ALBERT

Description: Hematopoietic stem cells (HSCs) are administered (i) to restore hematopoiesis and immunity in the course of hematopoietic stem-cell transplantation (HSCT), (ii) as a replacement for inherited blood disorders and bone marrow failure, (iii) to regenerate cells of alternative lineages for restorative medicine, and (iv) as a source for generating specific hematopoietic cells (e.g., T cells, NK cells, and dendritic cells). However, the widespread application of allogeneic HSCs for humans is hampered by their immune-mediated destruction by host T cells recognizing mismatched HLA or by HLA-specific antibodies. Despite pre-banking umbilical cord blood (UCB) units and access to adult donors through the National Marrow Donor Program (NMDP), finding a suitable HLA-matched product is challenging for many recipients, especially those from ethnic minorities who are under-represented in the donor pool. The available donor pool would be markedly increased if donor HSCs were edited to eliminate expression of the HLA-A locus. Indeed, modeling from NMDP shows that the chance of an African American recipient finding a HLA-matched donor increases from 18% to 73% when matched for HLA-B, C and DR, instead of HLA-A, B, C and DR. We have previously shown that engineered zinc finger nucleases (ZFNs) can disrupt HLA-A expression in genetically edited T cells (Blood 2013). To extend this proof-of-concept to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus. CD34+lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB. Electro-transfer of in vitro transcribed mRNA encoding the HLA-A-specific ZFN generated 30% HLA-Aneg HSCs after one week ex vivo culture with defined cytokines (FLT3-L, SCF, TPO, and IL-6) and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1). As expected, SR1 treatment maintained greater numbers of CD34+ cells (also CD34posCD38neg) in culture compared to controls. DNA sequence analysis revealed that HLA-Aneg HSCs encode the expected nucleotide changes at the ZFN target site. An in vivo engraftment assay, using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, demonstrated that HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg hematopoietic cells (Figure). Thus, disruption of HLA-A expression in HSCs provides an appealing approach to increasing the chances for of finding HLA-matched donors and may broaden the clinical application of allogeneic HSCT. Furthermore, the ability to genetically edit HSCs has implications for (i) preventing immune-mediated recognition of HLA-disparate HSC and (ii) preventing immune mediated recognition of self-antigens. Engraftment of HLA-A2neg HSCs was evaluated in vivo. Data shown are flow-cytometry analysis of bone marrow obtained from NSG mice 16 weeks after HSC injection. HSC engraftment and HLA-A2 expression in NSG mice injected with un-modified HSCs (left panel) and HSCs treated with the HLA-A specific ZFNs (right panel) are shown. Data are gated on human CD45 positive cells. Figure Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Figure. Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Disclosures: Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

Description: Abstract 3766 Cell therapy by infusion of T cells can reconstitute immunity to combat pathogens and malignancies. However, the time required to manufacture T cells with the desired properties and in sufficient numbers ex vivo is often incompatible with the treatment window for patients. Furthermore, autologous T cells from patients with advanced disease may have compromised function and be tolerant to desired antigens. A potential solution would be an approach to infuse allogeneic T cells that avoids immune-mediated rejection caused by host T cells recognizing disparate major or minor histocompatibility antigens on the infused cells. To broaden the application of T cell therapy, we investigated whether HLA gene expression can be disrupted by designer zinc-finger nucleases (ZFNs). ZFNs comprise a zinc finger DNA binding domain designed to bind a specific DNA sequence fused to the cleavage domain of Fok I endonuclease. Since FokI dimerization is required to introduce a double strand break (DSB), we generated ZFN pairs that flank the intended DNA target sequences in the required spatial conformation. Cellular repair of the DSB by error-prone non-homologous end joining allows disruption of HLA gene expression. As an initial proof of concept experiment, transfection of ZFN pairs designed to target exon 3 of the HLA-A locus into the human kidney cell line HEK293 resulted in 10% genetic modification of the HLA-A loci. We generated clones of HEK293 cells that showed deletion or insertion mutations within the ZFN binding site of one or both HLA-A alleles leading to early termination of translation. These HLA-Anull HEK293 clones evaded HLA-A-restricted lysis by T cell clones, even after interferon-γ and TNF-α treatment was used to upregulate HLA expression. Since only transient expression of ZFNs is needed to disrupt a target gene, we tested the ability to disrupt HLA-A gene expression by electro-transfer of in vitro-transcribed ZFN mRNA into primary T cells. We show that a single administration of the mRNA encoding the ZFNs targeting HLA-A could render over 40% of primary T cells HLA-A negative. We enriched the HLA-Anull population by paramagnetic bead separation to obtain a pool of T cells 〉90% of which lack HLA-A expression. An attractive potential clinical application for HLAnull allogeneic T cells is to redirect their specificity independent of HLA via expression of a chimeric antigen receptor (CAR) targeting CD19. Thus, we eliminated HLA-A expression from CD19-specific CAR+ T cells and demonstrated that they (i) evade HLA-A-restricted lysis by T cell clones, and (ii) specifically lysed CD19+ tumor targets. Finally, to further improve this T cell product and eliminate potential deleterious immune mediated recognition by the endogenous T cell receptor (TCR) on allogeneic CAR+ T cells, we used ZFN pairs targeting the TCR α or the TCR β locus. Transient expression of these ZFNs resulted in permanent disruption of endogenous TCR expression and a highly enriched αβ TCRnull cell population could be generated by paramagnetic bead selection. These data support our plans to develop allogeneic T cells as “off-the-shelf” biologics that can be infused on demand as “drugs”. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment.

Description: Minor histocompatibility antigens (mHAgs) with expression limited to hematopoietic cells represent attractive targets for immunotherapy to induce selective graft-versus-leukemia (GVL) reactions. Here we report the identification of a novel mHAg which is recognized by an HLA-B*4403-restricted CTL clone. Microsatellite allele image analysis of two DNA pools generated from CTL-defined mHAg positive and mHAg negative groups was performed using microsatellite markers set at 100 kb intervals within the segment initially mapped by two-point genetic linkage analysis and detailed mapping of the chromosomal recombinant points. This approach defined a 0.53 Mbp region of chromosome 18q21–22 containing 12 candidate genes potentially encoding the mHAg, although the target gene could not be identified. Subsequently, cDNA expression cloning studies demonstrated that the CTL epitope of interest was encoded by a novel allelic splice variant of XM_209104, hereafter designated as XM_209104-av. Indeed, this gene was found to lie within the region predicted by microsatellite allele image analysis. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by one intronic single nucleotide polymorphism (SNP) located in the consensus 5′ splice site adjacent to an exon. To our knowledge, this is the first example of a mHAg controlled by a SNP located in a region other than coding sequences. Because the CTL lysed also HLA-B*4402 positive, mHAg positive B-LCLs, this novel epitope peptide can bind to not only HLA-B*4403 but also HLA-B*4402 which is a relatively common HLA-B allele in Caucasian populations. Finally, the finding that the novel XM_209104-av showed low or no expression in normal tissues including resting hematopoietic cells, but significantly higher expression in primary acute leukemia cells, especially those of myeloid lineage, suggest that this novel epitope may be an attractive therapeutic target for immunotherapy not only as a minor H antigen but also as a leukemia-associated antigen.

Description: ABO incompatibility between donor and recipient is not a barrier for successful allogeneic hematopoietic stem cell transplantation, but conflicting data still exist concerning its influence on transplant outcome, graft-versus-host disease (GVHD), relapse, and survival. We retrospectively analyzed the data of patients who underwent UR-BMT through the Japan Marrow Donor Program between January 1993 and September 2005, with complete data on ABO-blood group compatibility, age, and gender in donors and recipients. A total of 4,970 patients were transplanted with marrow from ABO-matched (M; n=2,513, 50.6%), major incompatible (MA; n=1,254, 25.2%), minor incompatible (MI; n=1,081, 21.8%), and bidirectional incompatible donors (IA; n=122, 2.5%), and were followed up over a median period of 325 days. Among these four groups, excluding age, there was no significant difference in the gender of patients and donors, number of transplantations, conditioning regimen, GVHD prophylaxis, and performance status before transplantation by the likelihood ratio test. The 5-year overall survival of any ABO-incompatible group was significantly lower compared to an identical group (Wilcoxon test, p

Description: Minor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

Description: Here we report the identification of a novel human leukocyte antigen (HLA)-B44–restricted minor histocompatibility antigen (mHA) with expression limited to hematopoietic cells. cDNA expression cloning studies demonstrated that the cytotoxic T lymphocyte (CTL) epitope of interest was encoded by a novel allelic splice variant of HMSD, hereafter designated as HMSD-v. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by 1 intronic single-nucleotide polymorphism located in the consensus 5′ splice site adjacent to an exon. Both HMSD-v and HMSD transcripts were selectively expressed at higher levels in mature dendritic cells and primary leukemia cells, especially those of myeloid lineage. Engraftment of mHA+ myeloid leukemia stem cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull mice was completely inhibited by in vitro preincubation with the mHA-specific CTL clone, suggesting that this mHA is expressed on leukemic stem cells. The patient from whom the CTL clone was isolated demonstrated a significant increase of the mHA-specific T cells in posttransplantation peripheral blood, whereas mHA-specific T cells were undetectable in pretransplantation peripheral blood and in peripheral blood from his donor. These findings suggest that the HMSD–v–encoded mHA (designated ACC-6) could serve as a target antigen for immunotherapy against hematologic malignancies.

Description: Background T-cell receptors (TCRs) can be used to redirect the specificity of T-cells for human application. This has particular appeal for the targeting of neoantigens. However, efficient identification, cloning, and characterization of antigen (Ag)-specific TCRs is needed to enable the timely adoptive transfer of T-cells genetically modified to express therapeutic TCRs. We have harnessed next generation sequencing (NGS) to identify desirable TCRs. This approach enables us to simultaneously identify hundreds of Ag-specific TCRs, along with the expression of genes to characterize functional and phenotypical values of individual Ag-specific T-cells (e.g. related to cytotoxicity, exhaustion, etc...). To take advantage of NGS technology, a sister high-throughput technology was developed to evaluate harvested TCRs. A reporter system was implemented using (immortalized) Jurkat T-ALL cells genetically modified to (i) enforce expression of CD8aβ, (ii) conditionally express GFP under minimal elements of NR4A1 promoter, and (iii) prevent expression of both endogenous TCRα and β chains. Sequenced CDR3 regions were coded within DNA plasmids from Sleeping Beauty (SB) transposons as TCR Vα and Vβ libraries that were expressed on the reporter cell to identify both TCR specificity as well as TCR avidity. Thus, we implemented (i) a TCR cloning system based on CDR3 sequencing of Ag-specific T-cells identified by NGS and (ii) a novel reporter cell based on the TCR-mediated induction of GFP expression. As a proof-of-concept to evaluate the entire platform, we used HLA-A2-restricted CMV peptide (NLVPMVATV: CMV/A2) and NY-ESO-1 peptide (SLLMWITQC: NY-ESO-1/A2) as model Ags. Results The reporter cell was initially genetically modified with either high- or low-avidity TCRs against a NY-ESO-1/A2. Upon stimulation with HLA-A2+ 721.221 immortalized B cells, it was found that the expression of GFP positively correlated with the avidity of TCRs (Figure). Next, we isolated naïve Ag-specific T-cells from umbilical cord blood using CMV/A2 tetramer. Single CMV/A2-specific CD8+ T-cells were sorted and their TCRαβ CDR3 sequences were amplified by reverse transcription and PCR with bar-coded probes. The pooled PCR products were sequenced in MiSeq Sequencer (illumina) to obtain TCRαβ CDR3 regions and analyzed in silico using IMGT (International Immunogenetics Information System). The efficiency of identifying individual TCRαβ pairs was 65% to 76% (using 96-well plate or 384-well plates, respectively). Individual SB-derived DNA transposons expressing TCRαβ constructs were synthesized by Gibson assembly and electroporated, with SB transposase, into the reporter cell. These cells were co-cultured with HLA-A2+ 721.221 stimulator cells loaded with graded doses of cognate peptide. The percentage and intensity of GFP expression was evaluated by high-throughput flow-cytometer (IntelliCyt) which revealed high-avidity Ag-specific TCRαβs. Conclusion Our new high-throughput system can identify and characterize, based on specificity and avidity, Ag-specific TCRαβs within a week. This system will be used to generate neoantigen-specific TCRαβs for human application. Disclosures Zong: ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership, Patents & Royalties. Cooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria. McNamara:GeoMcNamara: Consultancy, Other: Consultant in immuno-oncology field; ZIOPHARM Oncology, Inc: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Torikai:intrexon: Equity Ownership, Patents & Royalties; ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership.

Description: Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR+ T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR+TCRneg T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

Description: Key Points Allogeneic-donor–derived cells can be genetically modified to eliminate expression of HLA-A. HLA-A disruption from donor cells is a step toward generating allogeneic cells as an off-the-shelf therapeutic.

Description: The BCR/ABL fusion protein transforms hematopoietic stem cells and causes chronic myeloid leukemia (CML). An increasing number of mature neutrophils is a characteristic feature in the chronic phase of CML. However, the mechanism by which stem cells transformed by Bcr/Abl differentiate mainly to mature neutrophils remains obscure. To investigate this mechanism, we compared the gene expression profile of CML neutrophils with that of normal neutrophils by microarray analysis. The genes encoding neutrophil granule proteins were upregulated in CML neutrophils, and C/EBPα and C/EBPε, critical transcription factors that regulate granulocytic differentiation, were also upregulated in these neutrophils. On the contrary, the expression of c-jun, a transcriptional factor that contributes to monopoiesis, was downregulated in CML neutrophils. Differences in the expressions of these genes were confirmed by quantitative RT-PCR. A BCR/ABL tyrosine kinase inhibitor, imatinib, released the downregulation of c-jun expression in primary CML neutrophils, showing that Bcr/Abl inhibited the expression of c-jun. Next, to explore the roles of these transcriptional factors in the chronic phase of CML, we established sublines of KCL22, a cell line derived from CML blastic crisis, in which C/EBPα or C/EBPε expression was inducible (KCL22/α or KCL22/ε respectively). Overexpression of either C/EBP protein resulted in morphological changes, such as a reduction of the nuclear to cytoplasmic ratio, more condensed nuclear chromatin, and segmented nuclei, as well as the expression of differentiation specific markers including G-CSF receptor. These data indicate that C/EBPα/ε expression is sufficient to induce myeloid differentiation in BCR/ABL-positive CML cells. Imatinib treatment released the down regulation of c-jun in KCL22, KCL22/α, and KCL22/ε cells in a manner similar to that in primary cells. Interestingly, imatinib induces monocytic differentiation of KCL22/α cells instead of granulocytic differentiation. This effect of imatinib is independent from C/EBPα induction in KCL22/α cells. The monocytic differentiation and the inhibition of granulocytic differentiation in KCL22/α cells were accompanied by c-jun upregulation. To investigate whether these effects on differentiation of KCL22/α cells depend on releasing the downregulation of c-jun expression with imatinib, we knocked down c-jun expression with siRNA in KCL22/α cells after induction of C/EBPα protein and with imatinib treatment. In the fraction of KCL22/α cells with segmented nuclei, the G-CSF receptor increased when c-jun expression was inhibited with siRNA, indicating that the level of c-jun expression controlled the differentiation fate of CML cells. These findings suggest that Bcr/Abl promotes neutrophil differentiation through downregulation of c-jun accompanied by elevated expressions of C/EBPα/ε.