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PDF file, 111K, Supplementary Table S1. Samples from 14 patients that received autologous TILs were studied. Supplementary Table S2.TCR Vα primers (the first 50 nt) and probes (the last 50 nt)used for DTEA analysis. Supplementary Table S3. TCR Vβ primers (the first 50 nt) and probes (the last 50 nt)used for DTEA analysis. Supplementary Table S4. House keeping genes used in DTEA analysis. Supplementary Table S5. DTEA can detect the low-frequency TCRVβ genes that cannot be readily identified by classical cloning.

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.

PDF file, 264K, Supplementary Fig. S1. TCR Va and Vb gene expression in total RNA from TILs patient 303 and patient 232. Supplementary Fig S2. Global measurement of TCR Va (A-C) and TCR Vb (D-F) gene expression in patient 231 including in vitro rapid expanded TIL (A and D) and PBMC (B-C and E-F) collected after TIL infusion. Supplementary Fig S3. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 228 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S4. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 106 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S5. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 172 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S6. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 152 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion. Supplementary Fig S7. Global measurement of TCR Va (A-D) and TCR Vb (E-H) gene expression in patient 188 including in vitro rapid expanded TIL (A and E) and PBMC (B-D) and (F-H) collected after TIL infusion.

Supplementary Figure 1 from Reprogramming CD19-Specific T Cells with IL-21 Signaling Can Improve Adoptive Immunotherapy of B-Lineage Malignancies

BACKGROUND/PURPOSE Accurate assessment of malignant T-cell clones in patients with leukemic cutaneous T-cell lymphoma (L-CTCL) is crucial for diagnosis, treatment, and monitoring disease. Although multiple approaches to quantitate malignant T-cell clones have been reported, a cost-effective assay with broad coverage is not available. We report a NanoString-nCounter-Technology-based direct TCR expression assay (DTEA) that was previously developed to quantify both TCR-Vα and TCR-Vβ usages after adoptive immunotherapy. This study was performed to test the effectiveness of DTEA in assessing malignant T-cell clones in L-CTCL patients. METHODS Total RNAs extracted from peripheral blood mononuclear cells of patients before starting extracorporeal photopheresis (ECP) (n = 15) and during therapy at 3 months and 6 months (n = 12) were used for DTEA, with customized probes for 45 TCR-Vα and 46 TCR-Vβ family members. RESULTS At baseline, DTEA detected TCR-Vβ clones in all 15 patients (100%) compared to flow cytometry that detected TCR-Vβ clones in 9 of 13 patients (69.2%). In addition to predominant TCR-Vβ clones, DTEA also detected additional TCR-Vβ clones in 8 of 15 patients (53.3%). Furthermore, DTEA simultaneously identified clonal TCR-Vα usages, which allowed us to pair TCR-Vα and TCRVβ usages by malignant T-cells and identify diversified clonotypes. Changes in the relative frequencies of clonal TCR-Vβ and TCRVα usages over therapy were consistent with patients' clinical responses. CONCLUSIONS Our results indicate that DTEA can effectively assess malignant T-cell clones by detecting clonal TCR-Vα and TCR-Vβ usages. By providing a global view of TCR repertoires, DTEA may also help us understand the origin(s) of malignant T-cells and pathogenesis of CTCL.

Although immunotherapy has achieved impressive durable clinical responses, many cancers respond only temporarily or not at all to immunotherapy. To find novel, targetable mechanisms of resistance to immunotherapy, patient-derived melanoma cell lines were transduced with 576 open reading frames, or exposed to arrayed libraries of 850 bioactive compounds, prior to co-culture with autologous tumor-infiltrating lymphocytes (TILs). The synergy between the targets and TILs to induce apoptosis, and the mechanisms of inhibiting resistance to TILs were interrogated. Gene expression analyses were performed on tumor samples from patients undergoing immunotherapy for metastatic melanoma. Finally, the effect of inhibiting the top targets on the efficacy of immunotherapy was investigated in multiple preclinical models. Aurora kinase was identified as a mediator of melanoma cell resistance to T-cell-mediated cytotoxicity in both complementary screens. Aurora kinase inhibitors were validated to synergize with T-cell-mediated cytotoxicity in vitro. The Aurora kinase inhibition-mediated sensitivity to T-cell cytotoxicity was shown to be partially driven by p21-mediated induction of cellular senescence. The expression levels of Aurora kinase and related proteins were inversely correlated with immune infiltration, response to immunotherapy and survival in melanoma patients. Aurora kinase inhibition showed variable responses in combination with immunotherapy in vivo, suggesting its activity is modified by other factors in the tumor microenvironment. These data suggest that Aurora kinase inhibition enhances T-cell cytotoxicity in vitro and can potentiate antitumor immunity in vivo in some but not all settings. Further studies are required to determine the mechanism of primary resistance to this therapeutic intervention. Electronic supplementary material The online version of this article (10.1007/s00262-020-02748-9) contains supplementary material, which is available to authorized users.

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αβ T-cell receptors (TCRαβ) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαβs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαβ genes as Sleeping Beauty transposons and the determination of the introduced TCRαβs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Purpose: The human endogenous retrovirus (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) expressed on melanoma but not normal cells. This study was designed to engineer a chimeric antigen receptor (CAR) on T-cell surface, such that they target tumors in advanced stages of melanoma. Experimental Design: Expression of HERV-K protein was analyzed in 220 melanoma samples (with various stages of disease) and 139 normal organ donor tissues using immunohistochemical (IHC) analysis. HERV-K env–specific CAR derived from mouse monoclonal antibody was introduced into T cells using the transposon-based Sleeping Beauty (SB) system. HERV-K env–specific CAR+ T cells were expanded ex vivo on activating and propagating cells (AaPC) and characterized for CAR expression and specificity. This includes evaluating the HERV-K–specific CAR+ T cells for their ability to kill A375-SM metastasized tumors in a mouse xenograft model. Results: We detected HERV-K env protein on melanoma but not in normal tissues. After electroporation of T cells and selection on HERV-K+ AaPC, more than 95% of genetically modified T cells expressed the CAR with an effector memory phenotype and lysed HERV-K env+ tumor targets in an antigen-specific manner. Even though there is apparent shedding of this TAA from tumor cells that can be recognized by HERV-K env–specific CAR+ T cells, we observed a significant antitumor effect. Conclusions: Adoptive cellular immunotherapy with HERV-K env–specific CAR+ T cells represents a clinically appealing treatment strategy for advanced-stage melanoma and provides an approach for targeting this TAA on other solid tumors. Clin Cancer Res; 21(14); 3241–51. ©2015 AACR.

Purpose: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. Experimental Design: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. Results: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts. Conclusions: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

Purpose: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates of up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as “feeders” to support a rapid-expansion protocol. Here, we optimized a platform to propagate TIL to a clinical scale using K562 cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand, and membrane-bound IL-15 to function as artificial antigen-presenting cells (aAPC) as an alternative to using PBMC feeders. Experimental Design: We used aAPC or g-irradiated PBMC feeders to propagate TIL and measured rates of expansion. The activation and differentiation state was evaluated by flow cytometry and differential gene expression analyses. Clonal diversity was assessed on the basis of the pattern of T-cell receptor usage. T-cell effector function was measured by evaluation of cytotoxic granule content and killing of target cells. Results: The aAPC propagated TIL at numbers equivalent to that found with PBMC feeders, whereas increasing the frequency of CD8 + T-cell expansion with a comparable effector-memory phenotype. mRNA profiling revealed an upregulation of genes in the Wnt and stem-cell pathways with the aAPC. The aAPC platform did not skew clonal diversity, and CD8 + T cells showed comparable antitumor function as those expanded with PBMC feeders. Conclusions: TIL can be rapidly expanded with aAPC to clinical scale generating T cells with similar phenotypic and effector profiles as with PBMC feeders. These data support the clinical application of aAPC to manufacture TIL for the treatment of melanoma.

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 now have developed this molecular strategy to render cytotoxic T cells specific for fungi. We adapted the pattern-recognition receptor Dectin-1 to activate T cells via chimeric CD28 and CD3-ζ (designated "D-CAR") upon binding with carbohydrate in the cell wall of Aspergillus germlings. T cells genetically modified with the Sleeping Beauty system to express D-CAR stably were propagated selectively on artificial activating and propagating cells using an approach similar to that approved by the Food and Drug Administration for manufacturing CD19-specific CAR(+) T cells for clinical trials. The D-CAR(+) T cells exhibited specificity for β-glucan which led to damage and inhibition of hyphal growth of Aspergillus in vitro and in vivo. Treatment of D-CAR(+) T cells with steroids did not compromise antifungal activity significantly. These data support the targeting of carbohydrate antigens by CAR(+) T cells and provide a clinically appealing strategy to enhance immunity for opportunistic fungal infections using T-cell gene therapy.

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity.

CAR-redirected T cells have demonstrated clinical effectiveness in early phase clinical trials, with persistence of adoptively transferred CD19-specific T cells correlated with positive outcomes. Notwithstanding the successes for some hematological malignancies, CAR-T targets a limited number of cell-surface antigens that curtails their appeal for solid tumors. This can be overcome by TCR gene transfer with specificity for intracellular tumor-associated antigens such as NY-ESO-1 expressed by hematologic malignancies and solid tumors. The predominant technologies for both CAR- and TCR-redirection of T cells utilize viral genetic modification as well as require a lengthy period of in vitro propagation with resultant deleterious differentiation to achieve clinically relevant cell numbers. A major impediment with current TCR-T is the high-cost and lengthy time associated with a viral-based manufacture of a library of TCRs that can address a multitude of desired targets and match HLA restriction to meet the need to infuse personalized TCR-T products with multiple specificities in each recipient. The Sleeping Beauty (SB) platform is the most clinically advanced non-viral gene transfer technology and overcomes the issues of scalability with viral based manufacture of TCR-T. We initially showed in pre-clinical models that co-expression of membrane-bound interleukin-15 (mbIL15) enhanced in vivo persistence of CAR-T (PMID: 27849617). This technology has been recently advanced to produce CD19-specific T cells in ≤ 2 days after electro-transfer of DNA plasmids using so-called "rapid personalized manufacture" (RPM). This was based on the SB system to stably co-express CAR and mbIL15 with a kill switch (HER1t). We have now adapted these technologies to address current limitations for T-cell therapy by using the RPM process to very rapidly generate TCR-modified T cells. The rationale for RPM of TCR-T is based on: (i) SB to genetically modify resting T cells thus eliminating the need to propagate cells prior to, or after, genetic modification, (ii) introduction of TCR to redirect T-cell specificity to tumor-associated antigens, (iii) mbIL15-HER1t to support T-cell persistence and enable selective elimination to increase safety, and (iv) manufacture within two days of gene transfer which limits T-cell differentiation and decreases time to manufacture. Mononuclear cells were electroporated with SB-derived DNA plasmids expressing (a) HLA A2-restricted NY-ESO-1-specific TCR or (b) the TCR and mbIL15-HER1t in separate plasmids. Following electroporation, cells were directly (unpropagated) injected into NSG (immunocompromised) mice bearing established HLA A2+ NY-ESO-1+ tumor. Administration of RPM TCR-mbIL15 T cells exhibited superior anti-tumor activity compared with RPM TCR T cells (Figure). Though engraftment of TCR+ T cells was not significantly different between the two groups, the RPM TCR-mbIL15 T cell-treated mice exhibited increased frequency of CD27+TCR+ T cells (p = 0.035, n = 6-7, Mann Whitney test), a phenotype that is correlated with improved therapeutic responses in human subjects. The RPM technology can thus be adapted to co-express TCR with mbIL15 (and HER1t), which can now be scaled to provide a cost-effective approach to manufacturing a multitude of TCR-T products from a library of TCRs with the necessary complexity to manage the range of specificities and HLA restrictions to treat multiple patients. Disclosures Hurton: • M.D. Anderson Cancer Center: Patents & Royalties; Intrexon: Patents & Royalties: US 9,629,877 B2 ; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties: US 9,629,877 B2 . Zhang:Intrexon: Patents & Royalties: US 9,629,877 B2; Ziopharm Oncology: Patents & Royalties: US 9,629,877 B2. Deniger:Ziopharm Oncology: Employment, Equity Ownership. Olivares:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1; Intrexon: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1. Cooper:CytoSen: Equity Ownership; Targazyme: Equity Ownership; MD Anderson Cancer Center: Patents & Royalties; Sangamo BioSciences: Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; City of Hope: Patents & Royalties; Ziopharm Oncology: Employment, Equity Ownership, Other: Contracted research, Patents & Royalties; Secure Transfusion Services: Equity Ownership; CellChorus: Equity Ownership. Singh:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2; Intrexon: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2.

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in10(9) CAR(+)γδ T cells from10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19 artificial antigen presenting cells was used to numerically expand CD3 T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3 T cells expressed CAR. CAR T cells specifically killed CD19 target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR Vα and TCR Vβ repertoire. Quantitative fluorescence in situ hybridization revealed that CAR T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19 B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).

Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

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 BCR peptide-specific CD4+ T-cell epitopes derived from BCR heavy and light chain variable region sequences. Detailed analysis revealed these CD4+ T-cell epitopes stimulated normal donors' and patients' Th1 CD4+ T cells to directly recognize the autologous tumors by secretion of IFNγ, indicating the epitopes are processed and presented by tumor cells. One BCR peptide-specific CD4+ T cell line was also cytotoxic and lysed autologous tumor cells through the perforin pathway. Sequence analysis of the epitopes revealed that 10 were shared by multiple primary patients' tumors, and 16 had the capacity to bind to more than one HLA DRB1 allele. T cells stimulated by shared epitopes recognized primary tumors expressing the same sequences on multiple HLA DRB1 alleles. In conclusion, we identified 17 BCR-derived CD4+ T-cell epitopes with promiscuous HLA DRB1 binding affinity that are shared by up to 36% of patients, suggesting a strategy to overcome the requirement for individual preparation of therapeutic agents targeting idiotype.

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

A deeper mechanistic understanding of tumour angiogenesis regulation is needed to improve current anti-angiogenic therapies. Here we present evidence from systems-based miRNA analyses of large-scale patient data sets along with in vitro and in vivo experiments that miR-192 is a key regulator of angiogenesis. The potent anti-angiogenic effect of miR-192 stems from its ability to globally downregulate angiogenic pathways in cancer cells through regulation of EGR1 and HOXB9. Low miR-192 expression in human tumours is predictive of poor clinical outcome in several cancer types. Using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes, we show that miR-192 delivery leads to inhibition of tumour angiogenesis in multiple ovarian and renal tumour models, resulting in tumour regression and growth inhibition. This anti-angiogenic and anti-tumour effect is more robust than that observed with an anti-VEGF antibody. Collectively, these data identify miR-192 as a central node in tumour angiogenesis and support the use of miR-192 in an anti-angiogenesis therapy., The formation of blood vessels in tumours, angiogenesis, is a promising target for therapy. Here, the authors show that microRNA192 has anti-angiogenic functions and negatively regulates EGR1 and HOXB9, and that delivery of this microRNA to tumours in vivo can reduce angiogenesis and tumour growth.

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

Cancer-associated fibroblasts (CAFs) are a major cellular component of tumor microenvironment in most solid cancers. Altered cellular metabolism is a hallmark of cancer, and much of the published literature has focused on neoplastic cell-autonomous processes for these adaptations. We demonstrate that exosomes secreted by patient-derived CAFs can strikingly reprogram the metabolic machinery following their uptake by cancer cells. We find that CAF-derived exosomes (CDEs) inhibit mitochondrial oxidative phosphorylation, thereby increasing glycolysis and glutamine-dependent reductive carboxylation in cancer cells. Through 13C-labeled isotope labeling experiments we elucidate that exosomes supply amino acids to nutrient-deprived cancer cells in a mechanism similar to macropinocytosis, albeit without the previously described dependence on oncogenic-Kras signaling. Using intra-exosomal metabolomics, we provide compelling evidence that CDEs contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates that are avidly utilized by cancer cells for central carbon metabolism and promoting tumor growth under nutrient deprivation or nutrient stressed conditions. DOI: http://dx.doi.org/10.7554/eLife.10250.001, eLife digest Cancer cells behave differently from healthy cells in many ways. Healthy cells rely on structures called mitochondria to provide them with energy via a process that requires oxygen. However cancer cells don’t rely on this process, and instead release energy by breaking down sugars outside of the mitochondria. This may explain why cancer cells are able to thrive even when little oxygen is available. Cancer cells also interact with neighboring cells called fibroblasts, which are a major part of a tumor’s microenvironment, and recruit them into the tumors. The fibroblasts communicate with cancer cells, in part, by releasing chemical messengers packaged into tiny bubble-like structures called exosomes. Recent studies have suggested that these exosomes may help cancer cells to thrive, but there are many questions remaining about how they might do this. Now, Zhao et al. show that the fibroblasts smuggle essential nutrients to cancer cells via the exosomes and disable oxygen-based energy production in cancer cells. First, exosomes released by cancer-associated fibroblasts from people with prostate cancer were collected and marked with a green dye. Next, the green-labeled exosomes were mixed with prostate cancer cells, and shown to be absorbed by the cells. Oxygen-based energy release was dramatically reduced in the exosome-absorbing cells, and sugar-based energy release increased. Next, Zhao et al examined the contents of the exosomes, and found that they contain the building blocks of proteins, fats, and other important molecules. Next, the experiments revealed that both prostate cancer and pancreatic cancer cells deprived of nutrients can use these smuggled resources to continue to grow. Importantly, this process did not involve the protein Kras, which previous studies had show helps cancer cells absorb nutrients. These findings suggest that preventing exosomes from smuggling resources to starving cancer cells might be an effective strategy to treat cancers. DOI: http://dx.doi.org/10.7554/eLife.10250.002

Natural killer (NK) cells are a subset of cytotoxic lymphocytes that play a critical role in innate immune surveillance against infections and tumors through cytokine secretion and target cell lysis. NK cells function without any need for prior antigen exposure. Thus, more recently NK cells are considered a promising source of lymphocytes for adoptive tumor therapy. However, because NK cells represent only a small lymphocyte fraction, expand poorly ex vivo, and have limited life spans, clinical scale generation of NK cells for tumor immunotherapy was a challenging issue. To overcome this challenge, numerous expansion platforms have been developed. However, ex vivo expansion of NK cells could lead to proliferation-induced senescence. Telomeres at the end of chromosomes play a crucial role in maintaining the integrity of the chromosome and are lost at each cell division in somatic cells and have emerged as important cellular elements in aging and cancer. Because telomere length is known to decrease in adult human NK cells and is associated with proliferation-induced senescence, it is important to determine the effect of NK cell expansion systems on telomere length. In this chapter, a detailed protocol is provided to analyze the telomere length of expanded NK cells using a commercially available Flow FISH kit.

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.

Improving the therapeutic efficacy of T cells expressing a chimeric antigen receptor (CAR) represents an important goal in efforts to control B-cell malignancies. Recently an intrinsic strategy has been developed to modify the CAR itself to improve T-cell signaling. Here we report a second extrinsic approach based on altering the culture milieu to numerically expand CAR+ T cells with a desired phenotype, for the addition of interleukin (IL)-21 to tissue culture improves CAR-dependent T-cell effector functions. We used electrotransfer of Sleeping Beauty system to introduce a CAR transposon and selectively propagate CAR+ T cells on CD19+ artificial antigen-presenting cells (aAPC). When IL-21 was present, there was preferential numeric expansion of CD19-specific T cells which lysed and produced IFN-γ in response to CD19. Populations of these numerically expanded CAR+ T cells displayed an early memory surface phenotype characterized as CD62L+CD28+ and a transcriptional profile of naïve T cells. In contrast, T cells propagated with only exogenous IL-2 tended to result in an overgrowth of CD19-specific CD4+ T cells. Furthermore, adoptive transfer of CAR+ T cells cultured with IL-21 exhibited improved control of CD19+ B-cell malignancy in mice. To provide coordinated signaling to propagate CAR+ T cells, we developed a novel mutein of IL-21 bound to the cell surface of aAPC that replaced the need for soluble IL-21. Our findings show that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR+ T cells for effective immunotherapy. Cancer Res; 71(10); 3516–27. ©2011 AACR.

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Chimeric antigen receptor (CAR)-modified T-cell therapy targeting CD19 induces high response rates in patients with relapsed or refractory B-cell lymphomas. However, about 60% of patients experience primary or secondary resistance after CD19-targeted CAR T-cell therapy and a major of cause of failure appears to be due to loss of CD19 expression on the tumor. Therefore, novel targets for adoptive T-cell therapeutic approaches are needed to further improve clinical outcome in these patients. T-cell leukemia/lymphoma antigen1 (TCL1) is an oncoprotein that is overexpressed in multiple B-cell malignancies including follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). Importantly, it has restricted expression in only a subset of B cells among normal tissues. We previously identified a TCL1-derived HLA-A2-binding epitope (TCL170-79 SLLPIMWQLY) that can be used to generate TCL1-specific CD8+ T cells from peripheral blood mononuclear cells of both HLA-A2+ normal donors and lymphoma patients. More importantly, we showed that the TCL1-specific CD8+ T cells lysed autologous primary lymphoma cells but not normal B cells (Weng et al. Blood 2012). To translate the above discovery into clinic, we cloned the T-cell receptor (TCR) alpha and beta chains from a TCL1-specific CD8+ T-cell clone and showed that this TCL1-TCR could be transduced into polyclonal donor T cells using a lentiviral system with a transduction efficiency of >40% as determined by TCL170-79 tetramer positive T cells. Furthermore, we demonstrated that the TCL1-TCR-transduced T cells recognized T2 cells pulsed with TCL170-79 peptide producing IFN- γ >8 ng/ml and IL-2 >350 ng/ml but were not reactive to control HIV-Gag peptide (IFN- γ 10 ng/ml) suggesting it has moderate to high avidity. Importantly, TCL1-TCR-transduced T cells lysed HLA-A2+ (up to 43% lysis of Mino and 25% lysis of Jeko-1 at 40:1 Effector:Target ratio) but not HLA-A2- lymphoma cell lines (5.5% lysis of HLA A2- Raji and 2.3% lysis of Daudi at 40:1 Effector:Target ratio). TCL1-TCR-transduced T cells were also cytotoxic to HLA-A2+ primary lymphoma tumor cells (up to 48% lysis of CLL, 43% lysis of FL, 41% lysis of DLBCL, 46% lysis of splenic marginal zone lymphoma, and 11% lysis of MCL at 40:1 Effector:Target ratio) but not normal B cells derived from the same patients. Lastly, TCL1-TCR transduced T cells showed high efficacy in in vivo models. Adoptive transfer of the TCL1-TCR-tranduced T cells significantly reduced lymphoma tumor growth and extended survival in Mino mantle cell lymphoma cell line xenograft model (48% survival in TCL1-TCR-T treated group vs. 12.5% survival in control group at 10 weeks n=7-8 mice/group; P=0.02). Collectively, our data suggest that the high expression in B-cell tumors, restricted expression in normal tissues, and presence of an immunogenic CD8 T-cell epitope, make TCL1 a target for T cell-based therapeutic approaches in multiple B-cell malignancies. Our results also demonstrate that the TCL1-specific TCR-transduced T cells may serve as a novel adoptive immunotherapy approach for the treatment of patients with various B-cell malignancies (including FL, MCL, DLBCL, CLL). Acknowledgments: This study is supported by MD Anderson Moon Shot Program and CPRIT and the National Natural Science Foundation of China Grant (No. 81570189) Disclosures Neelapu: Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees.

T-cell leukemia/lymphoma Antigen1 (TCL1) is a B-cell oncoprotein that is overexpressed in multiple forms of B-cell lymphoma. In our previous study, we identified a HLA*A0201-restricted T cell epitope from TCL1 that stimulated CD8+ T cells to specifically lyse lymphoma cells. In this study, we have cloned the TCL170-79 peptide-specific T-cell receptor (TCR) gene that after lentivirus transduction into donor T cells can redirect the specificity of T cells to lyse T2 cells pulsed with TCL170-79 but not control peptide. The TCR-transduced CD8+ T cells efficiently lysed HLA-0201+, TCL1-expressing human lymphoma cell lines and primary lymphoma cells, but not normal B cells. Adoptive transfer of TCR-transduced T cells into a mantle cell lymphoma xenograft mouse model inhibited the tumor growth and significantly extended the survival of mice. In addition, we found that TCL1 is expressed in multiple solid tumors. The TCR-transduced T cells lysed colon and liver tumor cells but not normal human cells in vitro as well as in a mouse xenograft model. Collectively, our data demonstrate that TCL1-specific TCR-based immunotherapy is highly effective against human B-cell lymphomas and non-hematological tumors. Citation Format: Jinsheng Weng, Kelsey E. Moriarty, Yong Pan, Flavio Egidio Baio, Hua Wang, Hiroki Torikai, Sourindra N. Maiti, Fuliang Chu, Xiaoyun Cheng, Swathi Karri, Deepshika Medapalli, Laurence J. N. Cooper, Sattva S. Neelapu. A TCL1-specific T-cell receptor redirects T cells against B-cell lymphomas and non-hematological tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2566.

Nonviral integrating vectors can be used for expression of therapeutic genes. piggyBac (PB), a transposon/transposase system, has been used to efficiently generate induced pluripotent stems cells from somatic cells, without genetic alteration. In this paper, we apply PB transposition to express a chimeric antigen receptor (CAR) in primary human T cells. We demonstrate that T cells electroporated to introduce the PB transposon and transposase stably express CD19-specific CAR and when cultured on CD19(+) artificial antigen-presenting cells, numerically expand in a CAR-dependent manner, display a phenotype associated with both memory and effector T cell populations, and exhibit CD19-dependent killing of tumor targets. Integration of the PB transposon expressing CAR was not associated with genotoxicity, based on chromosome analysis. PB transposition for generating human T cells with redirected specificity to a desired target such as CD19 is a new genetic approach with therapeutic implications.

The recognition and removal of apoptotic cells is critical to development, tissue homeostasis, and the resolution of inflammation. Many studies have shown that phagocytosis is regulated by signaling mechanisms that involve distinct ligand-receptor interactions that drive the engulfment of apoptotic cells. Studies from our laboratory have shown that the plasma protein β-2-glycoprotein 1 (β2GP1), a member of the short consensus repeat superfamily, binds phosphatidylserine-containing vesicles and apoptotic cells and promotes their bridging and subsequent engulfment by phagocytes. The phagocyte receptor for the protein/apoptotic cell complex, however, is unknown. Here we report that a member of the low density lipoprotein receptor-related protein family on phagocytes binds and facilitates engulfment of β2GP1-phosphatidylserine and β2GP1-apoptotic cell complexes. Using recombinant β2GP1, we also show that β2GP1-dependent uptake is mediated by bridging of the target cell to the phagocyte through the protein C- and N-terminal domains, respectively.

beta-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved beta-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance.

Mismatch of human leukocyte antigens (HLA) adversely impacts the outcome of patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). This translates into the clinical requirement to timely identify suitable HLA-matched donors which in turn curtails the chances of recipients, especially those from a racial minority, to successfully undergo alloHSCT. We thus sought to broaden the existing pool of registered unrelated donors based on analysis that eliminating the expression of the HLA-A increases the chance for finding a donor matched at HLA-B, -C and -DRB1 regardless of a patient’s race. Elimination of HLA-A expression in HSC was achieved using artificial zinc finger nucleases designed to target HLA-A alleles. Significantly, these engineered HSCs maintain their ability to engraft and reconstitute hematopoiesis in immunocompromised mice. This introduced loss of HLA-A expression decreases the need to recruit large number of donors to match with potential recipients and has particular importance for patients whose HLA repertoire is under-represented in the current donor pool. Furthermore, the genetic engineering of stem cells provides a translational approach to HLA-match a limited number of third-party donors with a wide number of recipients.

Adoptive transfer of autologous ex vivo expanded tumor-infiltrating lymphocytes (TIL) is a highly successful cell therapy approach in the treatment of late-stage melanoma. Notwithstanding the success of this therapy, only very few centers worldwide can provide it. To make this therapy broadly available, one of the major obstacles to overcome is the complexity of culturing the TIL. Recently, major efforts have been deployed to resolve this issue. The use of the Gas-permeable flask (G-Rex) during the REP has been one application that has facilitated this process. Here we show that the use of this new device is able to rescue poor TIL growth and maintain clonal diversity while supporting an improved mitochondrial function.

T cells modified with chimeric antigen receptors (CARs) targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1) is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28) or CD137 (designated ROR1RCD137) and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL)-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString) and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

Renal failure is a frequent and costly complication of many chronic diseases, including diabetes and hypertension. One common feature of renal failure is glomerulosclerosis, the pathobiology of which is unclear. To help elucidate this, we generated a mouse strain carrying the missense mutation Wt1 R394W, which predisposes humans to glomerulosclerosis and early-onset renal failure (Denys-Drash syndrome [DDS]). Kidney development was normal in Wt1(+/R394W) heterozygotes. However, by 4 months of age 100% of male heterozygotes displayed proteinuria and glomerulosclerosis characteristic of DDS patients. This phenotype was observed in an MF1 background but not in a mixed B6/129 background, suggestive of the action of a strain-specific modifying gene(s). WT1 encodes a nuclear transcription factor, and the R394W mutation is known to impair this function. Therefore, to investigate the mechanism of Wt1 R394W-induced renal failure, the expression of genes whose deletion leads to glomerulosclerosis (NPHS1, NPHS2, and CD2AP) was quantitated. In mutant kidneys, NPHS1 and NPHS2 were only moderately downregulated (25 to 30%) at birth but not at 2 or 4 months. Expression of CD2AP was not changed at birth but was significantly upregulated at 2 and 4 months. Podocalyxin was downregulated by 20% in newborn kidneys but not in kidneys at later ages. Two other genes implicated in glomerulosclerosis, TGFB1 and IGF1, were upregulated at 2 months and at 2 and 4 months, respectively. It is not clear whether the significant alterations in gene expression are a cause or a consequence of the disease process. However, the data do suggest that Wt1 R394W-induced glomerulosclerosis may be independent of downregulation of the genes for NPHS1, NPHS2, CD2AP, and podocalyxin and may involve other genes yet to be implicated in renal failure. The Wt1(R394W) mouse recapitulates the pathology and disease progression observed in patients carrying the same mutation, and the mutation is completely penetrant in male animals. Thus, it will be a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.

Non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system has been successfully tested in humans to express a chimeric antigen receptor (CAR) to redirect T-cell specificity to CD19. This system has been modified to (i) improve the design of the CD19-specific CAR and (ii) reduce the time in culture to 14 days. Our previous clinical trials infused T cells expressing a 2nd generation CAR (designated CD19RCD28) with an IgG4-Fc stalk that activated via chimeric CD28 and CD3ζ. To evaluate the length of extracellular domain on function, we tested four CD19-specific CARs with two long [IgG4-Fc (CD19RCD28) and EQ (L235E and N297Q) mutant IgG4-Fc (CD19R*CD28)], medium (CD8α hinge, CD19RCD8CD28) and short (12aa IgG1 hinge, CD19R12aaCD28) stalks which all signaled through chimeric CD28 and CD3ζ endodomains. Generation of our T cells is based on electro-transfer of CARs coded by the SB system and antigen-specific stimulation through activating and K562-derived propagating cells (AaPC) in the presence of exogenous cytokines. After electro-transfer of SB-derived DNA plasmids, T cells were selectively propagated with either a new two-weekly (2x) or standard four-weekly (4x) additions of AaPC. All genetically modified T cells were capable of specific lysis of CD19+ tumor targets and producing IFN-γ in response to CD19+ stimulator cells. Serial killing was tested using massively parallel microscopy to observe single T cells and we observed that CDl9RCD8CD28+ T cells exhibited superior ability to partake in multiple killing events. CAR+ T cells were further tested in vivo for their ability to control CD19+ leukemia in a mouse model of minimal residual disease as well as established disease (Figure A and BFigure A and B). We found that T cells expressing modified CARs (CD19R*CD28, CD19RCD8CD28, CD19R12aaCD28) with reduced ability to bind to Fc gamma receptors (FcγR) were able to control leukemia more efficiently in mice compared to T cells expressing CD19RCD28. The CD19RCD8CD28 CAR was superior in controlling disease in the model of minimal residual disease compared with the CAR design evaluated in our prior clinical trials. T cells expressing CD19R*CD28 and CD19RCD8CD28 were then evaluated in 2x stimulation cycle. Both the 4x CAR+ T cells had similar CAR expression (>70%) whereas the 2x CAR+ T cells exhibited reduced CAR expression (~40%). The 2x CAR+ T cells expressed markers associated with less differentiated state of naive-like and memory T cells when compared to 4x CAR+ T cells, which was supported by measurement of mRNA species using bar-coded probes. The efficacy of the CAR+ T cells was tested in mice bearing established CD19+ leukemia and we observed superior survival in mice receiving the 2x CAR+ T cells compared with the 4x CAR+ T cells (Figure CFigure C). These data depict that length of extracellular domain and its associated binding to FcγR improves T-cell effector functions and that decreasing the time in culture can improve control of leukemia in vivo. These data support the use of cDl9RCD8CD28 testing in a next-generation clinical trial (IND# 16474).View Large Image | Download PowerPoint Slide

Myelodysplastic syndromes are a heterogeneous group of clonal bone marrow hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Chromosomal abnormalities and gene mutations have been shown to have essential roles in pathogenesis and correlate with prognosis. Molecular markers, however, are not integrated into currently used prognostic systems. The goal of this study is to identify plasma microRNAs useful for classification and risk stratification of myelodysplastic syndromes. We applied a novel, high-throughput digital quantification technology (NanoString) to profile microRNA expression in plasma samples of 72 patients with myelodysplastic syndromes and 12 healthy individuals. We correlated these results with overall survival. In patients with myelodysplastic syndromes associated with a diploid karyotype, we identified and validated a 7-microRNA signature as an independent predictor of survival with a predictive power of 75% accuracy (P=0.008), better than those of the International Prognostic Scoring Systems and the MD Anderson Prognostic Lower Risk Prognostic Model. We also identified differentially expressed plasma microRNAs in patients with myelodysplastic syndromes versus healthy individuals and between patients with myelodysplastic syndromes associated with different cytogenetic features. These results validate the utility of circulating-microRNA levels as noninvasive biomarkers that can inform the management of patients with myelodysplastic syndromes. Our findings also shed light on interactions of gene regulation pathways that are likely involved in the pathogenesis of myelodysplastic syndromes.

T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) for in vivo clinical applications. CAR-modified T cells have demonstrated redirected specificity and, in several clinical trials, potent anti-tumor activity. Manufacture, to date, is based upon gene transfer in cycling T cells followed by a period of tissue culture to achieve stable expression of introduced CARs. In contrast, we have adapted the non-viral-based Sleeping Beauty (SB) system to avoid the need for (i) T-cell activation and (ii) extended ex vivo tissue culture; thereby developing an approach whereby T cells can be both manufactured and delivered at multiple points-of-care (POC). This shortened culture decreases the time frame for manufacturing CAR+ T cells compared with current protocols for viral- or non-viral-based methodologies and is a foundation of our POC technology. Furthermore, reducing the ex vivo culture time preserves the memory and sustained persistence of CAR+ T cells by avoiding the differentiation programming induced by activation events typically required before or after gene transfer. We have previously demonstrated that co-expressing a membrane-bound version of interleukin-15 (mbIL15) significantly enhances the in vivo persistence of CAR+ T cells that are generated following 28-day culture after electro-transfer of SB derived DNA plasmids. Herein, we incorporated mbIL15 to generate POC CD19-specific CAR+ T cells. Peripheral blood mononuclear cells were genetically modified with mbIL15 and 2nd generation CAR coded from individual SB DNA plasmids and placed in culture for less than 2 days prior to adoptive transfer. NSG mice burdened by established and disseminated CD19+ leukemia were intravenously injected with just 7.5 x 105 CAR+ T cells, or an equivalent total T-cell dose of CARneg (unmodified or mock-treated) T cells. The mbIL15-CAR T-cell infusion yielded excellent disease-free survival, anti-tumor activity (Figure), and T-cell persistence. This approach to expediting the generation of genetically modified T cells enables the administration of CAR-modified naïve T cells and demonstrates that POC T cells have potent anti-tumor effects, even at a reduced CAR+ T-cell dose. This improvement to non-viral gene transfer and T-cell production reduces the requirement for tissue culture and thus time to manufacture within a GMP facility which translates to improvements in scalability and reduced costs. In summary, these data provide a translational pathway to undertake clinical trials by rapidly infusing T cells after genetic modification using the SB system. Disclosures Hurton: Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Singh:Immatics: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Switzer:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Mi:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Maiti:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Su:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Huls:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Employment, Equity Ownership, Patents & Royalties. Champlin:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Cooper:Immatics: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.: Equity Ownership; Sangamo BioSciences: Patents & Royalties; Intrexon: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria.

Characterizing the genetic alterations leading to the more aggressive forms of oestrogen receptor-positive (ER+) breast cancers is of critical significance in breast cancer management. Here we identify recurrent rearrangements between the oestrogen receptor gene ESR1 and its neighbour CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal B tumours, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170-positive tumours. These fusions encode amino-terminally truncated CCDC170 proteins (ΔCCDC170). When introduced into ER+ breast cancer cells, ΔCCDC170 leads to markedly increased cell motility and anchorage-independent growth, reduced endocrine sensitivity and enhanced xenograft tumour formation. Mechanistic studies suggest that ΔCCDC170 engages Gab1 signalosome to potentiate growth factor signalling and enhance cell motility. Together, this study identifies neoplastic ESR1-CCDC170 fusions in a more aggressive subset of ER+ breast cancer, which suggests a new concept of ER pathobiology in breast cancer.

Splenic NK1.1+CD4+ T cells that express intermediate levels of TCRαβ molecules (TCRint) and the DX5 Ag (believed to identify an equivalent population in NK1.1 allelic negative mice) possess the ability to rapidly produce high quantities of immunomodulatory cytokines, notably IL-4 and IFN-γ, upon primary TCR activation in vivo. Indeed, only T cells expressing the NK1.1 Ag appear to be capable of this function. In this study, we demonstrate that splenic NK1.1-negative TCRintCD4+ T cells, identified on the basis of FcγR expression, exist in naive NK1.1 allelic positive (C57BL/6) and negative (C3H/HeN) mice with the capacity to produce large amounts of IL-4 and IFN-γ after only 8 h of primary CD3 stimulation in vitro. Furthermore, a comparison of the amounts of early cytokines produced by FcγR+CD4+TCRint T cells with NK1.1+CD4+ or DX5+CD4+TCRint T cells, simultaneously isolated from C57BL/6 or C3H/HeN mice, revealed strain and population differences. Thus, FcγR defines another subpopulation of splenic CD4+TCRint cells that can rapidly produce large concentrations of immunomodulatory cytokines, suggesting that CD4+TCRint T cells themselves may represent a unique family of immunoregulatory CD4+ T cells whose members include FcγR+CD4+ and NK1.1/DX5+CD4+ T cells.