scholarly journals Tumor Inflammation and Myeloid Derived Suppressor Cells Reduce the Efficacy of CD19 CAR T Cell Therapy in Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2885-2885 ◽  
Author(s):  
Michael D. Jain ◽  
Hua Zhao ◽  
Reginald Atkins ◽  
Meghan A Menges ◽  
Crystal R Pope ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
T Cell Therapy ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Introduction: Approximately 60% of Large B cell Lymphoma (LBCL) patients that receive CD19 CAR T cell therapy with axicabtagene ciloleucel (axi-cel) experience lymphoma progression (Locke et al. Lancet Oncol. 2019) and the likelihood of response to subsequent therapy is low (Spiegel, Dahiya et al. ASCO 2019). Target loss of CD19 is observed in less than a third of patients experiencing relapse. Alternative mechanisms of resistance to axi-cel are poorly understood. Lymphoma patients with elevated serum markers of systemic inflammation, such as ferritin and IL-6, have worse outcomes following axi-cel (Locke, Neelapu et al. Mol.Ther.2017; Faramand et al. ASH 2018). We hypothesized that suppressive monocytic myeloid derived suppressor cells (M-MDSCs), which are associated with worse chemotherapy outcomes in LBCL (Azzaoui et al. Blood 2016), and tumor driven inflammation may be present and responsible for decreased efficacy of axi-cel in LBCL. Methods: LBCL patients undergoing axi-cel treatment were enrolled onto prospective sample collection protocols. Patients were stratified for analysis into ongoing responders (complete response or partial response) or relapsed (progressive disease) after a minimum of 3 months follow-up (range 3 - 15 months). M-MDSCs, defined as a Lin-, CD11b+, CD33+, CD15-, CD14+, HLA-DRlow population, were sorted from leftover apheresis material after collection for axi-cel manufacture. M-MDSC ability to suppress proliferation of autologous T cells stimulated with CD3/CD28 coated beads was measured by 3H thymidine incorporation. Circulating peripheral blood M-MDSCs, quantified by % of live cells by flow cytometry, were measured at the time of apheresis and serially after axi-cel infusion until day 30. In vitro mouse experiments utilized a CD19-CD28 CAR and cytokine-induced bone marrow MDSCs (Thevenot et al. Immunity 2014). Cytokines were measured by ELISA and cytotoxicity against CD19 bearing cell lines used xCELLigence real-time cell analysis, as we have done previously (Li et al. JCI Insight 2018).Tumor biopsies were taken within 1 month prior to infusion of axi-cel. Limited gene expression profiling of tumor microenvironment (TME) genes used the Nanostring IO360 panel (770 genes). Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Results: First, we demonstrated that M-MDSCs sorted from patient apheresis material suppressed the proliferation of autologous T cells (n=6). We next enumerated M-MDSCs in the peripheral blood (n = 32). M-MDSC numbers initially decreased after lymphodepleting chemotherapy but recovered to baseline levels by day +10. The level of M-MDSCs following CAR T cell therapy strongly correlated with pre-CAR T baseline levels (R = 0.871, p <0.0001), suggesting that the number of M-MDSCs present during CAR T cell expansion is dependent on factors already present before therapy began. M-MDSC levels were significantly higher in patients who subsequently relapsed, both at baseline (p= 0.01) and after axi-cel (p=0.04), as compared to patients with durable response. Mouse MDSCs were able to suppress CAR T cell IFN-gamma excretion (p<0.0001) and cytotoxicity (p<0.0001) in vitro. To evaluate the role of the TME we interrogated limited set gene expression profiling on patient (n=27) pre-axi-cel tumor biopsies. By cell type scoring, the macrophage gene score was significantly higher in patients who relapsed after CAR T therapy (p <0.001). By differential gene expression and gene set enrichment, patients who relapsed had a significantly higher expression (p <0.01) of multiple genes indicative of chronic interferon (IFN) signaling including higher levels of OAS2, OAS3, IFI6 and IFIT1, as well as the IFN-stimulated macrophage gene SIGLEC-1/CD169. Conclusions: Systemic inflammatory myeloid cytokines, circulating M-MDSCs in the blood and chronic IFN in the TME all associate with LBCL relapse after axi-cel CAR T cell therapy. Our observations support that CAR T cells can be suppressed by baseline patient and tumor-related factors and strategies to overcome these factors should be targeted to improve patient outcomes. MDJ and HZ contributed equally. Disclosures Jain: Kite/Gilead: Consultancy. Bachmeier:Kite/Gilead: Speakers Bureau. Chavez:Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; AstraZeneca: Honoraria. Mullinax:Iovance: Research Funding. Davila:Celgene: Research Funding; GlaxoSmithKline: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; Atara: Research Funding; Bellicum: Consultancy; Adaptive: Consultancy; Anixa: Consultancy. Locke:Kite: Other: Scientific Advisor; Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy.

scholarly journals 124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A133-A133
Author(s):  
Cheng-Fu Kuo ◽  
Yi-Chiu Kuo ◽  
Miso Park ◽  
Zhen Tong ◽  
Brenda Aguilar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

scholarly journals Iomab with Adoptive Cellular Therapy (Iomab-ACT): A Pilot Study of 131-I Apamistamab Followed By CD19-Targeted CAR T-Cell Therapy for Patients with Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia or Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4810-4810
Author(s):  
Mark B. Geyer ◽  
Briana Cadzin ◽  
Elizabeth Halton ◽  
Peter Kane ◽  
Brigitte Senechal ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
Research Funding ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background: Autologous CD19-targeted chimeric antigen receptor-modified (CAR) T-cell therapy leads to complete responses (CR) in patients (pts) with (w/) relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL, &gt;80% CR rate) and diffuse large B-cell lymphoma (DLBCL, ~40-55% CR rate). However, following fludarabine/cyclophosphamide (Flu/Cy) conditioning and CAR T-cell therapy w/ a CD28 costimulatory domain (e.g. 19-28z CAR T-cells), rates of grade ≥3 ICANS and grade ≥3 cytokine release syndrome (CRS) in pts w/ R/R DLBCL and morphologic R/R B-ALL exceed 30%. CRS and ICANS are associated w/ considerable morbidity, including increased length of hospitalization, and may be fatal. Host monocytes appear to be the major reservoir of cytokines driving CRS and ICANS post-CAR T-cell therapy (Giavradis et al. and Norelli et al., Nature Medicine, 2018). Circulating monocytic myeloid-derived suppressor cells (MDSCs) may also blunt efficacy of 19-28z CAR T-cells in R/R DLBCL (Jain et al., Blood, 2021). The CD45-targeted antibody radioconjugate (ARC) 131-I apamistamab is being investigated at myeloablative doses as conditioning prior to hematopoietic cell transplantation in pts w/ R/R acute myeloid leukemia. However, even at low doses (4-20 mCi), transient lymphocyte and blast reduction are observed. Preclinical studies in C57BL/6 mice demonstrate low-dose anti CD45 radioimmunotherapy (100 microCi) transiently depletes &gt;90% lymphocytes, including CD4/CD8 T-cells, B-cells, NK cells, and T-regs, as well as splenocytes and MDSCs, w/ negligible effect on bone marrow (BM) hematopoietic stem cells (Dawicki et al., Oncotarget, 2020). We hypothesized a higher, yet nonmyeloablative dose of 131-I apamistamab may achieve more sustained, but reversible depletion of lymphocytes and other CD45 + immune cells, including monocytes thought to drive CRS/ICANS. We additionally hypothesized this approach (vs Flu/Cy) prior to CAR T-cell therapy would promote CAR T-cell expansion while reducing CSF levels of monocyte-derived cytokines (e.g. IL-1, IL-6, and IL-10), thus lowering the risk of severe ICANS (Fig 1A). Study design and methods: We are conducting a single-institution pilot study of 131-I apamistamab in lieu of Flu/Cy prior to 19-28z CAR T-cells in adults w/ R/R BALL or DLBCL (NCT04512716; Iomab-ACT); accrual is ongoing. Pts are eligible for leukapheresis if they are ≥18 years-old w/ R/R DLBCL (de novo or transformed) following ≥2 chemoimmunotherapy regimens w/ ≥1 FDG-avid measurable lesion or B-ALL following ≥1 line of multi-agent chemotherapy (R/R following induction/consolidation; prior 2 nd/3 rd gen TKI required for pts w/ Ph+ ALL) w/ ≥5% BM involvement and/or FDG-avid extramedullary disease, ECOG performance status 0-2, and w/ appropriate organ function. Active or prior CNS disease is not exclusionary. Pts previously treated w/ CD19-targeted CAR T-cell therapy are eligible as long as CD19 expression is retained. See Fig 1B/C: Post-leukapheresis, 19-28z CAR T-cells are manufactured as previously described (Park et al., NEJM, 2018). Bridging therapy is permitted at investigator discretion. Thyroid blocking is started ≥48h pre-ARC. 131-I apamistamab 75 mCi is administered 5-7 days pre-CAR T-cell infusion to achieve total absorbed marrow dose ~200 cGy w/ remaining absorbed dose &lt;25 cGy at time of T-cell infusion. 19-28z CAR T-cells are administered as a single infusion (1x10 6/kg, B-ALL pts; 2x10 6/kg, DLBCL pts). The primary objective is to determine safety/tolerability of 131-I apamistamab 75 mCi given prior to 19-28z CAR T-cells in pts w/ R/R B-ALL/DLBCL. Secondary objectives include determining incidence/severity of ICANS and CRS, anti-tumor efficacy, and 19-28z CAR T-cell expansion/persistence. Key exploratory objectives include describing the cellular microenvironment following ARC and 19-28z CAR T-cell infusion using spectral cytometry, as well as cytokine levels in peripheral blood and CRS. The trial utilizes a 3+3 design in a single cohort. If dose-limiting toxicity (severe infusion-related reactions, treatment-resistant severe CRS/ICANS, persistent regimen-related cytopenias, among others defined in protocol) is seen in 0-1 of the first 3 pts treated, then up to 6 total (up to 3 additional) pts will be treated. We have designed this study to provide preliminary data to support further investigation of CD45-targeted ARCs prior to adoptive cellular therapy. Figure 1 Figure 1. Disclosures Geyer: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals, Inc: Research Funding; Amgen: Research Funding. Geoghegan: Actinium Pharmaceuticals, Inc: Current Employment. Reddy: Actinium Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Berger: Actinium Pharmaceuticals, Inc: Current Employment. Ludwig: Actinium Pharmaceuticals, Inc: Current Employment. Pandit-Taskar: Bristol Myers Squibb: Research Funding; Bayer: Research Funding; Clarity Pharma: Research Funding; Illumina: Consultancy, Honoraria; ImaginAb: Consultancy, Honoraria, Research Funding; Ymabs: Research Funding; Progenics: Consultancy, Honoraria; Medimmune/Astrazeneca: Consultancy, Honoraria; Actinium Pharmaceuticals, Inc: Consultancy, Honoraria; Janssen: Research Funding; Regeneron: Research Funding. Sauter: Genmab: Consultancy; Celgene: Consultancy, Research Funding; Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Bristol-Myers Squibb: Research Funding; GSK: Consultancy; Gamida Cell: Consultancy; Novartis: Consultancy; Spectrum Pharmaceuticals: Consultancy; Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding. OffLabel Disclosure: 131-I apamistamab and 19-28z CAR T-cells are investigational agents in treatment of ALL and DLBCL

scholarly journals Single-Cell RNA Sequencing Identifies Expression Patterns Associated with Clinical Responses to Dual-Targeted CAR-T Cell Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Tyce Kearl ◽  
Ao Mei ◽  
Ryan Brown ◽  
Bryon Johnson ◽  
Dina Schneider ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Clinical Responses

INTRODUCTION: Chimeric Antigen Receptor (CAR)-T cell therapy is emerging as a powerful treatment for relapsed or refractory B cell lymphomas. However, a variety of escape mechanisms prevent CAR-T cell therapy from being more uniformly effective. To better understand mechanisms of CAR-T failure among patients treated with dual-targeted CAR-T cells, we performed single-cell RNA sequencing of samples from a Phase 1 trial (NCT03019055). The clinical trial used anti-CD20, anti-CD19 CAR-T cells for the treatment of relapsed/refractory B-cell non-Hodgkin Lymphoma. Clinical responses from this study are reported independently (Shah et al. in press in Nat Med). While robust clinical responses occurred, not all patients had similar outcomes. In single-antigen specific CAR-T cells, mechanisms of resistance include antigen down-regulation, phenotype switch, or PD-1 inhibition (Song et al. Int J Mol Sci 2019). However, very little is understood about the mechanisms of failure that are specific to dual-targeted CAR-T cells. Interestingly, loss of CD19 antigen was not observed in treatment failures in the study. METHODS: De-identified patient samples were obtained as peripheral blood mononuclear cells on the day of harvest ("pre" samples), at the peak of in vivo CAR-T cell expansion which varied from day 10 to day 21 after infusion ("peak" samples), and on day 28 post-infusion ("d28" samples). The CAR-T cell infusion product was obtained on day 14 of on-site manufacturing ("product" samples). All samples were cryopreserved and single cell preparation was performed with batched samples using 10X Genomics kits. Subsequent analysis was performed in R studio using the Seurat package (Butler et al. Nat Biotech 2018) with SingleR being used to identify cell types in an unbiased manner (Aran et al. Nat Immunol 2019). RESULTS: We found that distinct T cell clusters were similarly represented in the responder and non-responder samples. The patients' clinical responses did not depend on the level of CAR expression or the percentage of CAR+ cells in the infusion product. At day 28, however, there was a considerable decrease in the percentage of CAR+ cells in the responder samples possibly due to contracture of the CAR+ T cell compartment after successful clearance of antigen-positive cells. In all samples, the CAR-T cell population shifted from a CD4+ to a CD8+ T cell predominant population after infusion. We performed differentially-expressed gene analyses (DEG) of the total and CAR-T cells. In the pre samples, genes associated with T-cell stimulation and cell-mediated cytotoxicity were highly expressed in the responder samples. Since the responders had an effective anti-tumor response, we expected these pathways to also be enriched for in the peak samples; however, this was not the case. We hypothesize that differential expression of the above genes was masked due to homeostatic expansion of the T cells following conditioning chemotherapy. Based on the DEG results, we next interrogated specific genes associated with cytotoxicity, T cell co-stimulation, and checkpoint protein inhibition. Cytotoxicity-associated genes were highly expressed among responder CD8+ T cells in the pre samples, but not in the other samples (Figure 1). Few differences were seen in specific co-stimulatory and checkpoint inhibitor genes at any timepoint in the T cell clusters. We performed gene set enrichment analyses (GSEA). Gene sets representing TCR, IFN-gamma, and PD-1 signaling were significantly increased in the pre samples of the responders but not at later time points or in the infusion products. DISCUSSION: We found a correlation between expression of genes associated with T cell stimulation and cytotoxicity in pre-treatment patient samples and subsequent response to CAR-T cell therapy. This demonstrates that the existing transcriptome of T cells prior to CAR transduction critically shapes anti-tumor responses. Further work will discover biomarkers that can be used to select patients expected to have better clinical outcomes. Figure 1 Disclosures Johnson: Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Hari:BMS: Consultancy; Amgen: Consultancy; GSK: Consultancy; Janssen: Consultancy; Incyte Corporation: Consultancy; Takeda: Consultancy. Shah:Incyte: Consultancy; Cell Vault: Research Funding; Lily: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria; Verastim: Consultancy; TG Therapeutics: Consultancy; Celgene: Consultancy, Honoraria; Miltenyi Biotec: Honoraria, Research Funding.

scholarly journals Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 139 ◽  
Author(s):  
Jing Cui ◽  
Herui Wang ◽  
Rogelio Medina ◽  
Qi Zhang ◽  
Chen Xu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Therapeutic Potential ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

scholarly journals PTK7-Targeting CAR T-Cells for the Treatment of Lung Cancer and Other Malignancies

2021 ◽  
Vol 12 ◽  
Author(s):  
Yamin Jie ◽  
Guijun Liu ◽  
Lina Feng ◽  
Ying Li ◽  
Mingyan E ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Lung Cancers ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

In spite of impressive success in treating hematologic malignancies, adoptive therapy with chimeric antigen receptor modified T cells (CAR T) has not yet been effective in solid tumors, where identification of suitable tumor-specific antigens remains a major obstacle for CAR T-cell therapy due to the “on target off tumor” toxicity. Protein tyrosine kinase 7 (PTK7) is a member of the Wnt-related pseudokinases and identified as a highly expressed antigen enriched in cancer stem cells (CSCs) from multiple solid tumors, including but not limited to triple-negative breast cancer, non-small-cell lung cancer, and ovarian cancer, suggesting it may serve as a promising tumor-specific target for CAR T-cell therapy. In this study, we constructed three different PTK7-specific CAR (PTK7-CAR1/2/3), each comprising a humanized PTK7-specific single-chain variable fragment (scFv), hinge and transmembrane (TM) regions of the human CD8α molecule, 4-1BB intracellular co-stimulatory domain (BB-ICD), and CD3ζ intracellular domain (CD3ζ-ICD) sequence, and then prepared the CAR T cells by lentivirus-mediated transduction of human activated T cells accordingly, and we sequentially evaluated their antigen-specific recognition and killing activity in vitro and in vivo. T cells transduced with all three PTK7-CAR candidates exhibited antigen-specific cytokine production and potent cytotoxicity against naturally expressing PTK7-positive tumor cells of multiple cancer types without mediating cytotoxicity of a panel of normal primary human cells; meanwhile, in vitro recursive cytotoxicity assays demonstrated that only PTK7-CAR2 modified T cells retained effective through multiple rounds of tumor challenge. Using in vivo xenograft models of lung cancers with different expression levels of PTK7, systemic delivery of PTK7-CAR2 modified T cells significantly prevented tumor growth and prolonged overall survival of mice. Altogether, our results support PTK7 as a therapeutic target suitable for CAR T-cell therapy that could be applied for lung cancers and many other solid cancers with PTK7 overexpression.

PTK7-Targeting CAR T-cells for the Treatment of Lung Cancer and other Malignancies

2020 ◽  
Author(s):  
Yamin Jie ◽  
Guijun Liu ◽  
Lina Feng ◽  
Ying Li ◽  
Mingyan E ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Lung Cancers ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background: In spite of impressive success in treating hematologic malignancies, adoptive therapy with chimeric antigen receptor modified T cells (CAR T) has not yet been effective in solid tumors, where identification of suitable tumor-specific antigens remains a major obstacle for CAR T-cell therapy due to the “on target off tumor” toxicity. Protein tyrosine kinase 7 (PTK7) is a member of the Wnt-related pseudokinases and identified as a highly expressed antigen enriched in cancer stem cells (CSCs) from multiple solid tumors, including but not limited to triple-negative breast cancer, non-small cell lung cancer, and ovarian cancer, suggesting it may serve as a promising tumor-specific target for CAR T-cell therapy. Methods: In this study, we constructed 3 different PTK7-specific CAR (PTK7-CAR1/2/3) each comprising a humanized PTK7-specific single chain variable fragment (scFv), hinge and transmembrane (TM) regions of the human CD8α molecule, 4-1BB intracellular co-stimulatory domain (BB-ICD), and CD3ζ intracellular domain (CD3ζ-ICD) sequence, and then prepared the CAR T cells by lentivirus mediated transduction of human activated T cells accordingly, and sequentially evaluated their antigen-specific recognition and killing activity in vitro and in vivo.Results: T cells transduced with all 3 PTK7-CAR candidates exhibited antigen-specific cytokine production and potent cytotoxicity against naturally expressing PTK7-positive tumor cells of multiple cancer types without mediating cytotoxicity of a panel of normal primary human cells; meanwhile, in vitro recursive cytotoxicity assays demonstrated that only PTK7-CAR2 modified T cells retained effective through multiple rounds of tumor challenge. Using in vivo xenograft models of lung cancers with different expression level of PTK7, systemic delivery of PTK7-CAR2 modified T cells significantly prevented tumor growth and prolonged overall survival of mice. Conclusion: Altogether, our results support PTK7 as a therapeutic target suitable for CAR T-cell therapy that could be applied for lung cancers and many other solid cancers with PTK7 overexpression.

scholarly journals Clinical Experience of CAR T Cell Immunotherapy for Relapsed and Refractory Infant ALL Demonstrates Feasibility and Favorable Responses

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3869-3869 ◽  
Author(s):  
Colleen Annesley ◽  
Corinne Summers ◽  
Michael A. Pulsipher ◽  
Alan S. Wayne ◽  
Julie Rivers ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Lineage Switch ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Background: The youngest patients referred for CAR T cell therapy are those with relapsed or refractory (R/R) KMT2A-rearranged infant B-ALL. Infants with relapsed ALL following Interfant-99 therapy have a dismal reported 3-yr OS of 20.9%, indicating the need for novel therapies. Smaller patient size, heavily pre-treated disease and high leukemia burden are often characteristics of this subgroup of patients that pose unique challenges to apheresis and manufacture of a T cell product. Additionally, reports of KMT2A-rearranged leukemia undergoing lineage switch following CD19-targeting pressure raises concern for an increased risk of myeloid leukemia relapses after B-lineage targeted CAR T cell therapy in this population. Here we report our experience using CAR T cell immunotherapy for patients with R/R infant ALL enrolled on clinical trials PLAT-02 (NCT02028455) and PLAT-05 (NCT03330691). Methods: PLAT-02 is a phase 1/2 trial of CD19-specific (FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ) CAR T cells. PLAT-05 is a phase 1 trial of CD19xCD22 dual specific CAR T cells, transduced with two separate lentiviral vectors to direct the co-expression of the CD19-specific CAR above and a CD22-specific CAR (m971scFv:IgG4hinge-CH2(L235D)-CH3-CD28tm:4-1BB:ζ). Eligible subjects on both studies have R/R B-ALL, an absolute lymphocyte count ≥100 cells/µL, and were at least 1 year of age. In addition, subjects on PLAT-02 were ≥ 10kg, and ≥ 8kg on PLAT-05. For cell manufacture, apheresis products were immuno-magnetically selected for CD4 and CD8 cells. Selected T cells were activated with anti-CD3/CD28 beads, transduced, and grown in culture with homeostatic cytokines to numbers suitable for clinical use. Infant ALL subjects received a range of 5x105 to 10x106 CAR+ T cells/kg following lymphodepleting chemotherapy. Disease response assessments were required at Day 21 and Day 63 following CAR T cell infusion. Adverse events were graded according to CTCAEv4, except CRS which was graded according to 2014 Lee criteria. Results: Eighteen subjects with R/R infant ALL have enrolled on PLAT-02 (n=14) or PLAT-05 (n=4), with a median age of 22.5 months at enrollment (range: 14.5 - 40.1 months). Of these, 2 (11.1%) had primary refractory disease, 8 (44.4%) were in 1st relapse, 7 (38.9%) were in 2nd relapse and 1 (5.6%) was in 3rd or greater relapse. Ten subjects (55.6%) had an M2 marrow or greater at enrollment prior to apheresis, and 9/18 had a history of hematopoietic cell transplant (HCT). The mean ALC was 1309 cells/µL (range 253-6944). Successful CAR T cell products were manufactured in 17/18 subjects, including in 9/9 subjects with no prior history of HCT. Of these, 16/17 subjects with available products were infused, with a median follow up of 26.9 months. One subject died of disease complications prior to CAR T cell infusion. Of the 16 treated subjects, 1 is pending disease and toxicity assessments. The maximum grade of CRS was 3 and occurred in two of 15 evaluable subjects (13%) and neurotoxicity was limited to a maximum grade of 2. Fourteen of 15 (93.3%) achieved an MRD negative complete remission (MRD-CR) by Day 21. Of the 14 subjects with an MRD-CR, 6 went on to HCT with 1 subsequent CD19 negative relapse. Of the 8 subjects who did not proceed to HCT, 1 developed lineage switch at one month following CAR T cells, and 1 died of infectious complications with aplasia. A "wait and watch" approach was taken for the remaining 6 subjects, and 2 developed CD19+ relapse. The incidence of lineage switch among the infant ALL group was 1/15 (6.7%). The estimated 1-year LFS was 66.7% and 1-year OS was 71.4%. Conclusion: This is the largest reported cohort to date of R/R infant B-ALL subjects treated with CAR T cell therapy. We report successful manufacture and administration of a CAR T cell product in the significant majority of infant subjects. Toxicity and MRD-CR rates are comparable to that of non-infant ALL subjects. In our experience, subjects with R/R infant ALL are not at increased risk for lineage switch relapse compared with the entire study populations following B-antigen targeting CAR T cell immunotherapy. Numbers in this report are too small to make definitive conclusions about the value of consolidative HCT. However, the LFS of this cohort is remarkably higher when compared with historical controls. Future work is focused on overcoming feasibility issues for the smallest of subjects, to enable a larger number of these cases to access CAR T cell therapy. Disclosures Pulsipher: Amgen: Other: Lecture; Bellicum: Consultancy; Miltenyi: Research Funding; Medac: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Wayne:AbbVie: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding; Servier: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding. Jensen:Bluebird Bio: Research Funding; Juno Therapeutics, a Celgene Company: Research Funding. Gardner:Novartis: Honoraria.

scholarly journals The Lymphoma Tumor Microenvironment Influences Toxicity after CD19 CAR T Cell Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4105-4105 ◽  
Author(s):  
Michael D. Jain ◽  
Rawan Faramand ◽  
Verena Staedtke ◽  
Renyuan Bai ◽  
Sae Bom Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Research Funding ◽  
Myeloid Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Introduction: Of patients receiving CD19 CAR T cell therapy for large B cell lymphoma (LBCL), approximately 1 in 10 experience severe cytokine release syndrome (CRS) and 1 in 3 experience severe neurotoxicity. While CAR T cells trigger the onset of these toxicities, CRS and neurotoxicity are thought to occur as a consequence of activated myeloid cells amplifying cytokine and catecholamine release, thereby stimulating inflammation both systemically and at the blood-brain barrier. However, patient and tumor-related factors that account for differences in the amount of toxicity remain poorly understood. Methods: Serum cytokine levels were measured on an ELLA point of care device prior to lymphodepleting chemotherapy and throughout inpatient treatment with CD19 CAR T cell therapy (axicabtagene ciloleucel) for LBCL. Catecholamine levels were measured as we have previously reported. Tumor biopsies were taken within 1 month prior to infusion of CAR T cells. RNA expression was measured by RNAseq and/or a Nanostring IO360 panel consisting of 770 genes found in the tumor microenvironment (TME) in cancer. Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Mouse CAR T cell studies utilized mouse CD19-targeted CAR T cells derived from C57BL/6 splenocytes and cultured in vitro with myeloid cells and target cells to evaluate cytotoxicity and/or cytokine secretion. Elicited mouse macrophages were collected from peritoneal fluid 4 days after IP injection of 3% Brewer's thioglycollate medium. In vivo studies with mouse CD19-targeted CAR T cells were performed in IL2Ra-/- mice given cyclophosphamide as a pre-conditioning chemotherapy followed by adoptive transfer and analyses for CAR T cell and B cell persistence, as well as cytokines. Results: Of 58 patients undergoing CD19 CAR T cell therapy for LBCL, 8 (14%) had severe (grade 3 or higher) CRS and 16 (28%) had severe (grade 3 or higher) neurotoxicity. At baseline, peripheral blood levels of IL-6, IFN-γ, IL-15 and ferritin were significantly higher in patients who would subsequently experience severe CRS and severe neurotoxicity. Confirming our recent animal model of CRS we determined that peak serum catecholamine levels were higher in patients experiencing severe CRS. To identify if myeloid cells potentiate cytokine release we co-cultured CAR T cells with CD19 target and macrophages obtained from elicited mouse peritoneum. When these macrophages were added, IL-6 release from CAR T cells significantly increased compared to when macrophages were absent. Next, we studied the baseline TME in LBCL CAR T patients. Of 36 patients, 10 (27%) experienced severe neurotoxicity following CAR T cell therapy. By cell type score, the severe neurotoxicity group had a lower expression of genes associated with T cells overall and specifically Tregs. Also significantly lower in the severe neurotoxicity group were T cell genes including multiple subunits of CD3, CD3ζ, FOXP3, ICOS, CD62L and others. Association of increased T cell infiltration in the TME with low neurotoxicity raised the possibility that suppressive T cell subsets play a role in limiting toxicity post-CAR T cell therapy. To test this hypothesis, we injected CD19-targeted CAR T cells into an immune competent mouse model of Treg depletion (IL2Ra-/-) with established CD19+ leukemia. Treg deficient mice experienced a massive cytokine release after CAR T infusion and died prematurely due to CAR T toxicity compared to control mice with Tregs intact. Conclusions: Our observations suggest that the incidence of severe toxicity following CD19 CAR T cell therapy is influenced by baseline characteristics that are present prior to the infusion of CAR T cells. These include systemic inflammation characterized by high cytokine levels and a TME notable for a lack of infiltrating T cells. We posit a model whereby inflammation primes myeloid cells that are further activated upon CAR T cell infusion to release toxic amounts of cytokines and catecholamines. T cell subsets in the TME may modulate CAR T cells at the site of antigen encounter and prevent excessive CAR T activation. Reducing systemic inflammation or encouraging T cell infiltration into tumor prior to CAR T infusion are potential strategies for lowering the toxicity associated with CAR T therapy. Disclosures Jain: Kite/Gilead: Consultancy. Chavez:Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Novartis: Honoraria; Spectrum/Astrotech: Honoraria; Celgene/Juno: Honoraria; Kite/Gilead: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; AstraZeneca: Honoraria. Bachmeier:Kite/Gilead: Speakers Bureau. Mullinax:Iovance: Research Funding. Locke:Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy; Kite: Other: Scientific Advisor. Davila:Anixa: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Celgene: Research Funding; Atara: Research Funding; Bellicum: Consultancy.

scholarly journals Co-Expression of IL-7 Improves NKG2D-Based CAR T Cell Therapy on Prostate Cancer by Enhancing the Expansion and Inhibiting the Apoptosis and Exhaustion

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1969 ◽  
Author(s):  
Cong He ◽  
Ying Zhou ◽  
Zhenlong Li ◽  
Muhammad Asad Farooq ◽  
Iqra Ajmal ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) T-cell therapy is a promising approach in treating solid tumors but the therapeutic effect is limited. Prostate cancer is a typical solid malignancy with invasive property and a highly immunosuppressive microenvironment. Ligands for the NKG2D receptor are primarily expressed on many cancer cells, including prostate cancer. In this study, we utilized NKG2D-based CAR to treat prostate cancer, and improved the therapeutic effect by co-expression of IL-7. The results showed that NKG2D-CAR T cells performed significantly increased cytotoxicity against prostate cancer compared to non-transduced T cells in vitro and in vivo. Moreover, the introduction of the IL-7 gene into the NKG2D-CAR backbone enhanced the production of IL-7 in an antigen-dependent manner. NKG2DIL7-CAR T cells exhibited better antitumor efficacy at 16 h and 72 h in vitro, and inhibited tumor growth in xenograft models more effectively. In mechanism, enhanced proliferation and Bcl-2 expression in CD8+ T cells, decreased apoptosis and exhaustion, and increased less-differentiated cell phenotype may be the reasons for the improved persistence and survival of NKG2DIL7-CAR T cells. In conclusion, these findings demonstrated that NKG2D is a promising option for CAR T-cell therapy on prostate cancer, and IL-7 has enhanced effect on NKG2D-based CAR T-cell immunotherapy, providing a novel adoptive cell therapy for prostate cancer either alone or in combination with IL-7.

scholarly journals TMOD-18. EXPLORING THE FACTORS LEAD TO SUCCESS OF CAR T-CELL THERAPY IN GLIOBLASTOMA WITH COMPUTATIONAL MODELING AND IN VITRO DATA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi266-vi266
Author(s):  
Prativa Sahoo ◽  
Xin Yang ◽  
Daniel Abler ◽  
Davide Maestrini ◽  
Vikram Adhikarla ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Solid Tumors ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Abstract Chimeric antigen receptor (CAR) T-cell therapy is an emerging targeted immunotherapy which has shown success in liquid cancers such as leukemias. CAR T-cells are also being used for the treatment of solid tumors such as glioblastoma, which is a primary brain tumor. Ongoing phase I trials have been designed to evaluate CAR T-cell dosing, scheduling, and route of administration in order to understand and improve the efficacy of CAR T-cell therapy. A better understanding of factors leading to the success of CAR T-cell immunotherapy for solid tumors will be necessary to improve outcomes for patients with solid tumors and to advance the field of CAR T-cell immuno-oncology. Here we use mathematical model to explore factors in determining a successful response to CAR T-cell therapy: proliferation, persistence, and killing capacity of CAR T-cells. Using a novel in vitro experimental apparatus, we are able to measure the density of cancer cells over several days in 15 minute interval time resolution. This highly temporally resolved data provides a unique opportunity to confidently estimate parameters of the model and to provide insights into the dynamics of CAR T-cell proliferation, persistence, and killing capacity. Furthermore we explore the relationship between these factor with CAR T-cell dose level. We will show results from experiments using patient-derived cancer cell lines as well as cancer cells engineered to express specific levels of the target antigen (IL13Rα2) to quantitatively evaluate the roles of proliferation, persistence, and killing in cells with different levels of antigen expression. We will discuss the interpretation of the model parameters and demonstrate the clinical value of this analysis through an application of CAR T-cell treatment tailored to the dynamics of an individual patient’s cancer growth rate.

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