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The immune system is a complex network of checks and balances in constant flux. Quantitative characterization of the system dynamics, sampled via constituent components such as T cells, combined with mathematical modeling enabled us to obtain “statistical pattern recognition” of immune states and transitions over time. Here we show a statistical framework to characterize immune states for adoptive immunotherapy using serial infusions of activated polyclonal T cells into companion canines diagnosed with B-cell non-Hodgkin Lymphoma (NHL) post CHOP chemotherapy regimen as a model for human disease. We applied multiplexed gene profiling techniques to assess changes in gene expression data from 10 companion canine patient clinical samples and gene regulatory networks (GRN) information to build an “immune landscape” that predicts immune states, state transitions, and plasticity for immunomodulation (“reprogrammability”) over the course of immunotherapy regimens. The potency of immune modulation and immune surveillance, two critical parameters of efficacy through inferred state transitions, were evaluated by measuring distortions and shifts in the immune landscape of canine patients undergoing treatment through system dynamics, Adjusted Rand Index, Pearson Correlation, and ANOVA analyses. Adjust Rand Index validated hierarchical clustering analyses. Pearson correlations isolated genes that were up- or down-regulated with regard to single genes of interest. ANOVA analyses indicated gene expression difference between temporal samples. Applying such analyses to adoptive T-cell immunotherapy, allowed us to examine clinical/tumor remissions through the lens of immune state transitions, affording real-time improvements to therapeutic regimens. In developing a formal framework to capture the effects of molecular signatures and GRN over time to describe distinct immune states, we enhanced the clinical cancer immunologist's analytical arsenal (with a measuring tool/sensor) and helped bridge the gap between bench and bedside.

Meeting abstracts Adoptive cell therapy (ACT) with expanded autologous tumor infiltrating lymphocytes (TIL) has emerged to be a powerful salvage therapy for metastatic melanoma with response rates up to 50%. However, the generation of the TIL ACT product is technically challenging with current

The Pem gene encodes an atypical homeodomain protein, distantly related to Prd/Pax family members, that we demonstrate is regulated in a complex transcriptional and post-transcriptional manner. We show that the rat Pem genomic structure includes three 5′-untranslated (5′-UT) exons and four coding exons, three of which encode the homeodomain. Several alternatively spliced transcripts were identified, including one that skips an internal coding exon, enabling this mRNA to express a novel form of the Pem protein. Other alternatively spliced mRNAs were characterized that possess different 5′-UT regions, including a muscle-specific transcript. The different 5′-UT termini present in Pem transcripts conferred different levels of translatability in vitro. Two promoters containing multiple transcription initiation sites were identified: a distal promoter (Pd) in the first 5′-UT exon and a proximal promoter (Pp) located in the “intron” upstream of the first coding exon. The Pd was active in placenta, ovary, tumor cell lines, and to a lesser extent in skeletal muscle. In contrast, transcripts from the Pp were only detectable in testis and epididymis and were only expressed in epididymis in the presence of testosterone. To our knowledge no transcription factors have previously been identified that exhibit androgen-dependent expression in the epididymis.

A hallmark of homeobox genes is their high degree of sequence conservation in distantly related species. Here, we report the chromosomal localization, sequence, and expression pattern of an orphan homeobox gene, Pem, that encodes a homeodomain (HD) that has undergone a surprisingly high rate of evolutionary change. The N-terminal portion of the Pem HD, which includes the first two alpha-helices, exhibits only 44% sequence identity between rat Pem (r.Pem) and mouse Pem (m.Pem). This N-terminal subdomain exhibited an extremely high frequency of nonsynonymous substitutions, severalfold higher than other regions of the Pem protein. In contrast, the third helix, which is known to confer most of the base-specific contacts of HDs with DNA, was almost identical in r. Pem and m.Pem. Several lines of evidence suggested that the rat and mouse genes that we identified as Pem genes are true homologues: (1) the r.Pem and m.Pem genes both reside on the X chromosome; (2) they possess identical exon/intron splice junctions; (3) they both encode a distinctive motif upstream of the HD that is unique to Pem; and (4) the only m.Pem-like gene we were able to identify in the rat genome other than r.Pem was a pseudogene, r.Pem-ps, whose sequence and chromosomal localization indicated that it was derived by reverse transcription and reinsertion into the genome. The functional r.Pem gene is selectively expressed in placenta, testis, epididymis, and ovary. This expression pattern is of interest since other genes transcribed in reproductive tissue have also been shown to undergo high rates of sequence divergence. The high rate of amino acid substitutions in the N-terminal region of the Pem HD suggests the possibility of species-specific directional selection.

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

Functionally pleiotropic human γδ T cells are a unique set of immune cells that exhibit qualities of both innate and adaptive immune responses. In contrast to αβ T cells, γδ T cells recognize their ligands independent of MHC and are infrequent (1%–5% of T cells) in peripheral blood. Studies have shown that γδ T cells exhibit endogenous cytotoxicity towards tumor cells by directly recognizing a diverse array of Tumor Associated Antigen (TAA) and have the ability to present TAA to elicit an antitumor response. This broad recognition of antitumor activity is achieved because these cells express a diverse TCR γδ repertoire (combination of Vδ1, Vδ2, or Vδ3 with one of fourteen Vγ chains). Because of their characteristics γδ T cells are attractive targets for clinical manipulation and tumor immunotherapy. To obtain clinically significant numbers of cells for immunotherapy current ex vivo γδ T cells expansion protocol uses Zoledronic acid, an aminobisphosphonate that results only in selective propagation of Vγ9Vδ2. These cells however, showed clinical response against both solid and hematologic tumors. We have previously shown a novel ex vivo expansion protocol using artificial antigen presenting cells (aAPC), IL2 and IL21 assist aAPC in activating and expanding polyclonal γδ T-cells (Vδ1, Vδ2 and Vδ1neg Vδ2neg T cell subtypes) to clinically significant numbers. These expanded cell subsets secrete proinflammatory cytokines, lysed a broad range of malignancies and improved survival in ovarian cancer xenograft model. To better define the role of the subsets and their application in immunotherapy here we present high-resolution transcriptome, microRNA and secreted cytokine-chemokine signatures of ex vivo expanded subsets. Our data provide an atlas of transcriptional, epigenetic and secretome of three defined subsets, and subset-specific signatures. The data presented here will have a broader impact in understanding the immunobiology of γδ T cell subsets and facilitate improving the human application of these cells in cancer immunotherapy. Citation Format: Sourindra Maiti, Drew Deniger, Jianliang Dai, Laurence Cooper. Transcriptional and epigenetic signatures of ex vivo propagated three distinct TCR Vδ1, TCR Vδ2 and TCR Vδ3 cell subtypes with broad specificity for malignancies. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B028.

Myelosuppressive and ablative regimens are used to deplete lymphocytes in some patients before adoptive cell therapy. CD19-specific engineered T-cells have been used successfully to eliminate B-cells in patients with B-cell leukemias and lymphomas. Prolonged immunosuppression, including B-cell aplasia is associated with increased risk of invasive fungal infection (IFI). Thus, patients will benefit if bio-engineered T cells can control IFI as well as kill tumor cells. The Sleeping Beauty gene transfer system was used to render T cells to express a chimeric antigen receptor (CAR) on the cell surface to redirect specificity via Dectin-1. Upon binding with the β-1,3-glucan sugar moiety found in IFI, Dectin-1 CAR+ T cells signal via chimeric CD28 and CD3-ζ signaling domain. The activated T cells secrete perforin, granzyme and granulysin to damage hyphae and thus inhibit the hyphal growth of Aspergillus and Candida. The T cells also secrete IFN-γ to activate other immune cells to target IFI. To ready the Dectin-1 CAR-modified T cells for the human application, we have modified the Fc domain of the extracellular stalk to alleviate deleterious uptake by Fcγ receptors and thus in vivo loss of infused T cells. We have shown that the Dectin-1 CAR+ T cells do not kill the yeast form of Candida so normal commensals in the gut microbiota should not be affected. We have also demonstrated that Dectin-1 CAR+ T cells can control Aspergillus infection in the presence of immunosuppressive drugs at physiological concentrations. Finally, we have generated dual CAR+ T cells by co-expressing a CD19-specific CAR with Dectin-1 CAR rendering the resultant genetically modified T cells able to target both B-cell malignant cells as well as fungal hyphae. These dual CAR-T cells can thus control both leukemia and invasive fungal infections. These data provide a path forward for clinical trials testing Dectin-1 CAR+ T cells. Citation Format: Pappanaicken R. Kumaresan, Nathaniel Albert, Harjeet Singh, Simon Olivares, Sourindra N. Maiti, Tiejuan Mi, Helen Huls, Richard E. Champlin, Dimitrios P. Kontoyiannis, Laurence J.N. Cooper. Bioengineered Dectin-1 CAR+ T cells to control invasive fungal infection . [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A193.

Purpose: T-cell receptor (TCR) variable Vα and Vβ gene diversity is a surrogate biomarker for the therapeutic potential of adoptive immunotherapy and cellular immunity. Therefore, creating a straightforward, rapid, sensitive, and reliable method to view the global changes of both TCRVα and Vβ transcripts in heterogeneous populations of T cells is appealing. Experimental Design: We designed a “direct TCR expression assay” (DTEA) using a panel of customized bar-coded probes that simultaneously detects and quantifies 45 Vα and 46 Vβ transcripts in a nonenzymatic digital multiplexed assay from a small number of cells (104 cells) or as little as 100 ng of total RNA. Results: We evaluated DTEA on total RNA samples of tumor-infiltrating lymphocytes and peripheral blood obtained from patients with melanoma after adoptive T-cell therapy. DTEA detected a similar spectrum of the dominant patterns of TCRVβ gene usage as sequencing cloned TCRVβ CDR3 regions. However, DTEA was rapid, achieved a level of sensitivity to identify rare T-cell populations, and simultaneously tracked the full array of Vα and Vβ transcripts. Conclusions: DTEA can rapidly and sensitively track changes in TCRVα and Vβ gene usages in T-cell pools following immune interventions, such as adoptive T-cell transfer, and may also be used to assess impact of vaccination or reconstitution of T-cell compartment after hematopoietic stem cell transplantation. Clin Cancer Res; 18(17); 4733–42. ©2012 AACR.

Clinical observations reveal that an augmented pace of T-cell recovery after chemotherapy correlates with improved tumor-free survival, suggesting the add-back of T cells after chemotherapy may improve outcomes. To evaluate adoptive immunotherapy treatment for B-lineage non-Hodgkin lymphoma (NHL), we expanded T cells from client-owned canines diagnosed with NHL on artificial antigen presenting cells (aAPC) in the presence of human interleukin (IL)-2 and IL-21. Graded doses of autologous T cells were infused after CHOP chemotherapy and persisted for 49 days, homed to tumor, and significantly improved survival. Serum thymidine kinase changes predicted T-cell engraftment, while anti-tumor effects correlated with neutrophil-to-lymphocyte ratios and granzyme B expression in manufactured T cells. Therefore, chemotherapy can be used to modulate infused T-cell responses to enhance anti-tumor effects. The companion canine model has translational implications for human immunotherapy which can be readily exploited since clinical-grade canine and human T cells are propagated using identical approaches.

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.

It has been demonstrated that a chimeric antigen receptor (CAR) can directly recognize the CD19 molecule expressed on the cell surface of B-cell malignancies independent of major histocompatibility complex (MHC). Although T-cell therapy of tumors using CD19-specific CAR is promising, this approach relies on using expression vectors that stably integrate the CAR into T-cell chromosomes. To circumvent the potential genotoxicity that may occur from expressing integrating transgenes, we have expressed the CD19-specific CAR transgene from mRNA using a high throughput microelectroporation device. This research was accomplished using a microelectroporator to achieve efficient and high throughput non-viral gene transfer of in vitro transcribed CAR mRNA into human T cells that had been numerically expanded ex vivo. Electro-transfer of mRNA avoids the potential genotoxicity associated with vector and transgene integration and the high throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression. We fabricated and tested a high throughput microelectroporator that can electroporate a stream of 2 x 10(8) primary T cells within 10 min. After electroporation, up to 80% of the passaged T cells expressed the CD19-specific CAR. Video time-lapse microscopy (VTLM) demonstrated the redirected effector function of the genetically manipulated T cells to specifically lyse CD19+ tumor cells. Our biomedical microdevice, in which T cells are transiently and safely modified to be tumor-specific and then can be re-infused, offers a method for redirecting T-cell specificity, that has implications for the development of adoptive immunotherapy.

To address B-lineage acute lymphoblastic leukemia (B-ALL), we use a bioengineering approach to achieve high-efficient non-viral gene transfer of tumor-specific CAR+ T cells for on-demand use. Specifically, we have developed CD19-specific chimeric antigen receptors (CARs) to redirect the specificity of T cells for B-cell malignancies. We introduce CARs via a non-viral gene transfer: electroporation. We have fabricated and tested a series of high throughput micro-electroporation devices (HitMeDs) with the end result that a HitMeD can electroporate a large number (≫109) human T cells within short time period (≪4 hours). We connect the HitMeDs to a BTX pulse generator and a monitoring system. We have adapted HitMeDs to the electro-transfer of CAR mRNA which avoids the potential genotoxicity associated with vector integration. After electroporation, 70% to 80% of the T cells express CD19 -specific CAR.

A total of 160 Escherichia coli positive for F165 fimbrial antigen and isolated from diarrheic and septicemic animals, were examined for the presence of the pap, afa, and sfa/foc operons or related nucleotide sequences using colony hybridization. Most isolates shared DNA sequences with the pap operon sequences alone or in association with afa or sfa. Thus, our results indicate that F165-positive E. coli from diseased animals share DNA sequences with operons coding for adhesins important in human extra-intestinal disease and that multiple adhesin systems are often found in single isolates. However, 20% of the F165-positive isolates did not show any homology with the probes representing the three adhesin systems, suggesting that one of the operons responsible for F165 production could be different from the pap, sfa/foc, and afa operons.

Beta2-glycoprotein 1 (beta2GP1), a 50 kDa serum glycoprotein that binds anionic phospholipid-containing membranes, plays a regulatory role in physiology and pathology. The protein is a member of the short consensus repeat (SCR) superfamily containing four typical repeating domains and an aberrant fifth domain constructed into an SCR-like core at the C-terminus. To investigate the contribution of the individual domains to the binding of beta2GP1, a series of sequential domain-deleted recombinant protein fragments were generated and assessed for their interaction with PS-containing vesicles. Spectral analyses of lipid binding-dependent alterations in tryptophan emission spectra revealed that the (single) tryptophan residues of the individual domains underwent binding-dependent conformational alterations. Depending on the ionic strength, some domains moved from polar to nonpolar environments, while others moved from less polar to more polar environments. Analysis of a series of acrylamide quenching and resonance energy transfer experiments indicated that the binding of N-terminal domain 1 to PS membranes exists in two, ionic strength-dependent, conformations. At low ionic strengths, domain 1 bound to the vesicles and induced their precipitation and/or aggregation. At physiologic ionic strengths, domain 1 detached from the membrane surface while the remaining domains maintained their association with the membrane. Under these conditions, membrane-bound conformationally altered domain 1 projects away from the membrane surface, enabling it to interact with other proteins and/or cell surface ligands or receptors.

A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein beta-2-glycoprotein 1 (beta2GP1). beta2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of beta2GP1 to EC induced to expose PS. We also show that divalent 3G4-beta2GP1 complexes are required for enhanced binding, since 3G4 Fab' fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric beta2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of beta2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of beta2GP1 for anionic phospholipids through formation of multivalent 3G4-beta2GP1 complexes.

Accurate prognostication is essential for patients with myelodysplastic syndromes (MDS) to facilitate optimal patient management. Several systems for predicting the outcome of patients with MDS are commonly used in clinics, including the International Prognostic Scoring System (IPSS), revised IPSS, and a low risk MDS model developed by The University of Texas MD Anderson Cancer Center (MDA). We designed this study to evaluate and compare these three models in lower risk MDS patients in order to accurately identify patients with a poor prognosis who may need early therapeutic intervention. A total of 733 adult patients, median age 69 years (range, 24-91), who were diagnosed as lower risk MDS (IPSS low or intermediate-1) with complete cytogenetic evaluations at MDA were included in this study. Both IPSS and revised IPSS systems stratified the study cohort into groups with significantly different overall survival (OS) (P Because both IPSS models are heavily weighted toward cytogenetic features, but over half of MDS patients present with normal (diploid) cytogenetics, we compared the effectiveness of these models in risk stratification among diploid MDS patients. Without the effects of cytogenetics, all 3 models still stratified OS significantly (P Molecular markers hold promise for further risk stratification in diploid MDS patients. We assessed the prognostic significance of common gene mutations, such as NPM1, FLT3, DNMT3A, NRAS and KIT. We also identified a signature of plasma microRNAs of which expression levels can predict survival of diploid MDS patients more accurately than all three models. In summary, the revised IPSS system has improved the stratification of patients in the IPSS low and INT-1 categories. The MDA model improves the OS stratification in IPSS low and INT-1 groups, as well as the revised IPSS low group. All three systems effectively predict survivals in diploid MDS patients. Development of novel molecular markers will enable further risk stratification of MDS patients, especially in the diploid MDS group. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Kantarjian: ARIAD, Pfizer, Amgen: Research Funding. Cooper:InCellerate: Equity Ownership; Sangamo: Patents & Royalties; Targazyme: Consultancy; GE Healthcare: Consultancy; Ferring Pharmaceuticals: Consultancy; Fate Therapeutics: Consultancy; Janssen Pharma: Consultancy; BMS: Consultancy; Miltenyi: Honoraria.

Objectives: T cells can be genetically modified ex vivo to redirect specificity upon expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We employ non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in patient (pt)- or donor-derived T cells for patients with advanced B-cell malignancies. Methods: T cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of designer g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. Results: To date we have successfully manufactured product for 42 pts with multiply-relapsed ALL (n=19), NHL (n=17), or CLL (n=5) on 4 investigator-initiated trials at MD Anderson Cancer Center to administer thawed pt- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=5), allogeneic (n=21) or umbilical cord (n=4) hematopoietic cell transplantation (HCT), or for the treatment of active disease (n=12). Each clinical-grade T-cell product was subjected to a battery of in-process and final release testing. Adjuvant trials: Twelve pts have been infused with donor-derived CAR+ T cells following allogeneic HCT, including 2 pts with cord blood-derived T cells (ALL, n=10; NHL, n=2), beginning at a dose of 106 and escalating to 5x107 modified T cells/m2. Three pts, all with ALL, remain alive and in remission at median 5 months following T cell infusion. Five pts with NHL have been treated with pt-derived modified T cells following autologous HCT at a dose of 5x108 T cells/m2, and 4 pts remain in remission at median 12 months following T-cell infusions. Relapse trials: Thirteen pts have been treated for active disease (ALL, n=8; NHL, n=3; CLL, n=2) with pt or donor-derived (if prior allo-HCT) modified T cells at doses 106-5x107/m2, and 3 remain alive and in remission at median 3 months following T-cell infusions. No acute or late toxicities, including excess GVHD, have been noted. Conclusion: We report the first human application of the SB and AaPC systems to genetically modify clinical-grade cells. Furthermore, infusing CD19-specific CAR+ T cells in the adjuvant HCT setting and thus targeting minimal residual disease may provide an effective and safe approach for maintaining remission in pts at high risk for relapse. Next steps: The SB system serves as a nimble and cost-effective platform for genetic engineering of T cells. We are implementing next-generation clinical T-cell trials targeting ROR1, releasing T cells for infusion within days after electro-transfer of SB DNA plasmid coding for CAR and mRNA coding for transposase, and infusing T cells modified with CAR designs with improved therapeutic potential. Figure: Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Figure:. Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Disclosures No relevant conflicts of interest to declare.

Apoptotic cells and phagocytes have developed a diverse array of distinct ligand-receptor systems that drive the recognition and uptake of dying cells. Phagocytes recognize apoptotic cells either directly, by binding to specific ligands at their cell surface, or indirectly, by binding to bridging proteins that bind these ligands. Previous observations showed that the plasma bridging protein beta2GP1, binds PS containing vesicles, and enhances their binding and engulfment by phagocytes in vitro. In this study we show that apoptotic cells injected intravenously and intraperitonealy into syngeneic mice recruited the PS binding protein, beta2GP1. Examination of peritoneal exudates and spleen thin sections showed that only the injected apoptotic cells picked up endogenous beta2GP1. Recovery of cells from the peritoneum showed that apoptotic cells bearing beta2GP1 were clustered around host peritoneal phagocytes. In addition, tissue sections from mice injected with Fas antibody showed colocalization of beta2GP1 with TUNEL-positive apoptotic cells. These results provide evidence that endogenous beta2GP1 binds apoptotic cells in vivo, suggesting that the protein plays an important physiologic role in the recognition of dying cells.

One mechanism by which normal cells become converted to tumor cells involves the aberrant transcriptional activation of genes that are normally silent. We characterize a promoter that normally exhibits highly tissue- and stage-specific expression but displays ubiquitous expression when cells become immortalized or malignant, regardless of their lineage or tissue origin. This promoter normally drives the expression of thePem homeobox gene in specific cell types in ovary and placenta but is aberrantly expressed in lymphomas, neuroblastomas, retinoblastomas, carcinomas, and sarcomas. By deletion analysis we identified a region between nucleotides −80 and −104 that was absolutely critical for the expression from this distal Pempromoter (Pem Pd). Site-specific mutagenesis and transfection studies revealed that this region contains two consensus Ets sites and a single Sp1 site that were necessary for Pem Pd expression. Gel shift analysis showed that Ets and Sp1 family members bound to these sites. Transfection studies demonstrated that the Ets family members Elf1 and Gabp and the Sp1 family members Sp1 and Sp3 transactivated the Pem Pd. Surprisingly, we found that Sp3 was a more potent activator of the Pem Pdthan was Sp1; this is unusual, because Sp3 is either a weak activator or a repressor of most other promoters. Activation by either Elf1 or Gabp required an intact Sp1 family member binding site, suggesting that Ets and Sp1 family members cooperate to activate Pem Pdtranscription. Expression from the Pem Pd (either transiently transfected or endogenous) depended on the Ras pathway, which could explain both its Ets- and Sp1-dependent expression in normal cells and its aberrant expression in tumor cells, in which ras protooncogenes are frequently mutated. We suggest that the Pem Pd may be a useful model system to understand the molecular mechanism by which a tissue-specific promoter can be corrupted in tumor cells.

Meeting abstracts Adoptive cell therapy using autologous TIL is a promising immunotherapy for metastatic melanoma. This study was aimed at finding predictive biomarkers in TIL patients, using IHC and gene expression analysis on original tumors used to expand TIL. The purpose was to find factors

s / Biol Blood Marrow Transplant 20 (2014) S128eS150 S132 CTL was characterized as to eps specificity and restricting HLA allele. The pool of CTL donors inherited HLA alleles in frequencies similar to those in caucasian and black populations, with certain alleles overrepresented. In 55% of the CTL lines, the ID T-cell response (TCr) was restricted (restr.) by 3 HLA alleles: A0201(25%), B0702(21%) and B 3501-11(9%); 45% of CTL lines were restr. by other class-I alleles, and 17/123(14%) were restr. by class-II alleles. Certain CMVpp65 15 mers contained overlapping eps presented by both class-I and class-II, which elicitedmore robust TCr. CTLs from all B0702+ donors (26/26) were restr. by B0702. In comparison, CTLs from 30/39(77%) A0201+ donors and 9/19 (47%) HLA B3501-11+ donors were restr. by A0201 and B3501-11 respectively. Donors coinheriting A0201 and B 0702 (9/9) universally demonstrated B0702 restr. ID TCr. In comparison, 11/12 (91.6%) donors coinheriting A0201 and B 4401-04 demonstrated HLA A0201 restr. CTLs. Strikingly, only 1/123 and 0/123 A1101+ and A0301+ donors respectively demonstrated CTLs restr. by these commonly inherited class eI alleles. This analysis thus indicates an immunodominance hierarchy for CMVpp65 eps and their presenting HLA alleles. In a series of 239 MUD, 137 MMUD, and 100 DUCB consecutive HSCT at MSKCC, we could identify an immediately available CMVpp65 specific CTL line matched with the patient at 2-3 alleles and restr. by a shared HLA allele in this GMP-CTL bank in 86%, 89% and 80% cases respectively. This CTL bank thus provides a clinical reagent for the treatment of CMV infections in HSCT recipients. The delineated immunodominance hierarchy for CMVpp65 may also facilitate selection of an appropriately restr. CTL line for treatment with predicted activity in the recipient. We can also successfully generate CMV-CTLs against subdominant CMVpp65 eps presented prevalent HLA alleles using a panel of artificial antigen presenting cells (AAPCs). Expansion of this bank with CTLs generated with AAPCs can broaden its applicability to almost all HSCT recipients.

RNA splicing generates two products in equal molar amounts, mature mRNAs and spliced introns. Although the mechanism of RNA splicing and the fate of the spliced mRNA products have been well studied, very little is known about the fate and stability of most spliced introns. Research in this area has been hindered by the widely held view that most vertebrate introns are too unstable to be detectable. Here, we report that we are able to detect all three spliced introns from the coding region of the Pem homeobox gene. By using a tetracycline (tet)-regulated promoter, we found that the half-lives of these Pem introns ranged from 9 to 29 min, comparable with those of short lived mRNAs such as those encoding c-fos and c-myc. The half-lives of the Pem introns correlated with both their length and 5' to 3' orientation in the Pem gene. Subcellular fractionation analysis revealed that spliced Pem introns and pre-mRNA accumulated in the nuclear matrix, high salt-soluble, and DNase-sensitive fractions within the nucleus. Surprisingly, we found that all three of the spliced Pem introns were also in the cytoplasmic fraction, whereas Pem pre-mRNAs, U6 small nuclear RNA, and a spliced intron from another gene were virtually excluded from this fraction. This indicates either that spliced Pem introns are uniquely exported to the cytoplasm for degradation or they reside in a unique soluble nuclear fraction. Our study has implications for understanding the regulation of RNA metabolism, as the stability of introns and the location of their degradation may dictate the following: (i) the stability of nearby mRNAs that compete with spliced introns for rate-limiting nucleases, (ii) the rate at which free nucleotides are available for further rounds of transcription, and (iii) the rate at which splicing factors are recycled.

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.

Companion canine cancer provides a clinically-relevant immunotherapy model for human malignancies because of their large size, intra-species genetic diversity, genetic similarity of tumors to human oncology, and spontaneously occurring tumors that develop despite an intact immune system. Canine B-lineage non-Hodgkin lymphoma (NHL) is genetically, molecularly, and physiologically similar to human NHL and the various treatment modalities can be interchanged with exception of antibody-based chemotherapies. Canine NHL represents 25% of all canine cancers diagnosed in the United States among the more than one million pet dogs diagnosed with cancer annually. As in humans, the common presentation is Stage III or Stage IV disease. It is rarely curable in the out-bred dog and even with aggressive chemotherapy the median survival rate is less than a year. Current standard-of-care (SOC) combination chemotherapy, CHOP, induces remission in approximately 85% of dogs. Thus, the companion canine provides a model for human T-cell therapy and addresses an unmet need of dogs diagnosed with NHL. Our initial canine trial (Sci Rep. 2012;2:249) demonstrated a survival advantage upon reconstitution of the immune system with autologous ex vivo-activated CD3+CD8+ T cells after CHOP for pet dogs with NHL. These T cells were propagated on engineered artificial antigen presenting cells (aAPC), which are also used for our human T-cell trials. Our updated survival data shows significant improvement in overall survival (p = 0.01) and tumor-free survival (p = 0.0008) compared with matched historical controls (Figure). We also report initial data on an additional trial infusing donor- or patient-derived canine T cells after allogeneic or autologous hematopoietic stem-cell transplantation (HSCT), respectively. The first allogeneic and autologous recipients with NHL safely received 2 escalating doses of activated T cells and achieved an anti-tumor response. As in our first trial, granzyme B expression in the propagated T cells continues to significantly correlate with prognosis. A third trial tests whether steroid-resistant T cells can persist in pet dogs with NHL receiving systemic dosing of corticosteroids. Fluorescent dyes were used to distinguish infused T cells and demonstrated that steroid-resistant activated T cells survived compared to infused control T cells. In all trials, single-cell studies are underway to determine genetic factors concordant with the survival of infused T cells and treated companion canines. Global peripheral blood T-cell gene signatures, as quantitated by nanostring, were measured at 3 hours, 7 to 14 days, and 35 to 42 days post infusion. The different gene patterns between infused dogs and post infusion time points may reflect changes in en vivo T-cell activation and companion canine prognosis. In conclusion, the add-back of activated CD8+ T cells in dogs with NHL can inform of the immunobiology of adoptive transfer of T cells in humans. Disclosures: No relevant conflicts of interest to declare.

Human endogenous retroviruses (HERVs) are ancient viruses forming 8% of human genome. One subset of HERVs, the HERV-K has recently been found to be expressed on tumor cells including melanoma, breast cancer and lymphoma but not on normal body cells. Thus, targeting HERV-K protein as a tumor associated antigen (TAA) may be a potential treatment strategy for tumors that are resistant to conventional therapies. One approach to improve therapeutic outcome is by infusing T cells rendered specific for such TAAs preferentially expressed on tumor cells. Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a chimeric antigen receptor (CAR) on T cells using gene therapy. This approach is currently being used in our clinical trials adoptively transferring CD19-specific CAR+ T cells into patients with B-lineage malignancies. Preliminary analysis of HERV-K env protein expression in 268 melanoma samples and 139 normal organ donor tissues using immunohistochemistry demonstrated antigen expression in tumor cells and absence of expression in normal organ tissues. The scFv region from a mouse monoclonal antibody to target HERV-K env was used to generate a CAR and cloned into Sleeping Beauty (SB) plasmid for stable expression in T cells. HERV-K-specific CAR+T cells were selectively propagated ex vivo on artificial antigen presenting cells (aAPC) using an approach already in our clinical trials. Indeed, after genetic modification of T cells and selection on HERV-K+ aAPC, over 95% of propagated T cells stably expressed the introduced HERV-K-specific CAR and exhibited redirected specificity for HERV-K+ melanoma (Figure 1). Further, the adoptive transfer of HERV-K-specific CAR+T cells killed metastatic melanoma in a mouse xenograph model. While we have chosen melanoma as our tumor model, this study has the potential to be applied to other malignancies, including lymphoma and myeloma due to restricted expression of HERV-K envelope (env) protein on these tumor cells. These data demonstrate that it is feasible to generate T cells expressing a HERV-K-specific CAR using a clinically-appealing approach as a treatment strategy for HERV-K env+ tumors. Disclosures: No relevant conflicts of interest to declare.

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10) T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

Abstract 4174 The first-in-human clinical trial using Sleeping Beauty (SB) transposition for T-cell immunotherapy is under way at the University of Texas MD Anderson Cancer Center. To understand potential genotoxic risks involved in our non-viral SB transposition-based immunotherapy, we employed high throughput sequencing (Illumina) and profiling (BlueBioU supercomputer) of the SB insertion sites in T cells. Our SB clinical vector inserted 99.999% of the time at expected TA dinucleotide sites, with 44% of the insertions localizing to intragenic loci and the remainder 56% intergenic. The vast majority (96.5%) of intragenic insertions are intronic while the majority (>60%) of intergenic transpositions fall within non-coding repeat regions. By linking microarray gene expression profile to the vector insertion profile, we find that the number of transcriptional start sites (TSS) hit is proportional to the expected ‘open' conformational loci for the starting population of quiescent T cells. Compared to immunotherapy infusing T cells genetically modified with retrovirus, the integration profiles for SB-modified T cells are favored as: 1) there are more insertions within intergenic regions (56% SB vs. 44% in retroviral), 2) insertions concentrated at TA sites which have a different genomic distribution profile than retroviral insertion sites, 3) TSS associated with quiescent T cells were favored reflecting the electro-transfer of SB DNA plasmids into non-proliferating T cells vs. retroviral protocols that require transduction of activated T cells. Scanning for potential danger loci (e.g. oncogene, tumor suppressor, miRNA etc.), SB integration profiles compare favorably with retroviral insertions. In addition, we identified potential “safe harbor” genetic loci for future targeted modifications. Opportunities in immunotherapy and other personalized medicine modalities will be fully realized when the actual and perceived risks are understood and perhaps mitigated. In the absence of targeted insertions by clinical vectors, the next best option is random integration with an evenly distributed insertion profile across the whole genome to minimize hot spot and clonal expansion of therapeutic T cells. We now seek to proactively profile the T-cell products prior to infusion and follow up sampling to help clinicians in data-driven decision making in a time-sensitive manner. Our methods and findings are consistent with the human application of SB system and the harnessing high throughput supercomputing resources to help clinicians mitigate potential risks. Disclosures: No relevant conflicts of interest to declare.

Meeting abstracts Clinical-grade T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) to redirect their specificity to target tumor-associated antigens in vivo. We have developed gene therapy approach to render T cells specific for invasive fungal infections (IFI)

Adoptive cell therapy using expanded autologous TIL is a promising immunotherapy for metastatic melanoma. However, we still know relatively little about the types of T cells in TIL mediating tumor regression, and there have been no biomarker studies on the actual tumors used to expand TIL to identify factors predictive of clinical response. The purpose of this study was to identify immune biomarkers in tumors used to expand TIL and determine their power to predict an eventual response to TIL therapy. We performed immunohistochemistry (IHC) for a number of parameters (e.g., CD8, CD4, CD3, FoxP3, and PD-1) on formalin-fixed paraffin-embedded (FFPE) tumor samples from 48 patients that expanded TIL and were eventually treated as well as FFPE samples from 39 patients from which TIL did not successfully expand. We found a correlation between the percentage of CD8+ cells in the original tumor by IHC and the CD8 content of the final expanded TIL product of these patients (p=0.046, r2=0.283, Spearman correlation coefficient). We also found highly significant differences in CD8, CD4 and CD3 staining in tumors between TIL growers and non-growers (p

Meeting abstracts The B cell receptor (BCR) expressed by a clonal B cell tumor is a tumor specific antigen (idiotype). However, the T cell epitopes within human BCRs which stimulate protective immunity still lack detailed characterization. In this study, we identified 17 potential CD4 T cell