Publications by authors named "Avery D Posey"

32 Publications

Combination Therapy for Solid Tumors: Taking a Classic CAR on New Adventures.

Cancer Cell 2020 Nov 15;38(5):621-623. Epub 2020 Oct 15.

Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA, USA. Electronic address:

CD19-specific CAR-T cell therapies are the gold standard of adoptive cellular immunotherapy for hematopoietic malignancies. In Science Translational Medicine, Park et al. develop an oncolytic vaccinia virus that introduces truncated CD19 expression in solid tumors, which are then eradicated by CD19-specific CAR-T cells in immunodeficient and immunocompetent mouse models.
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http://dx.doi.org/10.1016/j.ccell.2020.10.003DOI Listing
November 2020

Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.

Cell 2020 Oct 21;183(1):126-142.e17. Epub 2020 Sep 21.

Parker Institute for Cancer Immunotherapy, Stanford University School of Medicine, Stanford, CA, USA; Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.
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http://dx.doi.org/10.1016/j.cell.2020.08.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640763PMC
October 2020

Diversity Is a Strength of Cancer Research in the U.S.

Cancer Cell 2020 09;38(3):297-300

We stand against racism and discrimination in cancer research in the U.S. By sharing the stories of scientists from different ethnicities, identities, and national origins, we want to promote change through mentoring, active participation, and policy changes and to inspire the next generation of cancer researchers: we make better science together.
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http://dx.doi.org/10.1016/j.ccell.2020.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7489360PMC
September 2020

Single residue in CD28-costimulated CAR-T cells limits long-term persistence and antitumor durability.

J Clin Invest 2020 06;130(6):3087-3097

Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Chimeric antigen receptor-T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response.
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http://dx.doi.org/10.1172/JCI133215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260017PMC
June 2020

Approaches of T Cell Activation and Differentiation for CAR-T Cell Therapies.

Methods Mol Biol 2020 ;2086:203-211

Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Chimeric antigen receptor (CAR) T cell therapies are ex vivo manufactured cellular products that have been useful in the treatment of blood cancers and solid tumors. The quality of the final cellular product is influenced by several amenable factors during the manufacturing process. This review discusses several of the influences on cell product phenotype, including the raw starting material, methods of activation and transduction, and culture supplementation.
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http://dx.doi.org/10.1007/978-1-0716-0146-4_15DOI Listing
December 2020

A cellular antidote to specifically deplete anti-CD19 chimeric antigen receptor-positive cells.

Blood 2020 02;135(7):505-509

Center for Cellular Immunotherapies and.

Unintentional transduction of B-cell acute lymphoblastic leukemia blasts during CART19 manufacturing can lead to CAR19+ leukemic cells (CARB19) that are resistant to CART19 killing. We developed an anti-CAR19 idiotype chimeric antigen receptor (αCAR19) to specifically recognize CAR19+ cells. αCAR19 CAR T cells efficiently lysed CARB19 cells in vitro and in a primary leukemia-derived xenograft model. We further showed that αCAR19-CART cells could be used as an "antidote" to deplete CART19 cells to reduce long-term side effects, such as B-cell aplasia.
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http://dx.doi.org/10.1182/blood.2019001859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019191PMC
February 2020

Keeping the Engine Running: The Relevance and Predictive Value of Preclinical Models for CAR-T Cell Development.

ILAR J 2018 12;59(3):276-285

Department of Pathology and Laboratory Medicine, and Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania; and Parker Institute for Cancer Immunotherapy; and Corporal Michael J. Crescenz VA Medical Center, Philadelphia, Pennsylvania.

The cellular immunotherapy field has achieved important milestones in the last 30 years towards the treatment of a variety of cancers due to improvements in ex-vivo T cell manufacturing processes, the invention of synthetic T cell receptors, and advances in cellular engineering. Here, we discuss major preclinical models that have been useful for the validation of chimeric antigen receptor (CAR)-T cell therapies and also promising new models that will fuel future investigations towards success. However, multiple unanswered questions in the CAR-T cell field remain to be addressed that will require innovative preclinical models. Key challenges facing the field include premature immune rejection of universal CAR-T cells and the immune suppressive tumor microenvironment. Immune competent models that accurately recapitulate tumor heterogeneity, the hostile tumor microenvironment, and barriers to CAR-T cell homing, toxicity, and persistence are needed for further advancement of the field.
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http://dx.doi.org/10.1093/ilar/ilz009DOI Listing
December 2018

Engineering and Design of Chimeric Antigen Receptors.

Mol Ther Methods Clin Dev 2019 Mar 31;12:145-156. Epub 2018 Dec 31.

Cellular Immunotherapy Program, Massachusetts General Hospital Cancer Center, Boston, MA, USA.

T cells engineered with chimeric antigen receptors (CARs) have emerged as a potent new class of therapeutics for cancer, based on their remarkable potency in blood cancers. Since the first clinical reports of their efficacy emerged 7 years ago, investigators have focused on the mechanisms and properties that make CARs effective or toxic, and their effects on T cell biology. Novel CAR designs coupled with improvements in gene transfer technology, incorporating advances in gene editing, have the potential to increase access to engineered cell therapies, as well as improve their potency in solid tumors.
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http://dx.doi.org/10.1016/j.omtm.2018.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330382PMC
March 2019

Expanding the Therapeutic Window for CAR T Cell Therapy in Solid Tumors: The Knowns and Unknowns of CAR T Cell Biology.

Front Immunol 2018 26;9:2486. Epub 2018 Oct 26.

Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States.

A major obstacle for chimeric antigen receptor (CAR) T cell therapy in solid tumors is the lack of truly tumor-specific target antigens, which translates to the targeting of tumor-associated antigens (TAAs) overexpressed on tumors but shared with normal organs, raising safety concerns. In addition, expression of TAAs in solid tumors is particularly heterogeneous. In this regard, it is critical to deeply understand the sensitivity of CAR T cells, especially against low-density targets and the possible therapeutic window of antigen density targeted by CAR T cells. In this review, we discuss the recent findings of mechanisms of antigen recognition through CAR, including immunological synapse formation, and the impact of target antigen density for induction of distinct T cell functions. We also discuss rational strategies to adjust and expand the therapeutic window for effective and safe targeting of solid tumors by CAR T cell platforms.
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http://dx.doi.org/10.3389/fimmu.2018.02486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212550PMC
September 2019

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

Mol Ther Oncolytics 2018 Dec 28;11:20-38. Epub 2018 Aug 28.

Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor . Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested by co-culture with canine tumor cells and in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.
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http://dx.doi.org/10.1016/j.omto.2018.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174845PMC
December 2018

Driving cars to the clinic for solid tumors.

Gene Ther 2018 06 7;25(3):165-175. Epub 2018 Jun 7.

Center for Cellular Immunotherapies, Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

FDA approval of chimeric antigen receptor T cells (CART cells) is the culmination of several decades of technology development and interrogation of the properties of these gene therapies. CART cells exist as personalized "living drugs" and have demonstrated astounding anti-tumor efficacy in patients with leukemia and lymphoma. However, the future promise of CART efficacy for solid tumors, the greatest unmet burden, is met with a number of challenges that must be surmounted for effective immune responses. In this review, we discuss the next-generation developments of CARs to target solid tumors, including fine-tuned and combinational-targeting receptors. We consider the structural intricacies of the CAR molecules that influence optimal signaling and CART survival, and review pre-clinical cell-intrinsic and cell-extrinsic combinational therapy approaches.
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http://dx.doi.org/10.1038/s41434-018-0007-xDOI Listing
June 2018

Immunotherapy for Breast Cancer: Current and Future Strategies.

Curr Surg Rep 2017 Dec 10;5. Epub 2017 Oct 10.

Department of Surgery, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Boulevard, 10th floor South, Philadelphia, PA 19104, USA.

Purpose Of Review: The breast tumor microenvironment is immunosuppressive and is increasingly recognized to play a significant role in tumorigenesis. A deeper understanding of normal and aberrant interactions between malignant and immune cells has allowed researchers to harness the immune system with novel immunotherapy strategies, many of which have shown promise in breast cancer. This review discusses the application of immunotherapy to the treatment of breast cancer.

Recent Findings: Both basic science and clinical trial data are rapidly developing in the use of immunotherapy for breast cancer. The current clinical trial landscape includes therapeutic vaccines, immune checkpoint blockade, antibodies, cytokines, and adoptive cell therapy.

Summary: Despite early failures, the application of immunotherapeutic strategies to the treatment of breast cancer holds promise.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894864PMC
http://dx.doi.org/10.1007/s40137-017-0194-1DOI Listing
December 2017

Improving CART-Cell Therapy of Solid Tumors with Oncolytic Virus-Driven Production of a Bispecific T-cell Engager.

Cancer Immunol Res 2018 05 27;6(5):605-616. Epub 2018 Mar 27.

Center for Cellular Immunotherapies, Perelman School of Medicine, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

T cells expressing chimeric antigen receptors (CART) have shown significant promise in clinical trials to treat hematologic malignancies, but their efficacy in solid tumors has been limited. Oncolytic viruses have the potential to act in synergy with immunotherapies due to their immunogenic oncolytic properties and the opportunity of incorporating therapeutic transgenes in their genomes. Here, we hypothesized that an oncolytic adenovirus armed with an EGFR-targeting, bispecific T-cell engager (OAd-BiTE) would improve the outcome of CART-cell therapy in solid tumors. We report that CART cells targeting the folate receptor alpha (FR-α) successfully infiltrated preestablished xenograft tumors but failed to induce complete responses, presumably due to the presence of antigen-negative cancer cells. We demonstrated that OAd-BiTE-mediated oncolysis significantly improved CART-cell activation and proliferation, while increasing cytokine production and cytotoxicity, and showed an favorable safety profile compared with EGFR-targeting CARTs. BiTEs secreted from infected cells redirected CART cells toward EGFR in the absence of FR-α, thereby addressing tumor heterogeneity. BiTE secretion also redirected CAR-negative, nonspecific T cells found in CART-cell preparations toward tumor cells. The combinatorial approach improved antitumor efficacy and prolonged survival in mouse models of cancer when compared with the monotherapies, and this was the result of an increased BiTE-mediated T-cell activation in tumors. Overall, these results demonstrated that the combination of a BiTE-expressing oncolytic virus with adoptive CART-cell therapy overcomes key limitations of CART cells and BiTEs as monotherapies in solid tumors and encourage its further evaluation in human trials. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688490PMC
May 2018

Glycan-directed CAR-T cells.

Glycobiology 2018 09;28(9):656-669

Center of Cellular Immunotherapies, Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy.
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http://dx.doi.org/10.1093/glycob/cwy008DOI Listing
September 2018

Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation.

JCI Insight 2018 01 11;3(1). Epub 2018 Jan 11.

Center for Cellular Immunotherapies, Department of Pathology and Laboratory Medicine.

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.
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http://dx.doi.org/10.1172/jci.insight.96976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821198PMC
January 2018

CAR T-Cell Therapies in Glioblastoma: A First Look.

Clin Cancer Res 2018 02 20;24(3):535-540. Epub 2017 Nov 20.

Center for Cellular Immunotherapies and Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Glioblastoma is an aggressive malignancy with a poor prognosis. The current standard of care for newly diagnosed glioblastoma patients includes surgery to the extent, temozolomide combined with radiotherapy, and alternating electric fields therapy. After recurrence, there is no standard therapy and survival is less than 9 months. Recurrent glioblastoma offers a unique opportunity to investigate new treatment approaches in a malignancy known for remarkable genetic heterogeneity, an immunosuppressive microenvironment, and a partially permissive anatomic blood-brain barrier. Results from three first-in-man chimeric antigen receptor (CAR) T-cell trials targeting IL13Rα2, Her2/CMV, and EGFRvIII have recently been reported. Each one of these trials addresses important questions, such as T-cell trafficking to CNS, engraftment and persistence, tumor microenvironment remodeling, and monitoring of glioma response to CAR T cells. Objective radiologic responses have been reported. Here, we discuss and summarize the results of these trials and suggest opportunities for the field. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2871DOI Listing
February 2018

Safety and Efficacy of Intratumoral Injections of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer.

Cancer Immunol Res 2017 12 6;5(12):1152-1161. Epub 2017 Nov 6.

Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 10 or 3 × 10 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug-related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712264PMC
December 2017

Cancer Killers.

Sci Am 2017 Feb;316(3):38-43

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http://dx.doi.org/10.1038/scientificamerican0317-38DOI Listing
February 2017

Distinguishing Truncated and Normal MUC1 Glycoform Targeting from Tn-MUC1-Specific CAR T Cells: Specificity Is the Key to Safety.

Immunity 2016 11;45(5):947-948

Center for Cellular Immunotherapies and Parker Institute for Cancer Immunotherapy at the University of Pennsylvania, Philadelphia, PA, 19104-5156 USA. Electronic address:

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate potent clinical antitumor effects in a variety of blood cancers. However, clinical activity in solid tumors has been disappointing and toxicity has been a serious concern (Lamers et al., 2013; Morgan et al., 2010). We recently found that a CAR composed of a scFv antibody fragment specific for the Tn-glycoform of MUC1 had potent activity in preclinical models of blood cancer and adenocarcinoma (Posey et al., 2016).
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http://dx.doi.org/10.1016/j.immuni.2016.10.015DOI Listing
November 2016

Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma.

Immunity 2016 06;44(6):1444-54

Center for Cellular Immunotherapies, Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Pathology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. Electronic address:

Genetically modified T cells expressing chimeric antigen receptors (CARs) demonstrate robust responses against lineage restricted, non-essential targets in hematologic cancers. However, in solid tumors, the full potential of CAR T cell therapy is limited by the availability of cell surface antigens with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer-associated Tn glycoform of MUC1, a neoantigen expressed in a variety of cancers. Anti-Tn-MUC1 CAR T cells demonstrated target-specific cytotoxicity and successfully controlled tumor growth in xenograft models of T cell leukemia and pancreatic cancer. These findings demonstrate the therapeutic efficacy of CAR T cells directed against Tn-MUC1 and present aberrantly glycosylated antigens as a novel class of targets for tumor therapy with engineered T cells.
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http://dx.doi.org/10.1016/j.immuni.2016.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358667PMC
June 2016

Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells.

Immunity 2016 Feb;44(2):380-90

Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Chimeric antigen receptors (CARs) redirect T cell cytotoxicity against cancer cells, providing a promising approach to cancer immunotherapy. Despite extensive clinical use, the attributes of CAR co-stimulatory domains that impact persistence and resistance to exhaustion of CAR-T cells remain largely undefined. Here, we report the influence of signaling domains of coreceptors CD28 and 4-1BB on the metabolic characteristics of human CAR T cells. Inclusion of 4-1BB in the CAR architecture promoted the outgrowth of CD8(+) central memory T cells that had significantly enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis. In contrast, CAR T cells with CD28 domains yielded effector memory cells with a genetic signature consistent with enhanced glycolysis. These results provide, at least in part, a mechanistic insight into the differential persistence of CAR-T cells expressing 4-1BB or CD28 signaling domains in clinical trials and inform the design of future CAR T cell therapies.
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http://dx.doi.org/10.1016/j.immuni.2016.01.021DOI Listing
February 2016

Measurement of intracellular ions by flow cytometry.

Curr Protoc Cytom 2015 Apr 1;72:9.8.1-9.8.21. Epub 2015 Apr 1.

Abramson Family Cancer Research Institute, and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

Using flow cytometry, single-cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. Most earlier techniques for measuring cellular activation parameters determined the mean value for a population of cells, which did not permit optimal resolution of the responses. The flow cytometer is particularly useful for this purpose because it can measure ion concentrations in large numbers of single cells and thereby allows ion concentration to be correlated with other parameters such as immunophenotype and cell cycle stage. A limitation of flow cytometry, however, is that it does not permit resolution of certain complex kinetic responses such as cellular oscillatory responses. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation.
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http://dx.doi.org/10.1002/0471142956.cy0908s72DOI Listing
April 2015

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

Sci Transl Med 2015 Feb;7(275):275ra22

Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).
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http://dx.doi.org/10.1126/scitranslmed.aaa4963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467166PMC
February 2015

Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

Cancer Immunol Res 2015 Apr 19;3(4):356-67. Epub 2015 Jan 19.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials.
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http://dx.doi.org/10.1158/2326-6066.CIR-14-0186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390458PMC
April 2015

ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells.

Blood 2014 Aug 1;124(7):1070-80. Epub 2014 Jul 1.

Abramson Cancer Center and the Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;

With the notable exception of B-cell malignancies, the efficacy of chimeric antigen receptor (CAR) T cells has been limited, and CAR T cells have not been shown to expand and persist in patients with nonlymphoid tumors. Here we demonstrate that redirection of primary human T cells with a CAR containing the inducible costimulator (ICOS) intracellular domain generates tumor-specific IL-17-producing effector cells that show enhanced persistence. Compared with CARs containing the CD3ζ chain alone, or in tandem with the CD28 or the 4-1BB intracellular domains, ICOS signaling increased IL-17A, IL-17F, and IL-22 following antigen recognition. In addition, T cells redirected with an ICOS-based CAR maintained a core molecular signature characteristic of TH17 cells and expressed higher levels of RORC, CD161, IL1R-1, and NCS1. Of note, ICOS signaling also induced the expression of IFN-γ and T-bet, consistent with a TH17/TH1 bipolarization. When transferred into mice with established tumors, TH17 cells that were redirected with ICOS-based CARs mediated efficient antitumor responses and showed enhanced persistence compared with CD28- or 4-1BB-based CAR T cells. Thus, redirection of TH17 cells with a CAR encoding the ICOS intracellular domain is a promising approach to augment the function and persistence of CAR T cells in hematologic malignancies.
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http://dx.doi.org/10.1182/blood-2013-10-535245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133482PMC
August 2014

EHD1 mediates vesicle trafficking required for normal muscle growth and transverse tubule development.

Dev Biol 2014 Mar 17;387(2):179-90. Epub 2014 Jan 17.

Department of Medicine, The University of Chicago, Chicago, IL 60637, USA. Electronic address:

EHD proteins have been implicated in intracellular trafficking, especially endocytic recycling, where they mediate receptor and lipid recycling back to the plasma membrane. Additionally, EHDs help regulate cytoskeletal reorganization and induce tubule formation. It was previously shown that EHD proteins bind directly to the C2 domains in myoferlin, a protein that regulates myoblast fusion. Loss of myoferlin impairs normal myoblast fusion leading to smaller muscles in vivo but the intracellular pathways perturbed by loss of myoferlin function are not well known. We now characterized muscle development in EHD1-null mice. EHD1-null myoblasts display defective receptor recycling and mislocalization of key muscle proteins, including caveolin-3 and Fer1L5, a related ferlin protein homologous to myoferlin. Additionally, EHD1-null myoblast fusion is reduced. We found that loss of EHD1 leads to smaller muscles and myofibers in vivo. In wildtype skeletal muscle EHD1 localizes to the transverse tubule (T-tubule), and loss of EHD1 results in overgrowth of T-tubules with excess vesicle accumulation in skeletal muscle. We provide evidence that tubule formation in myoblasts relies on a functional EHD1 ATPase domain. Moreover, we extended our studies to show EHD1 regulates BIN1 induced tubule formation. These data, taken together and with the known interaction between EHD and ferlin proteins, suggests that the EHD proteins coordinate growth and development likely through mediating vesicle recycling and the ability to reorganize the cytoskeleton.
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http://dx.doi.org/10.1016/j.ydbio.2014.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987670PMC
March 2014

Ferlin proteins in myoblast fusion and muscle growth.

Curr Top Dev Biol 2011 ;96:203-30

Genomics and Systems Biology, Committee on Genetics, The University of Chicago, Chicago, Illinois, USA.

Myoblast fusion contributes to muscle growth in development and during regeneration of mature muscle. Myoblasts fuse to each other as well as to multinucleate myotubes to enlarge the myofiber. The molecular mechanisms of myoblast fusion are incompletely understood. Adhesion, apposition, and membrane fusion are accompanied by cytoskeletal rearrangements. The ferlin family of proteins is implicated in human muscle disease and has been implicated in fusion events in muscle, including myoblast fusion, vesicle trafficking and membrane repair. Dysferlin was the first mammalian ferlin identified and it is now known that there are six different ferlins. Loss-of-function mutations in the dysferlin gene lead to limb girdle muscular dystrophy and the milder disorder Miyoshi Myopathy. Dysferlin is a membrane-associated protein that has been implicated in resealing disruptions in the muscle plasma membrane. Newer data supports a broader role for dysferlin in intracellular vesicular movement, a process also important for resealing. Myoferlin is highly expressed in myoblasts that undergoing fusion, and the absence of myoferlin leads to impaired myoblast fusion. Myoferlin also regulates intracellular trafficking events, including endocytic recycling, a process where internalized vesicles are returned to the plasma membrane. The trafficking role of ferlin proteins is reviewed herein with a specific focus as to how this machinery alters myogenesis and muscle growth.
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http://dx.doi.org/10.1016/B978-0-12-385940-2.00008-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4464798PMC
August 2011

Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion.

J Biol Chem 2011 Mar 22;286(9):7379-88. Epub 2010 Dec 22.

Committee on Genetics, Genomics and Systems Biology, University of Chicago, Chicago, Illinois 60637, USA.

The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044994PMC
http://dx.doi.org/10.1074/jbc.M110.157222DOI Listing
March 2011