ABT-737 Sensitizes B Cell Tumors for Killing by CD19-Retargeted T Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4032-4032
Author(s):  
Hannah S C Karlsson ◽  
Camilla Lindqvist ◽  
Gabriella Paul-Wetterberg ◽  
Helena Jernberg Wiklund ◽  
Kenneth Nilsson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
B Cell ◽  
Cell Lines ◽  
Single Agent ◽  
Tumor Cell Lines ◽  
Tumor Cell Death ◽  
T Cell Therapy ◽  
Apoptosis Pathways

Abstract Abstract 4032 Introduction: T cells expressing tumor-targeting chimeric antigen receptors are showing promise in clinical trials for patients with B cell leukemia and lymphoma. However, increased levels of anti-apoptotic proteins, a common trait among B-cell tumors, may hamper treatment efficacy. ABT-737 is a small molecule inhibitor of anti-apoptotic proteins such as BCL-2, BCL-xL, BCL-w, and MCL-1, which induces apoptosis via the intrinsic apoptosis pathway in contrast to T-cells that utilize the extrinsic pathway controlled by death receptors and their ligands. ABT-737 has been shown to efficiently promote apoptosis in B-cell tumors as exemplified in models of pre-B-ALL. Recently, ABT-737 was shown to synergize with TRAIL to induce apoptosis. This prompted us to investigate if ABT-737 could be combined with T-cell therapy to enhance tumor cell death. Methods: PBMCs from healthy donors and patients with pre-B-ALL was genetically engineered with a second generation chimeric antigen receptor (CAR) targeting CD19 on B-cells. The T-cells and ABT-737 were tested both individually, and in combination, for their cytotoxic capacity in in vitro assays such as flow cytometry and the Caspase-Glo® 3/7 assay. The effects were studied in a panel of B-cell tumor cell lines (Daudi, U698, Karpas422, DG75, Nall-1) since they may exhibit different apoptosis resistance profiles. The expression of anti-apoptosis molecules in these cell lines was investigated by PCR. Results: PCR confirmed expression of BCL family proteins in the cell lines tested. CD19-targeting T-cells specifically induced apoptosis in CD19+ tumor cells. Similarly, but less efficiently, ABT-737 as single agent increased apoptosis in the various tumor cell lines. When combining T-cell and ABT-737 therapy, the tumor cell death was significantly increased to that of single agent treatment. The effect varied from additive to synergistic effects. The tumor cell lines did not change the level of antigen presenting molecules (MHC I and II), death receptors (Fas) or adhesion or costimulatory molecules (ICAM-I, CD80, CD86) upon ABT-737 treatment. Hence, the effect did not likely represent increased killing by enhanced physical interaction between T-cells and tumors but rather simultaneous engagement of both intrinsic and extrinsic apoptosis pathways. Conclusion: The apoptosis inducer ABT-737 is potently enhancing CD19-targeting T-cell therapy. By triggering both intrinsic and extrinsic apoptosis pathways also resistant tumors may succumb to treatment. Disclosures: Simonsson: Novartis, BMS, Merck, Pfizer: Consultancy, Honoraria.

Multiple Myeloma as Target for γδ T Cell Mediated Immunotherapy - Expression of the Putative Vγ9Vδ2-TCR Ligand F1-ATPase on Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3391-3391
Author(s):  
Volker Kunzmann ◽  
Judith Engert ◽  
Brigitte Kimmel ◽  
Martin Wilhelm ◽  
Hermann Einsele
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Surface Expression ◽  
Tumor Cell Lines ◽  
Myeloma Cells ◽  
F1 Atpase ◽  
Vγ9vδ2 T Cells

Abstract Activated Vγ9Vδ2 T cells, the major γδ T lymphocyte subset in humans, show cytolytic activity against various tumor cells. However, tumor antigens recognized by the TCR remained unkown so far. Recently, the ectopic surface expression of the F1-ATPase, normally expressed on the internal membrane of mitochondria, was implicated in tumor recognition of Vγ9Vδ2 T cells (Scotet E. et al., Immunity2005; 22:71–80). Surface expression of the a chain of the F1-ATPase (recognized by monoclonal antibody 7H10) strongly correlates with susceptibility of tumor cells against Vγ9Vδ2 T cell lysis. Different functions have been attributed to the ectopic expression of the F1-ATPase on the cell surface, including an immunoregulatory role induced by cell stress, receptor for angiostatin or regulation of lipoprotein transport through high-affinity apolipoprotein A-I binding. In this study we evaluated the surface expression of this F1-ATPase on hematopoetic tumor cell lines and on primary tumor cells from hematological malignancies. As already shown, the a subunit of F1-ATPase was clearly detected on several tumor cell lines which are consistently killed by activated Vγ9Vδ2 T cells (Daudi, K562, RPMI 8226), whereas the known Vγ9Vδ2 T cell resistant tumor cell lines (Raji, Jurkat) did not express detectable levels of the F1-ATPase. Analysis of 42 primary hematopoetic tumor cells (21 myeloma, 17 AML, 4 B-NHL) revealed frequent expression of F1-ATPase on primary myeloma cells (14/19 positive), whereas primary AML blasts (3/17 positive) and primary NHL cells (1/4 positive) expressed the putative Vγ9Vδ2-TCR ligand F1-ATPase less frequently. To further evaluate the functional role of F1-ATPase expression in Vγ9Vδ2 T cell mediated recognition of myeloma cells, cytotoxicity assays were performed. The mAb against the a subunit of F1-ATPase significantly decreased in vitro lysis of myeloma cells lines and primary myeloma cells by activated Vγ9Vδ2 T cells. These results suggests Vγ9Vδ2 TCR-dependent interactions between myeloma cells and Vγ9Vδ2 T cells and indicate that multiple myeloma should be considered as a major target for γδ T-cell mediated immunotherapy.

Constitutive Expression of CD40L by CAR-Modified Tumor Targeted T Cells Enhances Anti-Tumor Efficacy Both in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4120-4120 ◽  
Author(s):  
Kevin J. Curran ◽  
Beatrijs Seinstra ◽  
Yan Nikhamin ◽  
Raymond Yeh ◽  
Yelena Usachenko ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Cd40 Ligand ◽  
Tumor Cell Lines ◽  
Pro Inflammatory Cytokines

Abstract Abstract 4120 T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). Recent reports have demonstrated the effectiveness of CAR modified T cells in patients with relapsed or refractory malignancies. However, CAR modified T cells have yet to demonstrate the ability to recruit an endogenous anti-tumor response which would greatly enhance their therapeutic benefit. To overcome these limitations we have developed a bi-cistronic gamma-retroviral vector allowing for constitutive co-expression of a CD19-specific CAR (19–28z) and human CD40 ligand (CD40L; CD154). The CD40 ligand/CD40 system has been demonstrated to activate dendritic cells (DCs) and alter the phenotype of B cells (upregulation of co-stimulatory and adhesion molecules and secretion of pro-inflammatory cytokines) with subsequent stimulation of CD8+ T cell activation and proliferation. We now demonstrate T cells genetically modified to constitutively express CD40L undergo enhanced proliferation and up-regulated secretion of pro-inflammatory cytokines including GM-CSF and INF-g. Furthermore, T cells modified to constitutively express CD40L, upon co-culture, will alter the phenotype of CD40+ B cell tumor cell lines by enhancing the expression co-stimulatory molecules (CD80/CD86), adhesion molecules (CD54/CD58/CD70) and death receptors (CD95; Fas). These findings were similarly evident in primary patient tumor samples (e.g. CLL cells) when co-cultured with autologous T cells modified to constitutively express CD40L. We further demonstrate maturation of monocyte derived DCs with subsequent secretion of IL-12 following co-culture with autologous T cells modified to constitutively express CD40L. T cells transduced with the bi-cistronic 19–28z/CD40L vector showed enhanced in vitro cytotoxicity against a panel of CD19+ tumor cell lines. Furthermore, infusion of 19–28z/CD40L modified T cells enhances the survival of CD19+ tumor bearing immunodeficient mice (SCID/Beige) when compared to mice treated with T cells modified to express the anti-CD19 19–28z CAR alone. We conclude that further genetic modification of CAR targeted T cells to constitutively express the co-stimulatory CD40L may enhance the anti-tumor efficacy of this adoptive T cell therapy. Our data suggests this enhanced T cell efficacy may be due to both autocrine and paracrine mediated mechanisms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Development of a Novel MICA/B-Specific CAR As a Pan-Tumor Targeting Strategy for Off-the-Shelf, Cell-Based Cancer Immunotherapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Ryan Bjordahl ◽  
John Goulding ◽  
Mochtar Pribadi ◽  
Robert Blum ◽  
Chiawei Chang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Nk Cell ◽  
Tumor Cell Lines ◽  
Publicly Traded ◽  
Cell Immunotherapy

Surface expression of the HLA-I related molecules MICA and MICB (MICA/B) in response to oncogenic and cellular stress acts as a natural anti-cancer immunosurveillance mechanism. The recognition of MICA/B by the activating immunoreceptor NKG2D, which is expressed by natural killer (NK) and T cell subsets, is responsible for the removal of many transformed and virally infected cells. However, tumors frequently evade NKG2D-mediated immunosurveillance by proteolytic shedding of MICA/B, which can inhibit NKG2D function and promote tumor immune escape. Recently, we demonstrated that monoclonal antibodies targeting the conserved, membrane-proximal α3 domain of MICA/B can prevent MICA/B shedding and enhance NK cell anti-tumor efficacy. With the goal of leveraging the ubiquity of MICA/B expression on malignant cells, we have developed a novel chimeric antigen receptor targeting the α3 domain of MICA/B (CAR-MICA/B) and are currently evaluating application of CAR-MICA/B in an off-the-shelf NK cell immunotherapy platform for both solid and hematopoietic tumor indications. Optimization of CAR-MICA/B design was performed by primary T cell transduction using a matrix of CAR spacers and ScFv heavy and light chain orientations. Six candidate CAR-MICA/B designs were screened in vitro against a panel of tumor cell lines and in vivo against the Nalm6 leukemia cell line engineered to express MICA (Nalm6-MICA). All tested constructs demonstrated MICA-specific in vitro activation and cytotoxicity as well as in vivo tumor control (Figure 1A). Additional studies utilizing the optimal CAR-MICA/B configuration demonstrated MICA/B-specific reactivity against a panel of solid and hematopoietic tumor cell lines in vitro, including melanoma, renal cell carcinoma, and lung cancer lines (Figure 1B). Further, CAR-MICA/B T cells were superior to NKG2D-CAR T cells in clearing A2058 melanoma cells in an in vivo xenograft metastasis model (Figure 1C). Although MICA/B expression has primarily been studied in the context of solid tumors, moderate MICA/B mRNA expression was identified in a number of hematopoietic tumor cell lines, including acute myeloid leukemia (AML) and multiple myeloma (MM) lines. Following the confirmation of surface MICA/B protein expression on a selection of MM and AML cell lines, we utilized MICA/B CAR primary T cells to further demonstrate MICA/B-specific activation and cytotoxicity and to confirm CAR-MICA/B targeting of hematological malignancies (Figure 1D). To further advance CAR-MICA/B development, we introduced the CAR-MICA/B construct into an induced pluripotent stem cell (iPSC) line designed for production of off-the-shelf natural killer (NK) cell immunotherapies. Using a panel of tumor cell lines expressing MICA/B, CAR-MICA/B iPSC-derived NK (iNK) cells displayed specific MICA reactivity, resulting in enhanced cytokine production, degranulation, and CAR-mediated cytotoxicity compared to CAR-negative iNK control cells (Figure 1E). In addition to MICA/B-specific cytotoxicity mediated by CAR, iNK cells also mediated innate cytotoxicity against cancer cells through endogenous NKG2D and other NK cell activating receptors, highlighting the multifaceted targeting capacity of CAR iNK cells. In order to isolate CAR-directed cytotoxicity from the iNK cells' innate anti-tumor capacity, an in vivo proof of concept study was performed using mouse B16-F10 melanoma cells engineered to express human MICA. In this model, iNK expressing CAR-MICA/B significantly reduced B16-F10-MICA liver and lung metastases from CAR-MICA/B iNK cells compared to CAR negative control cells, with reductions of the number of metastases by 87% in the lung (p<0.0001) and 93% in the liver (p<0.006) for CAR-MICA/B iNK cells vs non-CAR controls (Figure 1F). Additionally, CAR-MICA/B iNK cells were effective at controlling Nalm6-MICA progression in a disseminated leukemia model, suggesting potential application against both hematopoietic and solid tumors. Ongoing work is focused on extending these studies into disease-specific models of endogenous MICA/B expression to further advance CAR-MICA/B iNK cells in both solid and hematologic cancers. In summary, these preclinical data support the development and translation of an off-the-shelf NK cell immunotherapy targeting the conserved α3 domain of MICA/B with potential therapeutic application to multiple hematopoietic and solid tumor types. Figure 1 Disclosures Bjordahl: Fate Therapeutics: Current Employment. Goulding:Fate Therapeutics: Current Employment. Blum:Fate Therapeutics: Current Employment. Chang:Fate Therapeutics: Current Employment. Wucherpfennig:Fate Therapeutics: Research Funding. Chu:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company; Roche Holding AG: Current equity holder in publicly-traded company. Chu:Fate Therapeutics, Inc: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Liu:Fate Therapeutics: Current Employment. Sikaroodi:Fate Therapeutics: Current Employment. Fong:Fate Therapeutics: Current Employment. Huffman:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

scholarly journals T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies

Blood ◽  
2011 ◽  
Vol 117 (16) ◽  
pp. 4304-4314 ◽  
Author(s):  
Donald R. Shaffer ◽  
Barbara Savoldo ◽  
Zhongzhen Yi ◽  
Kevin K. H. Chow ◽  
Sunitha Kakarla ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
B Cell ◽  
Hematologic Malignancies ◽  
T Cell Therapy ◽  
Positive Tumor Cell ◽  
Cell Therapies ◽  
Tumor Cell Killing ◽  
Cd27 Costimulation

AbstractT-cell therapy with genetically modified T cells targeting CD19 or CD20 holds promise for the immunotherapy of hematologic malignancies. These targets, however, are only present on B cell–derived malignancies, and because they are broadly expressed in the hematopoietic system, their targeting may have unwanted consequences. To expand T-cell therapies to hematologic malignancies that are not B cell–derived, we determined whether T cells can be redirected to CD70, an antigen expressed by limited subsets of normal lymphocytes and dendritic cells, but aberrantly expressed by a broad range of hematologic malignancies and some solid tumors. To generate CD70-specific T cells, we constructed a chimeric antigen receptor (CAR) consisting of the CD70 receptor (CD27) fused to the CD3-ζ chain. Stimulation of T cells expressing CD70-specific CARs resulted in CD27 costimulation and recognition of CD70-positive tumor cell lines and primary tumor cells, as shown by IFN-γ and IL-2 secretion and by tumor cell killing. Adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts. Therefore, CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies.

scholarly journals Expression of MyD88/CD40 Drives In Vivo Activation and Proliferation of Chimeric Antigen Receptor-Modified T Cells That Can be Effectively Regulated By Inducible Caspase-9

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 851-851
Author(s):  
Aaron Foster ◽  
Peter Chang ◽  
Pei-Yi Lin ◽  
Jeannette Crisostomo ◽  
Aruna Mahendravada ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Cell Lines ◽  
Tumor Control ◽  
Tumor Cell Lines ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Introduction: Efficacy of chimeric antigen receptor (CAR)-modified T cells is dependent on their in vivo survival and expansion following infusion. The addition of accessory molecules (e.g., costimulatory and cytokine genes) may improve CAR-T proliferation and potency, but may also increase toxicity of these next generation CAR-T cell therapies, suggesting that the incorporation of a built in "safety switch" would balance safety and efficacy in a single, controllable therapy. Here, we demonstrate that cytosolic coexpression of a MyD88/CD40-derived fusion protein dramatically enhances CAR-T activation, cytokine production, and proliferation in vivo, resulting in improved antitumor efficacy. Importantly, CAR-T cell numbers, elevated cytokine levels, and observed CAR-T-related toxicity could be controlled by titratable rimiducid administration to reduce or eliminate CAR-T cells by activating the inducible caspase-9 (iC9) suicide gene. Methods: Human T cells were activated with anti-CD3/CD28 and transduced with retrovirus encoding, iC9, a first generation CAR (with CD3ζ) targeting CD19, Her2 or PSCA, and a detached, fusion protein comprising signaling domains from MyD88 and CD40 (MC). For comparison, additional CARs were constructed without MC, with MyD88 or CD40 elements only, or with conventional CARs coexpressing CD28 within the CAR molecule (CAR.28.ζ). Transduced T cells were assessed in vitro for cytotoxicity, cytokine production and proliferation against tumor cell lines (CD19+: Daudi, Raji; Her2+: SK-BR-3; PSCA+: Capan-1, HPAC). In vivo antitumor efficacy of CAR-modified T cells was assessed using immunodeficient NSG mice engrafted with antigen-matched tumor cell lines (5x105 Raji, i.v.; 1x106 SK-BR-3, s.c; 2x106 HPAC, s.c.) followed by i.t. or i.v. injection of variable doses of T cells. Reduction or elimination of CAR-T cells was performed by i.p. injection of rimiducid (0 - 5 mg/kg). Tumor cell lines expressing luciferase or T cells co-transduced with luciferase-encoding vectors were used for bioluminescence imaging (BLI) to measure tumor growth or T cell expansion/elimination, respectively. Serum cytokine levels were assessed by blood draws and CAR-T cell frequency was measured by flow cytometry. Results: All CAR constructs were stably expressed in T cells (30-90%). CAR vectors coexpressing MC induced high IL-2 levels in vitro when exposed to target antigen+ tumor cells (CD19 = 4246 ± 52, Her2 = 2613 ± 1298, and PSCA = 3263 ± 1393 pg/ml per 1x105 T cells over 48 hrs) and corresponded to improved CAR-T cell proliferation and tumor elimination compared to control vectors. In NSG mice, MC costimulation resulted in >2,000-fold expansion of CD19-targeted CAR-T cells and complete tumor control for >100 days in 100% of mice engrafted with CD19+ Raji cells (p = 0.0002) following injection of 5x106 CAR-T cells, followed on day 7 with a single i.p. dose of rimiducid (5 mg/kg) to control toxicity. MC-enabled CAR-T cells were eliminated or partially reduced by rimiducid titrations, which corresponded to decreased cytokine (IL-6, IFN-γ, TNF-α) levels and restoration of health in animals showing signs of toxicity (e.g., ≥15% weight loss). For solid tumors, Her2-targeted, MC-enabled CAR-T cells showed a 150-fold in vivo expansion and compared favorably to first (Her2.ζ; p = 0.01) and second generation (Her2.28.ζ; p = 0.01) CARs, causing 100% elimination of SK-BR-3 tumors and enhanced survival for >60 days following i.t. injection (p = 0.0015). PSCA-targeted CARs expressing MC also drove complete and durable (>42 days) elimination of large (200 mm3) HPAC tumors in 100% of mice, after a single i.v. injection of 1x107 CAR-T cells followed on day 14 with a single 5 mg/kg i.p. rimiducid dose to reverse toxicity. Summary: Coexpression of MC, and the cell therapy safety switch "CaspaCIDe", in combination with a first generation CAR, together comprising the novel "CIDeCAR" platform technology, dramatically increases efficacy against a number of tumor targets by enhancing T cell engraftment and proliferation following infusion, while incorporating an effective, built-in safety mechanism. In three distinct tumor models, rimiducid administration promptly eliminated signs and symptoms of CAR toxicity without subsequent loss of tumor control. CIDeCAR technology may allow the development of safer and more effective CAR-T cell therapies for a range of difficult-to-treat liquid and solid tumors. Disclosures Foster: Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Lin:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Khalil:Bellicum Pharmaceuticals: Employment. Saha:Bellicum Pharmaceuticals: Employment. Shaw:Bellicum Pharmaceuticals: Employment. Morschl:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.

scholarly journals Heat Shock Protein Inhibitor 17-Allyamino-17-Demethoxygeldanamycin, a Potent Inductor of Apoptosis in Human Glioma Tumor Cell Lines, Is a Weak Substrate for ABCB1 and ABCG2 Transporters

2021 ◽  
Vol 14 (2) ◽  
pp. 107
Author(s):  
Nikola Pastvova ◽  
Petr Dolezel ◽  
Petr Mlejnek
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Tumor Cell ◽  
Cell Lines ◽  
Single Agent ◽  
Heat Shock Protein 90 ◽  
Genetic Alterations ◽  
Tumor Cell Lines ◽  
Cytotoxic Effects ◽  
Glioma Tumor

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and has a poor prognosis. Complex genetic alterations and the protective effect of the blood–brain barrier (BBB) have so far hampered effective treatment. Here, we investigated the cytotoxic effects of heat shock protein 90 (HSP90) inhibitors, geldanamycin (GDN) and 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), in a panel of glioma tumor cell lines with various genetic alterations. We also assessed the ability of the main drug transporters, ABCB1 and ABCG2, to efflux GDN and 17-AAG. We found that GDN and 17-AAG induced extensive cell death with the morphological and biochemical hallmarks of apoptosis in all studied glioma cell lines at sub-micro-molar and nanomolar concentrations. Moderate efflux efficacy of GDN and 17-AAG mediated by ABCB1 was observed. There was an insignificant and low efflux efficacy of GDN and 17-AAG mediated by ABCG2. Conclusion: GDN and 17-AAG, in particular, exhibited strong proapoptotic effects in glioma tumor cell lines irrespective of genetic alterations. GDN and 17-AAG appeared to be weak substrates of ABCB1 and ABCG2. Therefore, the BBB would compromise their cytotoxic effects only partially. We hypothesize that GBM patients may benefit from 17-AAG either as a single agent or in combination with other drugs.

scholarly journals Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes.

1993 ◽  
Vol 121 (5) ◽  
pp. 1141-1152 ◽  
Author(s):  
E A Wayner ◽  
S G Gil ◽  
G F Murphy ◽  
M S Wilke ◽  
W G Carter
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Basement Membrane ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Lymphokine Activated Killer Cells ◽  
Cutaneous Inflammation ◽  
Epidermal Basement Membrane

The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up-regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL, the infiltrating T cells could be stained with mAbs to alpha 3 or beta 1 and were localized in close proximity to the epiligrin-containing basement membrane. Infiltrating lymphocytes in malignant cutaneous B disease (CBCL) did not express alpha 3 beta 1 by immunohistochemical techniques and did not associate with the epidermal basement membrane. The present findings clearly define a function for alpha 3 beta 1 in T cells and strongly suggest that alpha 3 beta 1 interaction with epiligrin may be involved in the pathogenesis of cutaneous inflammation.

scholarly journals 221 CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid but not liquid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A234-A234
Author(s):  
Rebecca Larson ◽  
Michael Kann ◽  
Stefanie Bailey ◽  
Nicholas Haradhvala ◽  
Kai Stewart ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).

scholarly journals A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Jie Wang ◽  
Katarzyna Urbanska ◽  
Prannda Sharma ◽  
Mathilde Poussin ◽  
Reza Nejati ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Receptor ◽  
Brentuximab Vedotin ◽  
T Cell Lymphomas ◽  
T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

scholarly journals CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lena Andersch ◽  
Josefine Radke ◽  
Anika Klaus ◽  
Silke Schwiebert ◽  
Annika Winkler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Lines ◽  
T Cell Therapy ◽  
Retinoblastoma Cell ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

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