Publications by authors named "Rimas J Orentas"

53 Publications

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

Cell Rep 2021 Nov;37(8):110047

University of Toronto Musculoskeletal Oncology Unit, Sinai Health System; Department of Surgery, University of Toronto, Toronto, ON, Canada.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.
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http://dx.doi.org/10.1016/j.celrep.2021.110047DOI Listing
November 2021

Towards access for all: 1st Working Group Report for the Global Gene Therapy Initiative (GGTI).

Gene Ther 2021 Sep 8. Epub 2021 Sep 8.

Caring Cross, Gaithersburg, MD, USA.

The gene and cell therapy field saw its first approved treatments in Europe in 2012 and the United States in 2017 and is projected to be at least a $10B USD industry by 2025. Despite this success, a massive gap exists between the companies, clinics, and researchers developing these therapeutic approaches, and their availability to the patients who need them. The unacceptable reality is a geographic exclusion of low-and middle-income countries (LMIC) in gene therapy development and ultimately the provision of gene therapies to patients in LMIC. This is particularly relevant for gene therapies to treat human immunodeficiency virus infection and hemoglobinopathies, global health crises impacting tens of millions of people primarily located in LMIC. Bridging this divide will require research, clinical and regulatory infrastructural development, capacity-building, training, an approval pathway and community adoption for success and sustainable affordability. In 2020, the Global Gene Therapy Initiative was formed to tackle the barriers to LMIC inclusion in gene therapy development. This working group includes diverse stakeholders from all sectors and has set a goal of introducing two gene therapy Phase I clinical trials in two LMIC, Uganda and India, by 2024. Here we report on progress to date for this initiative.
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http://dx.doi.org/10.1038/s41434-021-00284-4DOI Listing
September 2021

Combining Immunocytokine and Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2 Murine Neuroblastoma.

Front Immunol 2021 19;12:668307. Epub 2021 Aug 19.

Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, United States.

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2 NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L . Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α , but induced cytokine release syndrome (CRS) . Infusing Perforin CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.
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http://dx.doi.org/10.3389/fimmu.2021.668307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417312PMC
October 2021

Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis.

Nat Med 2021 09 12;27(9):1544-1552. Epub 2021 Jul 12.

Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Washington, Seattle, WA, USA.

Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 ( NCT03500991 ), an ongoing phase 1 clinical trial at Seattle Children's evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients.
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http://dx.doi.org/10.1038/s41591-021-01404-8DOI Listing
September 2021

Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma.

Cancer Discov 2021 Nov 21;11(11):2884-2903. Epub 2021 May 21.

Canada's Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada.

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system X transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy. SIGNIFICANCE: Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy...
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http://dx.doi.org/10.1158/2159-8290.CD-20-1690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8563374PMC
November 2021

Self-driving armored CAR-T cells overcome a suppressive milieu and eradicate CD19 Raji lymphoma in preclinical models.

Mol Ther 2021 09 8;29(9):2691-2706. Epub 2021 May 8.

Translational Research and Development, Lentigen, a Miltenyi Biotec Company, 1201 Clopper Road, Gaithersburg, MD 20878, USA. Electronic address:

Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-PrkdcIl2rg/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor β receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-β on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion.
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http://dx.doi.org/10.1016/j.ymthe.2021.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8417504PMC
September 2021

Trispecific CD19-CD20-CD22-targeting duoCAR-T cells eliminate antigen-heterogeneous B cell tumors in preclinical models.

Sci Transl Med 2021 03;13(586)

Lentigen, a Miltenyi Biotec Company, Gaithersburg, MD 20878, USA.

A substantial number of patients with leukemia and lymphoma treated with anti-CD19 or anti-CD22 monoCAR-T cell therapy relapse because of antigen loss or down-regulation. We hypothesized that B cell tumor antigen escape may be overcome by a chimeric antigen receptor (CAR) design that simultaneously targets three B cell leukemia antigens. We engineered trispecific duoCAR-T cells with lentiviral vectors encoding two CAR open reading frames that target CD19, CD20, and CD22. The duoCARs were composed of a CAR with a tandem CD19- and CD20-targeting binder, linked by the P2A self-cleaving peptide to a second CAR targeting CD22. Multiple combinations of intracellular T cell signaling motifs were evaluated. The most potent duoCAR architectures included those with ICOS, OX40, or CD27 signaling domains rather than those from CD28 or 4-1BB. We identified four optimal binder and signaling combinations that potently rejected xenografted leukemia and lymphoma tumors in vivo. Moreover, in mice bearing a mixture of B cell lymphoma lines composed of parental triple-positive cells, CD19-negative, CD20-negative, and CD22-negative variants, only the trispecific duoCAR-T cells rapidly and efficiently rejected the tumors. Each of the monoCAR-T cells failed to prevent tumor progression. Analysis of intracellular signaling profiles demonstrates that the distinct signaling of the intracellular domains used may contribute to these differential effects. Multispecific duoCAR-T cells are a promising strategy to prevent antigen loss-mediated relapse or the down-regulation of target antigen in patients with B cell malignancies.
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http://dx.doi.org/10.1126/scitranslmed.abc6401DOI Listing
March 2021

Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Sci Rep 2021 01 11;11(1):389. Epub 2021 Jan 11.

Lentigen, a Miltenyi Biotec Company, Gaithersburg, MD, USA.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.
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http://dx.doi.org/10.1038/s41598-020-79698-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801692PMC
January 2021

Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice.

Gastroenterology 2020 06 12;158(8):2250-2265.e20. Epub 2020 Feb 12.

Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. Electronic address:

Background And Aims: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties.

Methods: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgc (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgc (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice.

Results: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active β-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice.

Conclusions: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.
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http://dx.doi.org/10.1053/j.gastro.2020.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282931PMC
June 2020

EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma.

Oncotarget 2019 Dec 17;10(66):7080-7095. Epub 2019 Dec 17.

Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, WA, U.S.A.

Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.
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http://dx.doi.org/10.18632/oncotarget.27389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925027PMC
December 2019

CD19 CAR T cell product and disease attributes predict leukemia remission durability.

J Clin Invest 2019 05 12;129(5):2123-2132. Epub 2019 Mar 12.

Background: Chimeric antigen receptor (CAR) T cells can induce remission in highly refractory leukemia and lymphoma subjects, yet the parameters for achieving sustained relapse-free survival are not fully delineated.

Methods: We analyzed 43 pediatric and young adult subjects participating in a Phase I trial of defined composition CD19CAR T cells (NCT02028455). CAR T cell phenotype, function and expansion, as well as starting material T cell repertoire, were analyzed in relation to therapeutic outcome (defined as achieving complete remission within 63 days) and duration of leukemia free survival and B cell aplasia.

Results: These analyses reveal that initial therapeutic failures (n = 5) were associated with attenuated CAR T cell expansion and/or rapid attrition of functional CAR effector cells following adoptive transfer. The CAR T products were similar in phenotype and function when compared to products resulting in sustained remissions. However, the initial apheresed peripheral blood T cells could be distinguished by an increased frequency of LAG-3+/TNF-αlow CD8 T cells and, following adoptive transfer, the rapid expression of exhaustion markers. For the 38 subjects who achieved an initial sustained MRD-neg remission, remission durability correlated with therapeutic products having increased frequencies of TNF-α-secreting CAR CD8+ T cells, and was dependent on a sufficiently high CD19+ antigen load at time of infusion to trigger CAR T cell proliferation.

Conclusion: These parameters have the potential to prospectively identify patients at risk for therapeutic failure and support the development of approaches to boost CAR T cell activation and proliferation in patients with low levels of CD19 antigen.

Trial Registration: ClinicalTrials.gov NCT02028455.

Funding: Partial funding for this study was provided by Stand Up to Cancer & St. Baldrick's Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113), RO1 CA136551-05, Alex Lemonade Stand Phase I/II Infrastructure Grant, Conquer Cancer Foundation Career Development Award, Washington State Life Sciences Discovery Fund, Ben Towne Foundation, William Lawrence & Blanche Hughes Foundation, and Juno Therapeutics, Inc., a Celgene Company.
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http://dx.doi.org/10.1172/JCI125423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486329PMC
May 2019

CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors.

Clin Cancer Res 2019 04 17;25(8):2560-2574. Epub 2019 Jan 17.

Department of Pediatrics, Stanford University School of Medicine, Palo Alto, California.

Purpose: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system.

Experimental Design: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models.

Results: B7-H3 CAR T cells mediate significant antitumor activity , causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3.

Conclusions: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8456711PMC
April 2019

A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia.

Front Oncol 2018 22;8:539. Epub 2018 Nov 22.

Lentigen, A Miltenyi Biotec Company, Gaithersburg, MD, United States.

Acute myeloid leukemia (AML) remains a challenging pediatric and adult disease. Given the elevated expression of the CD33 antigen on leukemic blasts, therapeutic approaches to AML now feature the approved antibody drug conjugate (Mylotarg, GO) and investigational CART cell approaches incorporating CD33-binding domains derived from humanized scFvs. We designed a functional chimeric antigen receptor utilizing a human targeting sequence, derived from a heavy chain variable domain, termed CAR33VH. Lentiviral-based expression vectors which encoded CAR constructs incorporating the novel binding domain (CAR33VH), or the My96 scFv control binder (My96CAR) in frame with a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3 zeta activation domain, were transduced into primary human CD4 and CD8 T cells, and CAR expression was confirmed by flow cytometry. CAR33VH, similar to My96CAR, demonstrated robust and specific cytotoxicity in short-term and long-term co-incubation killing assays against CD33 AML lines. In overnight cytokine release assays in which CAR T cells were challenged with the CD33 tumor cells HL-60, MOLM-14 and KG-1a, CAR33VH elicited IFN-gamma, TNF-alpha and IL-2. This was seen with CD33 cell lines, but not when CAR T were cultured alone. Studies with a CD33 cell line engineered to stably express the full length CD33 variant 1, or the naturally occurring CD33 splice variant 2, revealed that both CAR33VH and My96CAR, target the V domain of CD33, suggesting a similar therapeutic profile. Colony-formation assays utilizing peripheral blood CD34 hematopoietic stem cells treated with CAR33VH, My96CAR, or with an untransduced T cell control, yielded similar numbers of BFU-E erythroid and CFU-GM myeloid colonies, suggesting a lack of CAR-related overt toxicity. In an AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH and CARMy96 efficiently eliminated tumors. In conclusion, we demonstrate for the first time the feasibility and efficacy of employing human variable domain-only binder derived from a phage display library in an anti-AML CAR design. CAR33VH, comprised of a human heavy-chain variable fragment-only antigen binding domain, was efficient in tumor killing and , and showed comparable functionality to the scFv-based My96CAR.
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http://dx.doi.org/10.3389/fonc.2018.00539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262782PMC
November 2018

CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy.

Nat Med 2018 01 20;24(1):20-28. Epub 2017 Nov 20.

Department of Pediatrics and Standford Cancer Center, Stanford University, Stanford, California, USA.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 10 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19 or CD19 B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22 cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.
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http://dx.doi.org/10.1038/nm.4441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774642PMC
January 2018

Paired Expression Analysis of Tumor Cell Surface Antigens.

Front Oncol 2017 21;7:173. Epub 2017 Aug 21.

Genetics Branch, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Bethesda, MD, United States.

Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs) is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19) or antibody-based therapy (anti-CD20) in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling's -squared test (Hotelling-Lawley multivariate analysis of variance) for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues). We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK) with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK1, GPR173, or with one another provided the most promising paired-hits. We propose that targeting these markers together would increase the specificity and thereby the safety of CAR-based therapy for neuroblastoma.
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http://dx.doi.org/10.3389/fonc.2017.00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566986PMC
August 2017

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase.

Mol Ther 2017 09 1;25(9):2189-2201. Epub 2017 Jul 1.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. T cells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.
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http://dx.doi.org/10.1016/j.ymthe.2017.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589087PMC
September 2017

A tandem CD19/CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines.

J Immunother Cancer 2017 16;5:42. Epub 2017 May 16.

Lentigen Technology, Inc., 910 Clopper Rd., Gaithersburg, MD 20878 USA.

Background: Clinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated.

Methods: Antigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice.

Results: Tandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs.

Conclusion: Tandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion.
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http://dx.doi.org/10.1186/s40425-017-0246-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433150PMC
January 2018

Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas.

Cancer Immunol Res 2016 10 22;4(10):869-880. Epub 2016 Aug 22.

Pediatric Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland. Department of Pediatrics, Stanford University School of Medicine, Stanford, California.

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2 sarcoma and neuroblastoma cell lines in vitro However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2 sarcoma, despite effectively controlling GD2 neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869-80. ©2016 AACR.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050151PMC
http://dx.doi.org/10.1158/2326-6066.CIR-15-0230DOI Listing
October 2016

Emerging Immunotherapies for Cancer and Their Potential for Application in Pediatric Oncology.

Crit Rev Oncog 2015 ;20(3-4):315-27

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.

After decades of basic research, immune-based therapeutics for the treatment of cancer are showing evidence of efficacy in clinical trials; several immunotherapeutics already incorporated into standard treatment regimens. Intensive research is underway to improve the efficacy of immunotherapeutics and to expand the application of immunotherapy to a wider array of cancers. The therapeutic options that comprise immunotherapy for cancer are vast and span monoclonal antibodies, tumor vaccines, adoptive cellular therapies, as well as therapies aimed at reversing immunosuppression and enhancing immune reactivity globally and/or locally within the tumor microenvironment. In pediatric cancer, monoclonal antibodies have demonstrated efficacy in hematologic malignancies, and neuroblastoma and bispecific antibodies that activate resident T cells, as well as adoptive cell therapy, have shown recent exciting results for the treatment of acute lymphoblastic leukemia in childhood. This review discusses the basic principles of tumor immunology driving clinical development of new immunotherapies, describes immunotherapeutics with demonstrated efficacy and several currently in clinical trials, and highlight agents that seem to be most promising for the treatment of pediatric cancer.
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http://dx.doi.org/10.1615/critrevoncog.2015013861DOI Listing
June 2016

Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein.

Blood 2015 Jul 3;126(5):629-39. Epub 2015 Jun 3.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD;

Adoptive transfer of T cells genetically modified to express chimeric antigen receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated potent activity against relapsed/refractory B-lineage acute lymphoblastic leukemia (B-ALL). Not all patients respond, and CD19-negative relapses have been observed. Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important, suggesting that TSLPR would be an ideal immunotherapeutic target. We constructed short and long CARs targeting TSLPR and tested efficacy against CRLF2-overexpressing B-ALL. Both CARs demonstrated activity in vitro, but only short TSLPR CAR T cells mediated leukemia regression. In vivo activity of the short CAR was also associated with long-term persistence of CAR-expressing T cells. Short TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line induced leukemia cytotoxicity with efficacy comparable with that of CD19 CAR T cells. Short TSLPR CAR T cells also eradicated leukemia in 4 xenograft models of human CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-targeted immunotherapy for high-risk ALL.
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http://dx.doi.org/10.1182/blood-2014-11-612903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520878PMC
July 2015

4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors.

Nat Med 2015 Jun 4;21(6):581-90. Epub 2015 May 4.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.
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http://dx.doi.org/10.1038/nm.3838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458184PMC
June 2015

Bioinformatic description of immunotherapy targets for pediatric T-cell leukemia and the impact of normal gene sets used for comparison.

Front Oncol 2014 10;4:134. Epub 2014 Jun 10.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, MD , USA.

Pediatric lymphoid leukemia has the highest cure rate of all pediatric malignancies, yet due to its prevalence, still accounts for the majority of childhood cancer deaths and requires long-term highly toxic therapy. The ability to target B-cell ALL with immunoglobulin-like binders, whether anti-CD22 antibody or anti-CD19 CAR-Ts, has impacted treatment options for some patients. The development of new ways to target B-cell antigens continues at rapid pace. T-cell ALL accounts for up to 20% of childhood leukemia but has yet to see a set of high-value immunotherapeutic targets identified. To find new targets for T-ALL immunotherapy, we employed a bioinformatic comparison to broad normal tissue arrays, hematopoietic stem cells (HSC), and mature lymphocytes, then filtered the results for transcripts encoding plasma membrane proteins. T-ALL bears a core T-cell signature and transcripts encoding TCR/CD3 components and canonical markers of T-cell development predominate, especially when comparison was made to normal tissue or HSC. However, when comparison to mature lymphocytes was also undertaken, we identified two antigens that may drive, or be associated with leukemogenesis; TALLA-1 and hedgehog interacting protein. In addition, TCR subfamilies, CD1, activation and adhesion markers, membrane-organizing molecules, and receptors linked to metabolism and inflammation were also identified. Of these, only CD52, CD37, and CD98 are currently being targeted clinically. This work provides a set of targets to be considered for future development of immunotherapies for T-ALL.
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http://dx.doi.org/10.3389/fonc.2014.00134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050364PMC
June 2014

A transplantable TH-MYCN transgenic tumor model in C57Bl/6 mice for preclinical immunological studies in neuroblastoma.

Int J Cancer 2014 Mar 14;134(6):1335-45. Epub 2013 Sep 14.

Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; Department of Pediatric Oncology, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands.

Current multimodal treatments for patients with neuroblastoma (NBL), including anti-disialoganglioside (GD2) monoclonal antibody (mAb) based immunotherapy, result in a favorable outcome in around only half of the patients with advanced disease. To improve this, novel immunocombinational strategies need to be developed and tested in autologous preclinical NBL models. A genetically well-explored autologous mouse model for NBL is the TH-MYCN model. However, the immunobiology of the TH-MYCN model remains largely unexplored. We developed a mouse model using a transplantable TH-MYCN cell line in syngeneic C57Bl/6 mice and characterized the immunobiology of this model. In this report, we show the relevance and opportunities of this model to study immunotherapy for human NBL. Similar to human NBL cells, syngeneic TH-MYCN-derived 9464D cells endogenously express the tumor antigen GD2 and low levels of MHC Class I. The presence of the adaptive immune system had little or no influence on tumor growth, showing the low immunogenicity of the NBL cells. In contrast, depletion of NK1.1+ cells resulted in enhanced tumor outgrowth in both wild-type and Rag1(-/-) mice, showing an important role for NK cells in the natural anti-NBL immune response. Analysis of the tumor infiltrating leukocytes ex vivo revealed the presence of both tumor associated myeloid cells and T regulatory cells, thus mimicking human NBL tumors. Finally, anti-GD2 mAb mediated NBL therapy resulted in ADCC in vitro and delayed tumor outgrowth in vivo. We conclude that the transplantable TH-MYCN model represents a relevant model for the development of novel immunocombinatorial approaches for NBL patients.
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http://dx.doi.org/10.1002/ijc.28463DOI Listing
March 2014

Fibrocytes represent a novel MDSC subset circulating in patients with metastatic cancer.

Blood 2013 Aug 11;122(7):1105-13. Epub 2013 Jun 11.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

Fibrocytes are hematopoietic stem cell-derived fibroblast precursors that are implicated in chronic inflammation, fibrosis, and wound healing. Myeloid-derived suppressor cells (MDSCs) expand in cancer-bearing hosts and contribute to tumor immune evasion. They are typically described as CD11b⁺HLA-DR⁻ in humans. We report abnormal expansions of CD11b⁺HLA-DR⁺ myeloid cells in peripheral blood mononuclear fractions of subjects with metastatic pediatric sarcomas. Like classical fibrocytes, they display cell surface α smooth muscle actin, collagen I/V, and mediate angiogenesis. However, classical fibrocytes serve as antigen presenters and augment immune reactivity, whereas fibrocytes from cancer subjects suppressed anti-CD3-mediated T-cell proliferation, primarily via indoleamine oxidase (IDO). The degree of fibrocyte expansion observed in individual subjects directly correlated with the frequency of circulating GATA3⁺CD4⁺ cells (R = 0.80) and monocytes from healthy donors cultured with IL-4 differentiated into fibrocytes with the same phenotypic profile and immunosuppressive properties as those observed in patients with cancer. We thus describe a novel subset of cancer-induced myeloid-derived suppressor cells, which bear the phenotypic and functional hallmarks of fibrocytes but mediate immune suppression. These cells are likely expanded in response to Th2 immune deviation and may contribute to tumor progression via both immune evasion and angiogenesis.
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http://dx.doi.org/10.1182/blood-2012-08-449413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744987PMC
August 2013

Lessons learned from a highly-active CD22-specific chimeric antigen receptor.

Oncoimmunology 2013 Apr;2(4):e23621

Pediatric Oncology Branch; National Cancer Institute; Center for Cancer Research; National Institutes of Health; Bethesda, MD USA.

CD22 is an attractive target for the development of immunotherapeutic approaches for the therapy of B-cell malignancies. In particular, an m971 antibody-derived, second generation chimeric antigen receptor (CAR) that targets CD22 holds significant therapeutic promise. The key aspect for the development of such a highly-active CAR was its ability to target a membrane-proximal epitope of CD22.
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http://dx.doi.org/10.4161/onci.23621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654586PMC
April 2013

Reading the tea leaves of tumor-mediated immunosuppression.

Authors:
Rimas J Orentas

Clin Cancer Res 2013 Mar 21;19(5):955-7. Epub 2013 Feb 21.

Pediatric Oncology Branch, National Cancer Institute, CCR, NIH, Bethesda, Maryland 20892, USA.

Polyphenol E, available as Polyphenon E, is a green tea extract whose activity can be benchmarked to the presence of specific catechins such as epigallocatechin 3-gallate (EGCG). Herein, Polyphenon E is shown to reverse myeloid-derived suppressor cell activity, linking the activity of a natural product extract to cell-mediated immunity.
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http://dx.doi.org/10.1158/1078-0432.CCR-12-3792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594479PMC
March 2013

Identification of cell surface proteins as potential immunotherapy targets in 12 pediatric cancers.

Front Oncol 2012 17;2:194. Epub 2012 Dec 17.

Immunology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health Bethesda, MD, USA.

Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.
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http://dx.doi.org/10.3389/fonc.2012.00194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3523547PMC
December 2012

Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

Blood 2013 Feb 14;121(7):1165-74. Epub 2012 Dec 14.

Pediatric Oncology Branch, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD 20892, USA.

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.
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http://dx.doi.org/10.1182/blood-2012-06-438002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575759PMC
February 2013

Immunotherapy targets in pediatric cancer.

Front Oncol 2012 30;2. Epub 2012 Jan 30.

Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health Bethesda, MD, USA.

Immunotherapy for cancer has shown increasing success and there is ample evidence to expect that progress gleaned in immune targeting of adult cancers can be translated to pediatric oncology. This manuscript reviews principles that guide selection of targets for immunotherapy of cancer, emphasizing the similarities and distinctions between oncogene-inhibition targets and immune targets. It follows with a detailed review of molecules expressed by pediatric tumors that are already under study as immune targets or are good candidates for future studies of immune targeting. Distinctions are made between cell surface antigens that can be targeted in an MHC independent manner using antibodies, antibody derivatives, or chimeric antigen receptors versus intracellular antigens which must be targeted with MHC restricted T cell therapies. Among the most advanced immune targets for childhood cancer are CD19 and CD22 on hematologic malignancies, GD2 on solid tumors, and NY-ESO-1 expressed by a majority of synovial sarcomas, but several other molecules reviewed here also have properties which suggest that they too could serve as effective targets for immunotherapy of childhood cancer.
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http://dx.doi.org/10.3389/fonc.2012.00003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3355840PMC
August 2012
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