scholarly journals P09.05 Plasma CD27, a surrogate of intratumoral CD27-CD70 interaction, correlates with immunotherapy resistance in renal cancer

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A30.1-A30
Author(s):  
N Benhamouda ◽  
I Sam ◽  
N Epaillard ◽  
A Gey ◽  
A Saldmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Principal Investigator ◽  
Cell Phenotype ◽  
Metastatic Rcc ◽  
Research Grant

BackgroundCD70, a costimulatory molecule on antigen presenting cells, is known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). However, persistent interaction of CD27 and CD70 such as in chronic infection may exhaust the T cell pool and promote apoptosis. Surprisingly, our analysis based on TCGA database show that clear cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors. Despite the important clinical efficacy of immunotherapy by anti-PD-1 in RCC patients, the overall response to anti-PD1 remains modest. The relationship between the CD27-CD70 interaction in the RCC and the response to immunotherapy is still unclear.Materials and MethodsTo study the CD27 and CD70 expression in the tumor microenvironment (TME), FFPE tumor tissues from 25 RCC patients were analysed using multiplex in situ immunofluorescence. 10 fresh RCC tumor samples were collected to analyse the phenotype of CD27+ T cells by flow cytometry and 4 samples were proceeded for single-cell RNA-seq analysis. A cohort of metastatic RCC patients (n = 35) treated by anti-PD-1 were enrolled for the measurement of plasma sCD27 by ELISA and the survival analysis is also realized.ResultsIn the TME, we demonstrated that CD27+ T cells interact with CD70-expressing tumor cells. In fresh tumors from RCC patients, CD27+ T cells express higher levels of cleaved caspase 3 (a classical marker of apoptosis) than CD27- T cells. We confirmed the apoptotic signature (BAX, FASLG, BCL2L11, CYCS, FBXO32, LGALS1, PIK3R1, TERF1, TXNIP, CDKN2A) of CD27+ T cells by single-cell RNAseq analysis. CD27+T cells also had a tissue resident memory T cell phenotype with enriched gene expression of ITGAE, PRDM1, RBPJ and ZNF683. Moreover, CD27+T cells display an exhaustion phenotype with the expression of multiple inhibitory receptors gene signature (PDCD1, CTLA4, HAVCR2, LAG3, etc). Besides, intratumoral CD27-CD70 interaction significantly correlates with plasma sCD27 concentration in RCC (p = 0.0017). In metastatic RCC patients treated with anti-PD-1, higher levels of sCD27 predict poor overall survival (p = 0.037), while it did not correlate with inflammatory markers or clinical prognostic criteria.ConclusionsIn conclusion, we demonstrated that sCD27, a surrogate of T cell dysfunction in tumors likely induced by persistent interactions of CD27+T cells and CD70-expressing tumor cells, is a predictive biomarker of resistance to immunotherapy in mRCC. To our knowledge, this is the first report showing that a peripheral blood biomarker may reflect certain aspects of the tumor-host interaction in the tumor microenvironment. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be further extended to other types of tumors. CD70-CD27 interaction could thus be considered as a mechanism of tumor escape, but also a novel therapeutic target in cancers.Disclosure InformationN. Benhamouda: None. I. Sam: None. N. Epaillard: None. A. Gey: None. A. Saldmann: None. J. Pineau: None. M. Hasan: None. V. Verkarre: None. V. Libri: None. S. Mella: None. C. Granier: None. C. Broudin: None. P. Ravel: None. B. Jabla: None. N. Chaput: None. L. Albiges: None. Y. Vano: None. O. Adotevi: None. S. Oudard: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; SIRIC CARPEM, FONCER. E. Tartour: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Fondation ARC, INCA PLBio, Labex Immuno-Oncology, SIRIC CARPEM, FONCER, IDEX université de Paris, Inserm Transfert.

scholarly journals P01.08 Beyond PD-1: characterization of new checkpoints restricting function of cytotoxic lymphocytes infiltrating human carcinoma

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A11.2-A12
Author(s):  
AS Herbstritt ◽  
PU Prinz ◽  
M Maxwell ◽  
M Kadiyala ◽  
D Yan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Nk Cells ◽  
Solid Tumors ◽  
Principal Investigator ◽  
Tumor Control ◽  
Research Support ◽  
Part Time ◽  
Research Grant

BackgroundT and NK cells from human renal cell carcinoma (RCC) are functionally non-responsive. Analysis of the TCR signaling cascade required for effector function identified that proximal signaling molecules were activated whereas activation of downstream ERK was blocked. Further investigation showed increased diacylglycerol kinase alpha (DGK-α) levels in T and NK cells from the RCC tumor microenvironment (TME). These cells were refractory to stimulation showing no degranulation or IFN-γ production. Using a small molecule DGK–α inhibitor (R59022), the function of tumor-infiltrating lymphocytes was restored ex vivo. A correlation of high DGK-α and loss of function was also observed in an experimental mouse model of adoptive therapy where CAR T cells that had lost their activity after infiltrating into solid tumors were found to have increased DGK-α.1 Blockade of the Programmed cell death protein 1 (PD-1) with monoclonal antibodies is used in the clinic enabling some patients to achieve tumor control. However, not all patients respond. DGK-α activity is positioned downstream of PD-1 and should, if overactive, curb T cell function even if PD-1 inhibition is released. Thus, we hypothesize that dual inhibition of PD-1 and DGK–α might be required to fully unleash the T cell’s potential in the TME. Current DGK-α inhibitors are not suitable for clinical application. Therefore, we investigated alternative means using an RNA interference (RNAi) approach to target DGK-α alone as well as in combination with PD-1 in T and NK cells.Material and MethodsKnockdown is performed by RNAi using INTASYLTM compounds developed by Phio Pharmaceuticals. INTASYLTM compounds incorporate drug-like properties into the siRNA, resulting in enhanced uptake in the presence of serum with no need for further transfection reagents. Knockdown is analyzed by RT-qPCR and flow cytometry. Functional assays include cytotoxicity, degranulation and cytokine production in tumor mimicking environments.ResultsA tumor mimicking in vitro system was developed which allows for the demonstration of functional restoration or prevention of functional loss of cell activity. Using T cell/tumor cell co–cultures at high tumor cell density, functional suppression could be induced in T and NK cells comparable to those observed in the TME. Testing of DGK-α targeting INTASYLTM compounds, silencing of DGK-α was observed in human U2OS osteosarcoma cells. Using a fluorescently labeled compound, highly efficient transfection of human primary immune cells was seen. Combinations of PD-1 and DGK-α targeting compounds are being tested and evaluated for synergism in experimental models.ConclusionsStrong activity of specific T and NK cells is necessary for tumor control. Dual targeting of PD-1 and DGK-α may be required to fully enable T and NK cell reactivity in the TME. Current DGK-α inhibitors do not exhibit the desirable pharmacokinetic/pharmacodynamic (PK/PD) properties for clinical development. The tested self-delivering RNAi technology represents a promising approach to targeting intracellular immune checkpoints such as DGK-α.ReferenceMoon EK, Wang L-C, Dolfi DV, Wilson CB, Ranganathan R, Sun J, et al. Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 2014; 20(16):4262–73Disclosure InformationA.S. Herbstritt: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Phio Pharmaceuticals. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals. P.U. Prinz: None. M. Maxwell: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. M. Kadiyala: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. D. Yan: A. Employment (full or part-time); Significant; Phio Pharmaceuticals. E. Noessner: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Phio Pharmaceuticals. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; Phio Pharmaceuticals.

scholarly journals Innovative CAR-T Cell Therapy for Solid Tumor; Current Duel between CAR-T Spear and Tumor Shield

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2087
Author(s):  
Yuna Jo ◽  
Laraib Amir Ali ◽  
Ju A Shim ◽  
Byung Ha Lee ◽  
Changwan Hong
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Cell Immunotherapy ◽  
Car T Cell Therapy ◽  
Car T ◽  
T Cell Immunotherapy

Novel engineered T cells containing chimeric antigen receptors (CAR-T cells) that combine the benefits of antigen recognition and T cell response have been developed, and their effect in the anti-tumor immunotherapy of patients with relapsed/refractory leukemia has been dramatic. Thus, CAR-T cell immunotherapy is rapidly emerging as a new therapy. However, it has limitations that prevent consistency in therapeutic effects in solid tumors, which accounts for over 90% of all cancer patients. Here, we review the literature regarding various obstacles to CAR-T cell immunotherapy for solid tumors, including those that cause CAR-T cell dysfunction in the immunosuppressive tumor microenvironment, such as reactive oxygen species, pH, O2, immunosuppressive cells, cytokines, and metabolites, as well as those that impair cell trafficking into the tumor microenvironment. Next-generation CAR-T cell therapy is currently undergoing clinical trials to overcome these challenges. Therefore, novel approaches to address the challenges faced by CAR-T cell immunotherapy in solid tumors are also discussed here.

scholarly journals Double Strike Approach for Tumor Attack: Engineering T Cells Using a CD40L:CD28 Chimeric Co-Stimulatory Switch Protein for Enhanced Tumor Targeting in Adoptive Cell Therapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Luis Felipe Olguín-Contreras ◽  
Anna N. Mendler ◽  
Grzegorz Popowicz ◽  
Bin Hu ◽  
Elfriede Noessner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Intracellular Signaling ◽  
Adoptive Cell Therapy ◽  
Tumor Rejection ◽  
Target Cells ◽  
Type I

Activation of co-stimulatory pathways in cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) have proven to boost effector activity, tumor rejection and long-term T cell persistence. When using antigen-specific T cell receptors (TCR) instead of CARs, the lack of co-stimulatory signals hampers robust antitumoral response, hence limiting clinical efficacy. In solid tumors, tumor stroma poses an additional hurdle through hindrance of infiltration and active inhibition. Our project aimed at generating chimeric co-stimulatory switch proteins (CSP) consisting of intracellular co-stimulatory domains (ICD) fused to extracellular protein domains (ECD) for which ligands are expressed in solid tumors. The ECD of CD40L was selected for combination with the ICD from the CD28 protein. With this approach, it was expected to not only provide co-stimulation and strengthen the TCR signaling, but also, through the CD40L ECD, facilitate the activation of tumor-resident antigen-presenting cells (APCs), modulate activation of tumor endothelium and induce TCR-MHC independent apoptotic effect on tumor cells. Since CD28 and CD40L belong to different classes of transmembrane proteins (type I and type II, respectively), creating a chimeric protein presented a structural and functional challenge. We present solutions to this challenge describing different CSP formats that were successfully expressed in human T cells along with an antigen-specific TCR. The level of surface expression of the CSPs depended on their distinct design and the state of T cell activation. In particular, CSPs were upregulated by TCR stimulation and downregulated following interaction with CD40 on target cells. Ligation of the CSP in the context of TCR-stimulation modulated intracellular signaling cascades and led to improved TCR-induced cytokine secretion and cytotoxicity. Moreover, the CD40L ECD exhibited activity as evidenced by effective maturation and activation of B cells and DCs. CD40L:CD28 CSPs are a new type of switch proteins designed to exert dual beneficial antitumor effect by acting directly on the gene-modified T cells and simultaneously on tumor cells and tumor-supporting cells of the TME. The observed effects suggest that they constitute a promising tool to be included in the engineering process of T cells to endow them with complementary features for improved performance in the tumor milieu.

scholarly journals Galectin-9 expression defines exhausted T cells and impaired cytotoxic NK cells in patients with virus-associated solid tumors

2020 ◽  
Vol 8 (2) ◽  
pp. e001849
Author(s):  
Isobel Okoye ◽  
Lai Xu ◽  
Melika Motamedi ◽  
Pallavi Parashar ◽  
John W Walker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Solid Tumors ◽  
Mononuclear Cells ◽  
Cell Phenotype ◽  
Peripheral Blood Mononuclear ◽  
Effector Functions ◽  
Cell Functions ◽  
Cell Effector

BackgroundWe have previously reported that the upregulation of galectin-9 (Gal-9) on CD4+ and CD8+ T cells in HIV patients was associated with impaired T cell effector functions. Gal-9 is a ligand for T cell immunoglobulin and mucin domain-3, and its expression on T cells in cancer has not been investigated. Therefore, we aimed to investigate the expression level and effects of Gal-9 on T cell functions in patients with virus-associated solid tumors (VASTs).Methods40 patients with VASTs through a non-randomized and biomarker-driven phase II LATENT trial were investigated. Peripheral blood mononuclear cells and tumor biopsies were obtained and subjected to immunophenotyping. In this trial, the effects of oral valproate and avelumab (anti-PD-L1) was investigated in regards to the expression of Gal-9 on T cells.ResultsWe report the upregulation of Gal-9 expression by peripheral and tumor-infiltrating CD4+ and CD8+ T lymphocytes in patients with VASTs. Our results indicate that Gal-9 expression is associated with dysfunctional T cell effector functions in the periphery and tumor microenvironment (TME). Coexpression of Gal-9 with PD-1 or T cell immunoglobulin and ITIM domain (TIGIT) exhibited a synergistic inhibitory effect and enhanced an exhausted T cell phenotype. Besides, responding patients to treatment had lower Gal-9 mRNA expression in the TME. Translocation of Gal-9 from the cytosol to the cell membrane of T cells following stimulation suggests persistent T cell receptor (TCR) stimulation as a potential contributing factor in Gal-9 upregulation in patients with VASTs. Moreover, partial colocalization of Gal-9 with CD3 on T cells likely impacts the initiation of signal transduction via TCR as shown by the upregulation of ZAP70 in Gal-9+ T cells. Also, we found an expansion of Gal-9+ but not TIGIT+ NK cells in patients with VASTs; however, dichotomous to TIGIT+ NK cells, Gal-9+ NK cells exhibited impaired cytotoxic molecules but higher Interferon gamma (IFN-γ) expression.ConclusionOur data indicate that higher Gal-9-expressing CD8+ T cells were associated with poor prognosis following immunotherapy with anti-Programmed death-ligand 1 (PD-L1) (avelumab) in our patients’ cohort. Therefore, for the very first time to our knowledge, we report Gal-9 as a novel marker of T cell exhaustion and the potential target of immunotherapy in patients with VASTs.

scholarly journals New Generation Chimeric Antigen Receptor T-Cell Therapy (CoupledCAR) Induces High Rate Remissions in Solid Tumor

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4631-4631
Author(s):  
Lei Xiao
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Thyroid Cancer ◽  
T Cell ◽  
Cell Therapy ◽  
Solid Tumors ◽  
Tumor Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

New Generation Chimeric Antigen Receptor T-Cell Therapy ( CoupledCAR ) Induces High Rate Remissions in Solid Tumor Yu Liu1,Song Li2,Youli Luo3,Haixia Song4,Chengfei Pu5, Zhiyuan Cao 5, Cheng Lu5,Yang Hang5,Xi Huang5,Xiaogang Shen5 ,Xiaojun Hu3 , Renbin Liu1,Xiuwen Wang2,Junjie Mao3,Shihong Wei4 ,Zhao Wu5and Lei Xiao5* 1.The Third Affiliated Hospital, SUN YAT-SEN University 2.Qilu Hospital of Shandong University 3.The Fifth Affiliated Hospital, SUN YAT-SEN University 4.Gansu Procincial Cancer Hospital 5.Innovative Cellular Therapeutics *Corresponding to: Lei Xiao, [email protected] Chimeric antigen receptor (CAR) T cell therapy made significant progress for treating blood cancer such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges, such as physical barrier, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and cell expansion in vivo for treatment of solid tumors Conventional CAR T cell therapy showed weak CAR T expansion in patients and thus achieved no or little response for treating solid tumors. Here, we generated "CoupledCAR" T cells including an anti-TSHR CAR molecule. Compared with conventional CART cells,these "CoupledCAR" T cells successfully improved the expansion of CART cells more than 100 times and enhanced CAR T cells' migration ability, allowing the CAR T cells to resist and infiltrate the tumor microenvironment and killed tumor cells. To verify the effect of "CoupledCAR" T cells on solid tumors, we have completed several clinical trials for different solid tumors, including two patients with thyroid cancer. Immunohistochemistry (IHC) results showed that thyroid stimulating hormone receptors (TSHR) were highly expressed in thyroid cancer cells. In vitro co-culture experiments showed that TSHR CAR T cells specifically recognized and killed TSHR-positive tumor cells. Animal experiments showed that TSHR CAR T cells inhibited the proliferation of TSHR-positive tumor cells. Therefore, we designed "CoupledCAR" T cells expressing a binding domain against TSHR. Further,we did clinical trials of two group patients that were successfully treated using conventional TSHR CAR T cells and the "CoupledCAR" T cells, respectively. In the first group using conventional TSHR CAR T cells, patients showed weak cell expansion and less migration ability. In the group using TSHR "CoupledCAR" T cells, patients showed rapid expansion of CAR T cells and killing of tumor cells. One month after infusion (M1), the patient was evaluated as PR(Partial Response): the lymph node metastasis disappeared, and thoracic paratracheal tumors decreased significantly. Three months after infusion (M3), the patient was evaluated as a durable response, and the tumor tissue was substantially smaller than M1. Further, two patients with colonrectal cancer were enrolled in this trial and infused "CoupledCAR" T cells. One patient achieved PR and the other one achieved SD (Stable Disease). Therefore, "CoupledCAR" T cells can effectively promote expansion, migration and killing ability of CAR T cells in patients with thyroid cancer. "CoupledCAR" T cell technology is a technological platform, which may be used to treat other cancer types. Next, we are recruiting more patients with solid tumors in clinical trials using "CoupledCAR" T cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals The tumor vasculature an attractive CAR T cell target in solid tumors

Angiogenesis ◽  
2019 ◽  
Vol 22 (4) ◽  
pp. 473-475 ◽  
Author(s):  
Parvin Akbari ◽  
Elisabeth J. M. Huijbers ◽  
Maria Themeli ◽  
Arjan W. Griffioen ◽  
Judy R. van Beijnum
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Solid Tumors ◽  
Solid Tumor ◽  
Hematological Malignancies ◽  
Tumor Vasculature ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract T cells armed with a chimeric antigen receptor, CAR T cells, have shown extraordinary activity against certain B lymphocyte malignancies, when targeted towards the CD19 B cell surface marker. These results have led to the regulatory approval of two CAR T cell approaches. Translation of this result to the solid tumor setting has been problematic until now. A number of differences between liquid and solid tumors are likely to cause this discrepancy. The main ones of these are undoubtedly the uncomplicated availability of the target cell within the blood compartment and the abundant expression of the target molecule on the cancerous cells in the case of hematological malignancies. Targets expressed by solid tumor cells are hard to engage due to the non-adhesive and abnormal vasculature, while conditions in the tumor microenvironment can be extremely immunosuppressive. Targets in the tumor vasculature are readily reachable by CAR T cells and reside outside the immunosuppressive tumor microenvironment. It is therefore hypothesized that targeting CAR T cells towards the tumor vasculature of solid tumors may share the excellent effects of CAR T cell therapy with that against hematological malignancies. A few reports have shown promising results. Suggestions are provided for further improvement.

TAMI-27. METABOLIC MANIPULATION OF T CELLS TO TREAT BRAIN TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi203-vi204
Author(s):  
Guimei Tian ◽  
Linchun Jin ◽  
Devshri Doshi ◽  
Aida Karachi ◽  
Mariana Dajac ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Brain Tumors ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Disease Recurrence

Abstract INTRODUCTION Glioblastoma are a challenge for neuro-oncologists and current therapies are minimally effective. Standard-of- care treatment is almost inevitably followed by disease recurrence. Adoptive T cell transfer has emerged as a viable therapeutic for brain malignancies. While promising, the efficacy of this approach is often limited by a complex immunosuppressive tumor microenvironment. These complexities mean that more sophisticated T cell products are required. OBJECTIVES The brain tumor microenvironment provides local restraints via metabolic competition suppressing antitumor immunity, specifically inhibiting infiltration and tumoricidal functions of host and adoptively transferred tumor-reactive T cells. The overall goal of this project is to test new treatments to reverse immune dysfunction in cancer through the regulation of T cell metabolic signaling. We propose that modulating glucose pathway in T cells can potentiate their anti-tumor activity once adoptively transferred. METHODS T cells glucose metabolic pathway was modulated via glucose transporters overexpression. The functionality of metabolically modified T cells was investigated in murine and human models. RESULTS We demonstrated the existence of a competition for glucose between T cells and tumor cells, with tumor cells imposing glucose restriction mediating T cell hyporesponsiveness. Overexpression of glucose transporters such as Glut1 and Glut3 increased T cell glucose utilization and provide survival/growth advantage and enhanced T cell activation in glucose-restricted conditions. We also established that glucose transporter overexpression improves intratumoral infiltration of adoptively transferred T cells. CONCLUSION This project integrates fundamental concepts of tumor and immune metabolism in the design of immunotherapy and confirms that immunometabolism represents a viable target for new cancer therapy to treat brain tumors.

Multiparametric Analysis of Intra-Tumoral T-Cells in Hodgkin's Lymphoma Using Mass Cytometry (CyTOF)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1438-1438
Author(s):  
Jose Villasboas Bisneto ◽  
Stephen M Ansell
Keyword(s):  
T Cells ◽  
Data Analysis ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Progression ◽  
Single Cell ◽  
Hodgkin Lymphoma ◽  
Cell Subset ◽  
Mass Cytometry ◽  
Mean Frequency

Abstract Classic Hodgkin lymphoma (cHL) is characterized by a rich non-malignant immune infiltrate. T-cells are key components of the antitumoral immune response and studies characterizing subsets in cHL have yielded conflicting results. Initial studies suggested a predominance of TH2-polarized CD4+ T-cells, thought to allow tumor progression due to exhaustion and hypofunctionality. More recent data contest these findings, supporting the theory of tumor progression through evasion from a TH1-rich infiltrate that is potentially functional. The role of tumor evasion in cHL has been highlighted by compelling early clinical data with the use of PD-1 blockade in patients with advanced disease. A similar trial in patients with non-Hodgkin lymphoma (NHL) yielded far more modest results. Intrinsic differences in T-cell subpopulations in the tumor microenvironment may correlate to response to immune checkpoint inhibitor therapy. CyTOF or mass cytometry is a platform able to evaluate more than 45 simultaneous parameters on a single-cell level using nonradioactive nonbiological isotopes tagged to monoclonal antibodies. Measurements are made based on mass spectrometry, avoiding the hurdles of interference and spectral overlap experienced with fluorochromes. This constitutes an ideal tool for the study of the tumor microenvironment given its ability to assess a large number of parameters and resolve differences in a heterogeneous population. We hypothesize that the phenotype of intratumoral lymphocytes in cHL identifies T-cells that can effectively eradicate malignant cells. To test this hypothesis, we compared the phenotype of intratumoral T-cells in cHL to that of NHL and nodular lymphocyte-predominant Hodgkin Lymphoma (nlpHL). Tonsil and hyperplastic lymph node (LN) tissues were used as normal controls. Single-cell suspensions created from tumor specimens were stained with a metal-tagged antibody panel containing 31 surface markers and acquired on CyTOF. Multiparametric data analysis was performed on Cytobank using spanning-tree progression analysis of density-normalized events (SPADE) and t-Distributed Stochastic Neighbor Embedding (viSNE) algorithms. Inferential statistical analyses were performed with JMP®, Version 10.0.0 (SAS Institute Inc., Cary, NC, 1989-2007) using two-tailed tests and a 95% confidence interval. Cell subsets are expressed as percentages of parent population (CD45+CD3+CD19-). A total of 10 samples were studied (4 cHL, 1 nlpHL, 3 NHL, 1 tonsil, 1 LN). The total T-cell population ranged from 30.52 to 67.05% in cHL and 15.36 to 47% in NHL compared to 4.02% and 24.58% in tonsil and LN respectively. The CD4+ T-cell subset ranged from 58.05 to 35.3% in cHL, 50.03 to 82.61% in NHL and corresponded to 82.74% and 87.07% in tonsil and LN respectively. SPADE analysis identified two areas of asymmetric frequency of events amongst samples (figure 1 and 2). The CD4+ Tnaive subset (CD4+CD45RA+CCR7+) ranged from 7.8 to 31.2% of total T-cells in cHL compared to 10.7% in nlpHL, 0.17 to 3.02% in NHL and 6.2 to 6.7% in controls. The pooled mean frequency of CD4+ Tnaive subset was significantly higher in HL (cHL + nlpHL) compared to NHL (14.3% vs. 1.55%; p<0.05; figure 3A). The regulatory T-cell subset (Treg; CD25+CCR4+) ranged from 0.49 to 1.84% of total T-cells in HL compared to 9.3 to 21.04% in NHL, and 4.45 to 8.28% in controls. The pooled mean frequency of the Treg subset was significantly smaller in HL compared to NHL (1.28% vs. 16.23%; p<0.05; figure 3B). Our data supports the use of mass cytometry as a platform to study the tumor microenvironment in B-cell lymphomas. Multiparametric data analysis revealed significant differences in the intratumoral T-cell population between HL and NHL samples, namely in the CD4+ Tnaive and Treg subsets. Further validation in a larger sample is underway and will include panels to evaluate intracellular cytokine production and cell signaling pathways. Correlation between specific intratumoral T-cell phenotypic signatures and clinical outcomes may identify prognostic and predictive characteristics and provide insight to mechanisms of resistance to immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals 788 A novel T- lymphocyte binding aptamer assembled into a bispecifc compound for the treatment of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A823-A823
Author(s):  
Irit Carmi Levy ◽  
Erez Lavi ◽  
Neta Zilony Hanin ◽  
Zohar Pode ◽  
Karin Mizrahi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Tumor Targeting ◽  
Specific Antigen ◽  
Therapeutic Agents ◽  
List Type ◽  
Bispecific T Cell Engager

BackgroundT-cell engagers are bispecific molecules directed against the CD3 complex on one end and a tumor specific antigen on the other end, allowing a physical link of T cell to a tumor cell, resulting in tumor killing and immune activation. Bispecific molecules harnessing and redirecting T-cells towards tumor cells are a promising therapeutic agents. Aptamers are single stranded oligonucleotides with binding and recognition propensities similar to those of antibodies. Aptamers have a number of advantages over bispecific antibodies including shorter generation time and low immunogenicity. Thus, aptamers capable of targeting T cells would have great potential for use as anti-cancer therapeuticsMethodsSystematic evolution of ligands by exponential enrichment (SELEX) methodology was employed in order to identify a novel CD3e binding aptamer. CD3 binding aptamer was subsequently linked into a bispecific T cell engager structure with a tumor-targeting aptameric arm. The tumor-targeting aptamer is developed by Aummune's proprietary tailored therapeutic platform.1 based on identifying functional aptamer sequences capable of specifically killing targeted tumor cells and sparing healthy tissue .Exemplary bispecific aptamers were tested for T cell stimulation by flow cytometry. In vivo antitumor activity was investigated in syngeneic and in xenograft tumor models.ResultsWe have successfully identified a novel CD3e –targeting aptamer with a Kd of 31nM. A bispecific T cell engager comprised of this aptamer and a tumor-targeting aptamer induced a potent stimulation of T cells in vitro, resulting in CD69 upregulation and IFNg secretion.Next, the CD3e targeting aptamer was hybridized to tumoricidal aptamers identified by Aummune's platform (VS12) to target either the human colon carcinoma HCT116 cells or (VS32) the murine triple negative breast cancer 4T1 cells. Both bispecific entities (CS6-VS12 and CS6-VS32) effectively lead to inhibition of tumor growth in vivo and increased survival in the corresponding models.ConclusionsOur data above provide a proof-of-concept for Aummune's Bispecific Aptamer efficacy and provide a framework for the clinical development of this novel tailored immune therapeutic agents. Indeed, we are currently in the process of developing a first-in-human clinical study in subjects with solid tumors.ReferenceMamet N, et al, Commun Biol 2020.

scholarly journals Combining De-Glycosylating Agents with CAR-T Cells for Targeting Solid Tumors and Reducing Toxicity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4544-4544
Author(s):  
Beatrice Greco ◽  
Katia Paolella ◽  
Barbara Camisa ◽  
Valeria Malacarne ◽  
Laura Falcone ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Combined Approach ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Glycosylated Proteins

Abstract Background: The adoptive transfer of CAR-T cells have shown impressive results against B-cell malignancies, but still limited efficacy against solid tumors. The discovery of the key factors regulating the activity of CAR-T cells is required to improve their antitumor potency and modulate toxicities. Since solid tumors display a wide range of glycosylation alterations, including increased N-glycan branching, we hypothesized that peptidic epitopes may be masked by glycans from CAR-T cell targeting, especially in richly glycosylated proteins. Results: To investigate if sugar chains may be sterically hulking for CAR-T cell targeting, we generated N-glycosylation-defective pancreatic tumor cell lines. This aim has been achieved by knocking-out the expression of the glycosyltransferase Mgat5, a key enzyme involved in the process of N-glycan branching, using the CRISPR-Cas9 technology. As model antigens for CAR targeting, we focused on CD44v6 and CEACAM-5 (CEA) since they are both heavily glycosylated proteins over-expressed on a wide variety of solid tumors, including pancreatic adenocarcinoma. Strikingly, the impairment of N-glycosylation resulted in a dramatic increase of tumor targeting by both CD44v6 (4-fold, p<0,001) and CEA CAR-T cells (10-fold, p<0,001). This effect associated with improved CAR-T cell activation, suggesting more proficient antigen engagement. To exploit this mechanism in order to increase the efficacy of CAR-T cells against solid tumors, we sought to block tumor N-glycosylation with the clinical-grade glucose/mannose analogue 2-Deoxy-D-glucose (2DG). Similarly to genetically induced glycosylation blockade, treatment with 2DG also sensitized tumor cells to recognition by CAR-T cells, significantly increasing their elimination (CD44v6: 3-fold, p<0,01; CEA: 13-fold, p<0,001). Notably, 2DG alone proved to be ineffective as mono-therapy, suggesting a synergistic effect with CAR-T cells. To get more insights on this mechanism, we took advantage of previous studies reporting that 2DG interference with N-glycosylation can be reverted by the addition of exogenous mannose. Of notice, mannose did revert the synergy between 2DG and CAR-T cells (p<0,05), implying that blockade of N-glycosylation rather than glycolysis is the crucial mechanism involved. These findings were further confirmed by using the N-glycosylation inhibitor tunicamycin (CD44v6: 2,5-fold; CEA: 5-fold, p<0,01) and by Western blot, looking at the presence of de-glycosylated proteins on tumor cell surface after 2DG treatment. Next, we challenged the combined approach in a pancreatic adenocarcinoma xenograft mouse model. Accordingly with in vitro data, mice receiving CAR-T cells highly benefited from 2DG administration (5-fold less tumor at 7d, p<0,05), which conversely was unable to mediate any antitumor effect alone. Interestingly, improved antitumor activity was accompanied by a decrease in the frequency of CAR-T cells expressing one or more exhaustion and senescence markers, such as TIM-3, LAG-3, PD-1 and CD57 (SPICE software analysis, p=0,0105). Finally, thanks to metabolic deregulation (Warburg effect), 2DG is expected to selectively accumulate in cancer cells compared to healthy tissues, supporting the safety of the combined approach. Accordingly, we observed that the same doses of 2DG able to enhance tumor cell recognition by CAR-T cells failed to increase the elimination of healthy cells, such as keratinocytes. Conclusions: Our results indicate that i) the glycosylation status of tumor cells regulates the efficacy of CAR-T cells, especially when targeting highly glycosylated antigens, and ii) combining CAR-T cells with the de-glycosylation agent 2DG, which preferentially accumulates in tumor masses, may pave the way for a successful immunotherapy against solid tumors. Disclosures Bonini: Intellia Therapeutics: Research Funding. Bondanza:Novartis: Employment.

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