-A2 mice showed high viral titers in liver, spleen, lungs, and salivary glands as well as a dramatic virus-induced morbidity and mortality. In contrast, after intravenous injection of TCRNLV-transduced human CD4+/CD8+ T cells, viral infection was reduced in all tested organs, and mice showed improved survival. These findings suggest that the NLV peptide is processed and presented by stromal and parenchymal cells of the NSG-A2 mice and promise validity of the model for preclinical evaluation of adoptive transfer protocols, for instance cell therapy with TCR-engineered T cells alone or in combination with therapeutic vaccination. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3577 Poster Board III-514 Reactivation 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.

Description: Introduction: Patients (pts) with acute myeloid leukemia (AML) who are refractory to intensive frontline treatment have a dismal outcome. In case of ineligibility for allogeneic stem cell transplantation (HSCT), the median survival of chemo-refractory AML is about 2 months and less than 5% of these pts are alive after 1-year (retrospective analysis from the AMLSG database). To date, there is no universally accepted standard approach for the treatment of chemo-refractory AML in older pts. Several retrospective studies have assessed the role of hypomethylating agents in this patient group, but complete remission (CR) rates were disappointingly low (≤10%) when compared to first line treatment. The presented study represents a novel approach focusing on hematopoietic tissue reprogramming (i.e. anakoinosis) (ClinicalTrials.gov Identifier: NCT02942758). Methods: The initial dose-finding phase I of the study evaluated the combination of azacitidine (AZA) 75 mg/d s.c. for 7 days, repeated every 28-days, pioglitazone 45 mg/d p.o. continuously from day 1 and all-trans retinoic acid (ATRA). A modified 3+3 design has been used to establish the maximum-tolerated dose of ATRA. Patients have been enrolled at an ATRA dose of 45 mg/m²/d from day 1 to day 28 and 15 mg/m²/d continuously thereafter if no dose limiting toxicity (DLT) occurred until start of next cycle on day 29. The safety DLTs were defined as toxicities attributable to ATRA, expected or unexpected, except if these are likely associated with another cause. Eligible patients had confirmed diagnosis of AML refractory to induction therapy and were not eligible for further intensive induction therapy or were not immediate candidates for allogeneic HSCT. The severity of adverse events was graded using the Common Terminology Criteria for Adverse Events (CTCAE) V. 4.03. The response to treatment was evaluated using standard criteria defined by the expert panel on behalf of the European LeukemiaNet and international working group (IWG) response. Results: Ten pts were enrolled in the safety-run-in phase I (one pt withdrew informed consent on day 9 of cycle 1). Among all treated pts, the median age was 67 years (range, 62-76 years), and the majority of pts (70%) had an ECOG PS of 1 (see table 1). Two pts had secondary AML; another two pts had therapy-related AML (t-AML). Eight pts had a complex karyotype. Concerning safety, hematological adverse events (AEs) were the most common toxicities observed. Because pts with baseline cytopenia were included (leukopenia n=8; 80%; thrombocytopenia n=9; 90%), occurrences of many hematological AEs began before study drug initiation and were attributed to underlying hematologic disease. Common 3°/4° AEs included neutropenia (50%), anemia (50%), thrombocytopenia (30%), and infections (40%). 50% of pts experienced a serious AE; one 5° AE (gastric hemorrhage) occurred. No DLTs were observed. Five pts discontinued the study, with progressive disease (PD) or relapse being the most common reason for discontinuation. Concerning efficacy, 3 pts (30%) achieved a CR and one pt a long-lasting stable disease (14 months). Morphologic review showed signs of differentiation of blasts in responding pts, which has already been shown in in-vitro analysis. In line with this observation, one pt demonstrated resolution of fungal pneumonia during the study. Conclusions: In summary, the low-intensity, biomodulatory regimen of low-dose AZA, pioglitazone, and ATRA demonstrated a tolerable safety profile and encouraging signals for efficacy in pts with AML refractory to standard induction chemotherapy warranting further investigation. S.T. and A.R. contributed equally to this abstract as senior co-authors. Disclosures Paschka: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Abbvie: Other: Travel expenses; Amgen: Other: Travel expenses; Otsuka: Membership on an entity's Board of Directors or advisory committees; BMS: Other: Travel expenses, Speakers Bureau; Astex: Membership on an entity's Board of Directors or advisory committees, Travel expenses; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Other: Travel expenses; Takeda: Other: Travel expenses; Sunesis: Membership on an entity's Board of Directors or advisory committees. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AROG, Bristol Myers Squibb, Pfizer: Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria. Thomas:Celgene: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Medigene AG: Consultancy, Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Other: Travel support; Janssen: Other: Travel support.

Description: HLA-DPB1 antigens are mismatched in about 80% of allogeneic hematopoietic stem cell transplantations from HLA 10/10 matched unrelated donors and were shown to be associated with a decreased risk of leukemia relapse. We recently developed a reliable in vitro method to generate HLA-DPB1 mismatch-reactive CD4 T-cell clones from allogeneic donors. Here, we isolated HLA-DPB1 specific T cell receptors (TCR DP) and used them either as wild-type or genetically optimized receptors to analyze in detail the reactivity of transduced CD4 and CD8 T cells toward primary AML blasts. While both CD4 and CD8 T cells showed strong AML reactivity in vitro, only CD4 T cells were able to effectively eliminate leukemia blasts in AML engrafted NOD/SCID/IL2Rγc−/− (NSG) mice. Further analysis showed that optimized TCR DP and under some conditions wild-type TCR DP also mediated reactivity to non-hematopoietic cells like fibroblasts or tumor cell lines after HLA-DP upregulation. In conclusion, T cells engineered with selected allo-HLA-DPB1 specific TCRs might be powerful off-the-shelf reagents in allogeneic T-cell therapy of leukemia. However, because of frequent (common) cross-reactivity to non-hematopoietic cells with optimized TCR DP T cells, safety mechanisms are mandatory.