Targeting of CD19 By Tafasitamab Does Not Impair CD19 Directed Chimeric Antigen Receptor T Cell Activity in Vitro

CD19 directed chimeric antigen receptor T cell (CART) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia leading to the approval of two CART therapies. With the emergence of therapeutic anti-CD19 antibodies for the treatment of B cell malignancies, it remains to be elucidated whether such antibodies would interfere with the ability of CD19 targeting CARTs to exert their anti-tumor effect in a subsequent therapy. To address a part of this question, we investigated the potential for functional interference between the monoclonal anti-CD19 antibody tafasitamab (MOR208) and CD19 directed CART cells (CART19). CART19 cells were generated through lentiviral transduction of healthy donor T cells with a second generation CD19 CAR construct (FMC63-CD8h-CD8TM-41BBζ) which is similar to the construct used for the FDA-approved CART tisagenlecleucel. Tafasitamab, is an Fc-enhanced humanized monoclonal antibody which mediates antibody-dependent cellular toxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity. It is currently being studied in phase 2 and 3 clinical trials in diffuse large B-cell lymphoma (DLBCL) in combination with the immunomodulatory agent lenalidomide (L-MIND) and the chemotherapeutic drug bendamustine (B-MIND). As a first step we confirmed the relevance of the tested CD19-positive target cell lines, JEKO (mantel cell lymphoma), Ly7 (DLBCL) and NALM-6 (ALL) based on functional activity of tafasitamab and CART19. In a 24 hours ADCC (tafasitamab titration plus natural killer (NK) cells; Figure 1A) and T cell cytotoxicity assays (CART19, E:T titrations; data not shown) distinct activity was observed for both therapies on all tested cell lines. Secondly, we studied whether the observed CART19 activity may be influenced by tafasitamab in case of a direct CD19 binding competition between tafasitamab and the CAR. To test for such binding competition we incubated the CD19+ cell lines NALM6 or JEKO with up to 100 µg/ml tafasitamab, to saturate the receptors. Subsequent flow cytometry analysis using the FMC63 antibody (carrying the same CD19 binding domain as contained in CART19) failed to detect CD19 expression, indicating a direct binding competition between FMC63 and tafasitamab (Figure 1B). Next, to investigate the potential impact of such binding competition on CART19 cell effector functions, we co-cultured tafasitamab CD19+ JEKO cell line at increasing concentrations of up to 100µg/ml, and then added CART19 cells at different effector to target ratios to the cell culture. The presence of tafasitamab, binding to the CD19 antigen, did not affect important CART cell effector functions such as antigen specific killing (Figure 1C), degranulation (Figure 1D), cytokine production or proliferation of CART19 (Figure 1E). In summary, our studies indicate that CART19 continue to exhibit potent antigen specific effector functions despite presence of tafasitamab and the related competition for CD19 binding. Besides the presented in vitro work the questions of therapeutic sequencing of tafasitamab and CART19 is being studied in xenograft models and will be presented at the meeting. Disclosures Sakemura: Humanigen: Patents & Royalties. Cox:Humanigen: Patents & Royalties. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding; Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

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PRAME Expression Is Correlated with Treatment Outcome and Specific Features of the Tumor Microenvironment in Classical Hodgkin Lymphoma

Blood ◽

10.1182/blood-2019-129730 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1509-1509

Author(s):

Katsuyoshi Takata ◽

Lauren C. Chong ◽

Avinash Thakur ◽

Tomohiro Aoki ◽

Anja Mottok ◽

...

Keyword(s):

T Cell ◽

Tumor Microenvironment ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Sequencing Data ◽

And Function ◽

Inhibitor Treatment

Background: The tumor-associated antigen PRAME is over-expressed in several types of cancer and is currently investigated as a therapeutic target for T-cell immunotherapy. Our previous integrative genomic study in diffuse large B-cell lymphoma (DLBCL) identified PRAME deletion to be correlated with patient outcome and an immunologically "cold" tumor microenvironment. However, it remains an open question whether PRAME expression significantly contributes to differential treatment outcomes and tumor microenvironment crosstalk across various B-cell lymphoma subtypes. Material and Methods: We performed an immunohistochemical (IHC) screen in a large cohort of B-cell lymphomas (de novo DLBCL; N=347, follicular lymphoma (FL); N= 166, mantle cell lymphoma (MCL); N= 180), and classical Hodgkin lymphoma (HL); N= 166) to assess PRAME expression as a prognostic biomarker. Moreover, to investigate PRAME-expression associated tumor microenvironment composition and function, we correlated PRAME IHC results with single cell RNA sequencing data of more than 127,000 cells from 22 HL tissue specimens. Results: PRAME IHC analysis revealed frequent PRAME over-expression in HL (115/166, 69%), followed by DLBCL (104/319, 33%), FL (13/166, 8%), and MCL (14/180, 8%). Interestingly, only HL showed a significant treatment outcome correlation, whereas other B-cell lymphoma subtypes did not. Specifically, using a previously published HL cohort (Steidl et al, NEJM 2010) PRAME-negative Hodgkin Reed Sternberg (HRS) cells indicated significantly shorter overall survival (P = 0.008) and disease-specific survival (P = 0.042 ). To characterize PRAME-specific microenvironment composition and function in HL, we analyzed T-, B-, NK-cell, and macrophage subsets in PRAME-positive (17 of 22 cases) vs -negative (5 of 22 cases) tumor samples using single cell RNA sequencing data. From 22 expression-based microenvironment cell clusters that were annotated and assigned to a cell type based on gene expression, all three CD4 helper T-cell clusters were de-enriched in PRAME-negative samples, and the CD4 non-Treg proportion was significantly lower in PRAME-negative samples (P = 0.049). Strikingly, when focusing on phenotypic features of cells within the CD4 non-Treg T-cell cluster, CXCL13 was identified as the most up-regulated gene in PRAME-negative samples. When interrogating published HRS cell transcriptome data (Steidl et al, Blood 2012), immune response pathways including chemokine receptors and chemokine ligands were up-regulated in PRAME-negative HRS cell samples. Of specific interest, CXCR5, the cognate receptor for CXCL13, was significantly upregulated as a member of the chemokine pathway (P = 0.0086) in PRAME-negative HRS cell samples. These results suggest that crosstalk between CXCL13 (produced in the microenvironment) and CXCR5 (expressed on HRS cells) contributes to tumor maintenance in PRAME-negative HL. Finally, to explore potential therapeutic approaches for PRAME-negative HL cells, we focused on 3 HL-derived cell lines (L540, L591, DEV) with low PRAME expression and exposed these lines to DNMT or HDAC inhibitors. DNMT inhibitor treatment showed clear restoration of PRAME expression in a dose dependent manner, but no restoration was found by HDAC inhibitor treatment. To investigate the effect of DNA methylation in transcriptional regulation of PRAME in HL cells, we performed bisulfite sequencing in the PRAME CpG promoter region in PRAME down-regulated (L540, L591, DEV) and up-regulated (HD-LM2, KMH-2, L1236) cell lines and found hypermethylation in PRAME low vs high cell lines. Moreover, the CpG promoter region was significantly demethylated by DNMT inhibitor treatment in cell lines with low PRAME expression. Conclusion: We discovered that PRAME protein expression was correlated with outcome in HL and identified specific T-cell subsets in PRAME-negative patients. PRAME restoration by DNMT inhibitors might represent a new therapeutic avenue in combination with modern immunotherapies, such as PRAME-specific T-cell therapy or PD1 inhibition. Disclosures Scott: Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy. Steidl:Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

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The in Vitro Effect of Tin Derivatives on Cell Lines of Canine Osteosarcoma (D-17), B-Cell Lymphoma (CL-1) and T-cell Leukaemia (GL-1)

Journal of Comparative Pathology ◽

10.1016/j.jcpa.2013.11.122 ◽

2014 ◽

Vol 150 (1) ◽

pp. 105

Author(s):

D. Poradowski ◽

H. Pruchnik ◽

A. Pawlak ◽

R. Ciaputa ◽

M. Kandefer-Gola ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cell Leukaemia ◽

Canine Osteosarcoma ◽

Vitro Effect

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Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Blood ◽

10.1182/blood-2020-134664 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-11

Author(s):

Eugenio Gaudio ◽

Chiara Tarantelli ◽

Luciano Cascione ◽

Filippo Spriano ◽

Gaetanina Golino ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Advisory Committees ◽

Travel Grant

Introduction. CD22 is a cell surface marker expressed by the vast majority of normal and neoplastic B-cells. ADCT-602 is an antibody drug conjugate (ADC) composed of Emab-C220, an engineered version of the anti-CD22 humanized IgG1 antibody epratuzumab, site-specifically conjugated to SG3249, which includes the DNA minor groove crosslinking pyrrolobenzodiazepine (PBD) dimer SG3199 linked to the antibody via a protease-cleavable linker (Zammarchi et al, ASH 2016). ADCT-602 is currently being tested in a phase I/II clinical trial (NCT03698552) in recurrent or refractory B-cell acute lymphoblastic leukemia (B-ALL) patients. Here, we assessed its in vitro anti-lymphoma activity, also exploring for potential biomarkers and mechanisms of resistance. Methods. Fifty-seven human lymphoma cell lines were exposed to ADCT-602, an isotype-control ADC and the PBD dimer SG3199 as single agents for 96h, followed by MTT proliferation assay and IC50 calculation. Quantum Simply Cellular (QSC) microspheres were used for the quantitative determination of cellular CD22 antigen expression (Bangs Laboratories. Inc). Differences in IC50 values among lymphoma subtypes were calculated using the Wilcoxon rank-sum test. Statistical significance was defined by P values of 0.05 or less. Sensitivity analysis to ADCT-602 was performed by integrating different omics data, such as Illumina HT-12 microarray data (GSE94669), HTG EdgeSeq Oncology Biomarker Panel data (GSE103934) and DNA copy number variations. Results. The median IC50 for ADCT-602 was 200 pM (95%C.I, 90-400 pM) in 48 B-cell lymphoma lines (including three Hodgkin lymphoma cell lines), and 1850 pM in nine T-cell lymphoma lines (95%C.I, 700-15000 pM). ADCT-602 was more active in B- than in T-cell lymphomas, as expected based on the pattern of CD22 expression (P < 0.005). Focusing on B-cell lymphomas, ADCT-602 in vitro activity was not correlated with its target expression measured both at the cell surface protein level (absolute quantitation, n=48, r=0.06 P=ns) and at the RNA level (Illumina HT-12 arrays, n=42, r=0.28, P=ns; HTG EdgeSeq Oncology Biomarker Panel, n=36, r=0.24, P=ns). In vitro activity was stronger in marginal zone lymphoma (MZL) cell lines than other B-cell lymphoma models (median IC50s 62.5 vs 312.5 pM; P = 0.03) as well as in diffuse large B-cell lymphoma (DLBCL) cell lines with BCL2 and MYC translocations (DHT DLBCL) versus DLBCL with none or a single translocation (median IC50s 25 vs 400 pM, P = 0.03). No associations were seen with TP53 status or other histology (mantle cell lymphoma, DLBCL, DLBCL cell of origin). We then exploited the gene expression profiling data using the Illumina HT-12 microarray gene expression technology. Within all the B-cell lymphoma cell lines (sensitive, n= 25; resistant, n= 23) we identified 1.207 genes down-regulated (FC-) and 1,104 genes up-regulated (FC+) in resistant cell lines. To delineate the pathways associated with the different degrees of sensitivity to ADCT-602, we performed a gene set enrichment analysis (GSEA; GSEA hallmarks and c2.common pathways) on the pre-ranked limma data. Transcripts up-regulated in resistant cell lines were enriched of genes coding for proteins involved in respiratory electron transport, oxidative phosphorylation and proteasome. Conversely, transcripts up-regulated in the sensitive cell lines were enriched of genes coding for proteins involved in inflammation, chemokine signaling, p53 response, IL2/STAT5 signaling, hypoxia, TGF-beta and interferon response. Similar gene signatures were picked up using the HTG platform, which can be applied to formalin-fixed paraffin-embedded clinical specimens, despite the smaller number of investigated genes. Conclusion. ADCT-602 showed in vitro anti-tumor activity across a large panel of B-cell lymphoma models of various histology. Expression signatures and other features (MZL or DHT DLBCL histology), but not the expression levels of its target, were associated with different sensitivity to the ADC. Our data supports the clinical evaluation of ADCT-602 in patients with B-cell lymphoma in addition to B-ALL. Disclosures Zucca: Kite: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Research Funding; Celltrion Healthcare: Membership on an entity's Board of Directors or advisory committees. Stathis:PharmaMar: Other: Travel Grant; Member of the steering committee of the trial of this abstract: Other; Loxo: Honoraria, Other, Research Funding; Cellestia: Research Funding; Roche: Other, Research Funding; Novartis: Other, Research Funding; Bayer: Other, Research Funding; Merck: Other, Research Funding; Pfizer: Other, Research Funding; MEI Pharma: Other, Research Funding; ADC Therapeutcis: Other, Research Funding; Abbvie: Other: Travel Grant. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bertoni:ADC-Therapeutics: Research Funding; Bayer AG: Research Funding; Helsinn: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Nordic Nanovector ASA: Research Funding; Astra Zeneca: Other: travel grant; Amgen: Other: travel grant.

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Asparagine Synthetase Expression and L-Asparaginase Sensitivity in Aggressive Lymphomas

Blood ◽

10.1182/blood.v124.21.5494.5494 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5494-5494 ◽

Cited By ~ 1

Author(s):

Willy Berlier ◽

Karine Aguera ◽

Anne-Marie Chevrier ◽

Fanny Gallix ◽

Alexandra Traverse-Glehen ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

T Cell Lymphoma ◽

Large B Cell Lymphoma ◽

T Cell Lymphomas ◽

Large B Cell

Abstract L-asparaginase (L-ASPA) displays a strong clinical benefit in the treatment of acute lymphoblastic leukemia (ALL), where it is included in most of current chemotherapy regimen. L-ASPA depletes plasmatic asparagine (ASN), an amino acid essential for the proliferation of leukemic cells. Since these cells are deficient in asparagine synthetase (ASNS), they rely on external (plasmatic) source of ASN and can be starved to death by L-ASP treatment. Several studies evidenced the potential of ASN depletion to treat lymphomas. Indeed, many animal and human lymphoma cell lines have been shown to be sensitive to L-ASPA in vitro. In veterinary medicine, L-ASPA is routinely administered to treat effectively both feline and canine lymphomas (Wypig et al., 2013). L-ASPA regained attention in the treatment of human lymphomas since its adjunction in current chemotherapy regimens significantly improved the outcome of patients with NK/T cell lymphoma (Zou et al., 2014). Some studies also evidenced its benefit in combined chemo or monotherapy for the treatment of B-cell and T-cell lymphomas (Sun et al., 2006; Takahashi et al., 2010). In this study, we assessed the in vitro sensitivity to L-ASPA of 6 lymphoma cell lines and we analyzed ASNS expression in biopsies from 166 cases of lymphomas (130 B-cell lymphomas and 17 T-cell lymphomas). Sensitivity to L-ASPA (expressed as an IC50) was assessed in vitro by measuring the cell viability in the presence of various concentrations of E.coliL-ASPA. ASNS expression in biopsies (TMA, USBiomax, Rockville, MD) was assessed with a validated immunohistochemistry (IHC) method attributing a score to each tumor based on ASNS labeling intensity from 0 (no expression) to 3 (strong expression). Tumors expressing no/low ASNS (scores 0 and 1) were considered potentially sensitive to asparagine depletion. As shown in the following table, all cell lines were proved to be sensitive to L-ASPA. Their in vitrosensitivity exceeded cell lines MOLT-4 (ALL) and HL-60 (AML). Table 1Cell lineSensitivity to L-ASPA (IC50 in IU/mL)HuT-78 (Peripheral T-cell lymphoma,PTCL)0.11 ± 0.02Toledo (Diffuse large B-cell lymphoma, DLBCL)0.19 ± 0.03SU-DHL-8(Diffuse large B-cell lymphoma, DLBCL)0.10 ± 0.04SU-DHL-10(Diffuse large B-cell lymphoma, DLBCL)0.10 ± 0.01REC-1 (Mantle cell lymphoma, MCL)0.15 ± 0.03KHYG-1 (NK/T-cell lymphoma)0.16 ± 0.06MOLT-4 (acute lymphoid leukemia, ALL)0.19 ± 0.07HL-60 (acute myeloid leukemia, AML)0.23 ± 0.02 As shown in the following table, ASNS expression was null/low in 85% in the entire population of patients with B-cell lymphomas. Considering DLBCL, 63% of patients displayed no ASNS expression at all. ASNS expression was also null/low in 88% of patients with T-cell lymphomas (n=17). Table 2ASNS expression (IHC score)Type of lymphoma(% of cases)DLBCL (n=110)Others BCL (n=20)PTCL (n=3)Others TCL (n=14)MCL(n=3)Hodgkin (n=16)Negative (0)62,770,00,057,133,343,8Low positive (1)21,825,066,635,766,656,3Positive (2)7,35,033,37,10,00,0Highly positive (3)8,20,00,00,00,00,0 Globally, these results suggest that L-ASPA is potentially effective for the treatment of several lymphomas. Indeed, B-cell as well as T-cell lymphoma cell lines are sensitive to L-ASP in vitroand the majority of lymphoma tissues express no/low ASNS. Based on our results on ASNS expression in lymphoma biopsies, L-ASPA therapy may be beneficial for up to 85% of patients with DLBCL. Up to 90% of patients with other B-cell lymphomas or T-cell lymphomas may be sensitive to L-ASPA treatment as well. However, L-ASPA has only been used scarcely in the treatment of lymphomas despite promising clinical responses. Its well known serious side-effects (hypersensitivity, coagulation disorders, pancreatitis, and liver failure) render its use hazardous, particularly in older or frail patients. Therefore, the development of a new formulation of L-ASPA with safer profile has to be considered in order to allow the clinical development of L-ASPA in the treatment of aggressive lymphomas. Disclosures Berlier: ERYTECH: Employment, Equity Ownership. Aguera:ERYTECH: Employment. Chevrier:ERYTECH: Employment. Gallix:ERYTECH: Employment. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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MyD88 L265P Mutation-Specific TCR Gene Therapy for Treatment of B-Cell Lymphoma and Leukemia

Blood ◽

10.1182/blood-2020-142880 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 5-5

Author(s):

Özcan Çinar ◽

Peter Michael Kloetzel ◽

Caroline Anna Peuker ◽

Ulrich Keller ◽

Antonio Pezzutto ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Myd88 L265p Mutation ◽

T Cell Lines ◽

Myd88 L265p

Adoptive transfer of engineered T cells has shown remarkable success in hematopoietic malignancies. However, the current most common strategy of targeting lineage-specific antigens often leads to undesirable side effects and a high relapse rate. Therefore, novel treatment approaches are still needed. Oncogenic somatic mutations represent ideal targets because of tumor specificity: such (neo)antigens can be recognized by T cell receptors (TCR) in the context of MHC-peptide presentation. Here we have generated T cell lines from multiple healthy donors targeting one of the most common driver mutations found in B-cell lymphomas; a missense mutation on adaptor protein MyD88 changing leucine at position 265 to proline (L265P). T cell lines generated by autologous in vitro priming were reactive selectively against the predicted mutant epitope restricted to HLA-B7, but not against the corresponding wild-type peptide. Cloned TCRs from these lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with mutation-specific TCR (TCR-T cells) recognized and killed cell lines of diffuse large B-cell lymphoma characterized by intrinsic MyD88 L265P. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models, while initial safety screening did not indicate any sign of cross- or allo-reactivity risk. Taken together, our data suggest that mutation-specific TCRs can be used to target MyD88 L265P mutation, and hold promise for precision therapy for a significant subgroup of B-cell malignancies. Disclosures Keller: Bristol Myers Squibb: Honoraria, Other: Travel support, Speakers Bureau. Busse:Daiichi Sankyo: Other: Travel Support; Hexal: Honoraria, Research Funding; Roche: Honoraria; BMS: Honoraria; Novartis: Research Funding.

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The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 Chimeric Antigen Receptor T Cells (CART19) Improves Engraftment and Antitumor Responses Against Mantle Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.704.704 ◽

2015 ◽

Vol 126 (23) ◽

pp. 704-704

Author(s):

Marco Ruella ◽

Saad S Kenderian ◽

Olga Shestova ◽

Joseph A. Fraietta ◽

Sohail Qayyum ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

University Of Pennsylvania ◽

Tumor Control

Abstract Introduction: The bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrates considerable activity in mantle cell lymphoma (MCL). However, approximately 30% of patients do not respond to this treatment and the therapy invariably leads to drug resistance with a median response of 17.5 months. Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B-cell specific CD19 antigen (CART19) leads to dramatic clinical responses in the majority of patients with acute lymphoblastic leukemia and the activity in B cell lymphoma is currently being evaluated in clinical trials. Bulky disease, as sometimes seen in MCL, may impair T cell infiltration. The features of ibrutinib that make it an interesting addition to CART19 include its efficacy in reducing tumor masses and its ability to mobilize neoplastic B cells into the peripheral blood, thereby potentially exposing them to the killing activity of CART19. Therefore, we sought to investigate the combination of the two novel targeted therapies, ibrutinib and CART19 in MCL. Results: In vitro studies with established MCL cell lines and with a novel cell line (MCL-RL) showed a range of responses to ibrutinib with an IC50 ranging from 10 nM to 10 µM; MCL-RL was the most sensitive cell line evaluated with an IC50 of 10nM, similar to primary MCL. Both ibrutinib-sensitive and ibrutinib-resistant cell lines strongly activated CART19 in an antigen-specific manner as detected by CD107a degranulation, cytokine production and CFSE proliferation assays. Importantly, in vitro assays with MCL cell lines co-cultured with increasing doses of CART19 (E:T= 2:1, 1:1, 0.5:1, 0.25:1) combined with increasing concentrations of ibrutinib (0, 10, 100, 1000 nM) demonstrated strong additive tumor killing (Figure 1). Notably, supra-therapeutic doses of Ibrutinib (>/=1 uM) impaired cytokine production and T cell proliferation in vitro. In order to test this combination in vivo we established a novel MCL model, injecting i.v. luciferase-positive MCL-RL cells into NSG mice. This resulted in 100% MCL engraftment in liver and spleen, with eventual dissemination into lymph nodes and bone marrow. Treatment with three different doses of CART19 (0.5, 1 and 2 million cells/mouse) led to a dose dependent anti-tumor effect. A similar dose response to CART19 was also observed in the ibrutinib-resistant Jeko-1 cell line. We also treated MCL-RL xenografts with different doses (0, 25 and 125 mg/Kg/day) of ibrutinib, with a median overall survival respectively of 70, 81 and 100 days (p<0.001). Importantly, a direct in vivo comparison of the highest ibrutinib dose (125 mg/kg) and CART19 showed a significantly improved tumor control for mice treated with CART19. However, treatment with either CART19 or ibrutinib as single agents invariably led to late relapse. Therefore we sought to treat MCL-RL xenografts with the combination of CART19 and ibrutinib and compare it to the single agent activity. The combination resulted in significant improvement in tumor control compared to mice treated with the single agents with 80% of mice achieving long-term disease-free survival ( p=0.007 at day 110, representative mice shown in Figure 2A). Intriguingly, we found that mice treated with ibrutinib had higher numbers of circulating CART19 cells (Figure 2B). Conclusions: Combining CART19 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Maus:Novartis: Consultancy, Patents & Royalties, Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Patents & Royalties, Research Funding. Mato:Genentech: Consultancy; Pronai Pharmaceuticals: Research Funding; Celgene Corporation: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy. Schuster:Genentech: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Hoffman-LaRoche: Research Funding; Janssen: Research Funding; Gilead: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Kalos:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Gill:Novartis: Patents & Royalties, Research Funding. Wasik:Janseen and Novartis: Research Funding.

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Characterization of two new high-grade B-cell lymphoma cell lines with MYC and BCL2 rearrangements that are suitable for in vitro drug sensitivity studies

Leukemia & Lymphoma ◽

10.1080/10428194.2018.1508663 ◽

2018 ◽

Vol 60 (4) ◽

pp. 1043-1052

Author(s):

Marie-Sophie Dheur ◽

Hélène A. Poirel ◽

Geneviève Ameye ◽

Gaëlle Tilman ◽

Pascale Saussoy ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Drug Sensitivity ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

High Grade ◽

Sensitivity Studies

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Repurposing dasatinib for diffuse large B cell lymphoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905239116 ◽

2019 ◽

Vol 116 (34) ◽

pp. 16981-16986 ◽

Cited By ~ 5

Author(s):

Claudio Scuoppo ◽

Jiguang Wang ◽

Mirjana Persaud ◽

Sandeep K. Mittan ◽

Katia Basso ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Kinase Inhibitor ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Multikinase Inhibitor ◽

Large B Cell Lymphoma ◽

Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

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Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

Journal of Virology ◽

10.1128/jvi.77.3.2134-2146.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 2134-2146 ◽

Cited By ~ 195

Author(s):

Vicky M.-H. Sung ◽

Shigetaka Shimodaira ◽

Alison L. Doughty ◽

Gaston R. Picchio ◽

Huong Can ◽

...

Keyword(s):

Hepatitis C ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Culture System ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

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Anti-CD45 Mediated Cytolysis of Human T-Cell Lymphoma Cells.

Blood ◽

10.1182/blood.v104.11.4637.4637 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4637-4637

Author(s):

Gerald G. Wulf ◽

Anita Boehnke ◽

Bertram Glass ◽

Lorenz Truemper

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Cytolytic Activity ◽

T Cell Lymphoma ◽

Lymphoma Cell ◽

Lymphoma Cells ◽

Human T Cell

Abstract Anti-CD45 mediated cytoreduction is an effective means for T-cell depletion in rodents and humans. In man, the CD45-specific rat monoclonal antibodies YTH24 and YTH54 are IgG2b subclass, exert a predominantly complement-dependent cytolytic activity against normal T-lymphocytes, and have been safely given to patients as part of conditioning therapies for allogeneic stem cell transplantation. The efficacy of such antibodies against human lymphoma is unknown. Therefore, we evaluated the cytolytic activity of YTH24 and YTH54 by complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), as well as by direct apoptotic and antiproliferative effects, against a panel of Hodgkin disease (HD) and non-Hodgkin lymphoma (NHL) cell lines, and against primary specimens. Significant CDC activity (>50% cytolysis) of the antibodies YTH54 and YTH24 was observed against three of five T-cell lymphoma lines, but against only one of nine B-cell lymphoma lines and none of four HD cell lines. The combination of YTH54 and YTH24 induced ADCC in all T-cell lymphoma cell lines and three primary leukemic T-cell lymphoma specimens, but were ineffective in B-cell lymphoma and HD cell lines.There were only minor effects of either antibody or the combination on lymphoma cell apoptosis or cell cycle arrest. In summary, anti-CD45 mediated CDC and ADCC via the antibodies YTH24 and YTH54 are primarily effective against lymphoma cells with T-cell phenotype, and may be an immunotherapeutic tool for the treatment of human T-cell lymphoma.

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