SILAC Phosphoproteomics Reveals Unique Signaling Circuits in CAR-T Cells and the Inhibition of B Cell-Activating Phosphorylation in Target Cells

Abstract Background The unprecedented efficacy of chimeric antigen receptor T (CAR-T) cell immunotherapy of CD19+ B-cell malignancies has opened a new and useful way for the treatment of malignant tumors. Nonetheless, there are still formidable challenges in the field of CAR-T cell therapy, such as the biodistribution of CAR-T cells in vivo. Methods NALM-6, a human B-cell acute lymphoblastic leukemia (B-ALL) cell line, was used as target cells. CAR-T cells were injected into a mice model with or without target cells. Then we measured the distribution of CAR-T cells in mice. In addition, an exploratory clinical trial was conducted in 13 r/r B-cell non-Hodgkin lymphoma (B-NHL) patients, who received CAR-T cell infusion. The dynamic changes in patient blood parameters over time after infusion were detected by qPCR and flow cytometry. Results CAR-T cells still proliferated over time after being infused into the mice without target cells within 2 weeks. However, CAR-T cells did not increase significantly in the presence of target cells within 2 weeks after infusion, but expanded at week 6. In the clinical trial, we found that CAR-T cells peaked at 7–21 days after infusion and lasted for 420 days in peripheral blood of patients. Simultaneously, mild side effects were observed, which could be effectively controlled within 2 months in these patients. Conclusions CAR-T cells can expand themselves with or without target cells in mice, and persist for a long time in NHL patients without serious side effects. Trial registration The registration date of the clinical trial is May 17, 2018 and the trial registration numbers is NCT03528421.

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5-Aza-2'-Deoxycytidine Promotes Cytotoxic Effect of CART-Cells on Leukaemia Cells

Blood ◽

10.1182/blood.v124.21.5812.5812 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5812-5812

Author(s):

Alla Dolnikov ◽

Swapna Rossi ◽

Ning Xu ◽

Guy Klamer ◽

Sylvie Shen ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Target Cells ◽

Car T Cells ◽

Anti Tumour Activity ◽

Car T ◽

Pre Treatment ◽

Tumour Activity

Abstract T cells modified to express CD19-specific chimeric antigen receptors (CAR) have shown anti-tumour efficacy in early phase clinical trials in patients with relapsed and refractory B-cell malignancies. In addition to direct cytotoxicity, chemotherapeutic drugs can have an immunomodulatory effect both through enhancing the tumour-specific immune response and increasing the tumour’s susceptibility to immune mediated destruction. Hence, combining immunomodulatory chemotherapy and CAR T-cells is an attractive approach for eliminating tumours, particularly in advanced stages. 5-aza-2'-deoxycytidine (5-AZA) is a hypomethylating agent that induces terminal differentiation, senescence or apoptosis in haematological malignancies. Here, we have explored a CAR-based immunotherapy combined with 5-AZA to maximise the effect of the CAR T-cells against CD19+ B-cell leukaemia. A second generation CAR including CD3zeta and the CD28 co-stimulatory domain was cloned into the PiggyBac-transposon vector and was used to generate CAR19 -T cells. Cord blood -derived mononuclear cells (MNC) were transfected with CAR19-transposon/transposase plasmids and expanded with CD3/28 beads for 2 weeks in the presence of 20ng/ml IL2 and 10ng/ml IL7. CAR19 T-cells efficiently induced cytolysis of CD19+ leukaemia cells in vitro and exhibited anti-tumour activity in vivo in a xenograft mouse model of leukaemia. Pre-treatment with 5-AZA produced greater leukaemia cell cytolysis in vitro and maximised anti-tumour activity of CAR19 T-cells in vivo against xenograft primary leukaemia compared to 5-AZA or CAR19 T-cells alone. In vitro analysis revealed that pre-treatment with 5-AZA up-regulates CD19 expression in leukaemia cells and improves CAR19 T-cell recognition of target cells increasing the formation of effector/ target cell conjugates and target cell cytolysis. Therefore using 5-AZA pre-treatment can be particularly useful for B-cell leukaemias with reduced expression of CD19. We have also demonstrated that pre-treatment of target cells with 5-AZA potentiates the effect of CAR19 T-cells used at low dose or low effector:target (E:T) suggesting that even small numbers of CAR19 T-cells can mediate a potent antitumor effect when combined with 5-AZA pre-treatment of target cells. This is particularly important for patients receiving limited numbers of CAR T-cells or for patients with large leukaemic burden. In addition, we speculate that the enhanced cellular cytotoxicity produced by 5-AZA-conditioning may allow the infusion of decreased numbers of CAR19 T-cells. Disclosures No relevant conflicts of interest to declare.

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CAR T cell therapy in B-cell acute lymphoblastic leukaemia: Insights from mathematical models

10.1101/2020.03.18.20038257 ◽

2020 ◽

Cited By ~ 1

Author(s):

Odelaisy León-Triana ◽

Soukaina Sabir ◽

Gabriel F. Calvo ◽

Juan Belmonte-Beitia ◽

Salvador Chulián ◽

...

Keyword(s):

Mathematical Model ◽

T Cells ◽

T Cell ◽

Side Effects ◽

B Cell ◽

Target Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T

AbstractImmunotherapies use components of the patient immune system to selectively target cancer cells. The use of CAR T cells to treat B-cell malignancies – leukaemias and lymphomas– is one of the most successful examples, with many patients experiencing long-lasting complete responses to this therapy. This treatment works by extracting the patient’s T cells and adding them the CAR group, which enables them to recognize and target cells carrying the antigen CD19+, that is expressed in these haematological tumors.Here we put forward a mathematical model describing the time response of leukaemias to the injection of CAR T-cells. The model accounts for mature and progenitor B-cells, tumor cells, CAR T cells and side effects by incorporating the main biological processes involved. The model explains the early post-injection dynamics of the different compartments and the fact that the number of CAR T cells injected does not critically affect the treatment outcome. An explicit formula is found that provides the maximum CAR T cell expansion in-vivo and the severity of side effects. Our mathematical model captures other known features of the response to this immunotherapy. It also predicts that CD19+ tumor relapses could be the result of the competition between tumor and CAR T cells analogous to predator-prey dynamics. We discuss this fact on the light of available evidences and the possibility of controlling relapses by early re-challenging of the tumor with stored CAR T cells.

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Novel Humanised ROR1 Chimeric Antigen Receptors for the Treatment of Haematological Malignancies

Blood ◽

10.1182/blood.v128.22.3361.3361 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3361-3361

Author(s):

Satyen Harish Gohil ◽

Marco Della Peruta ◽

Solange R Paredes-Moscosso ◽

Micaela Harrasser ◽

Gordon Weng-Kit Cheung ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Cell Lines ◽

Target Cell ◽

Reciprocal Relationship ◽

Target Cells ◽

Single Chain ◽

Car T Cells ◽

Solid Malignancies ◽

Car T

Abstract Introduction: Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) is a surface antigen expressed on a range of haematological and solid malignancies including Chronic Lymphocytic Leukaemia (CLL). Although expressed during embryogenesis, its virtual absence on normal adult tissues makes it an attractive target for immunotherapy, especially with Chimeric Antigen Receptor modified T-cells (CAR T-cells). We have generated novel fully humanised ROR1CAR constructs for the treatment of CLL and other ROR1 positive malignancies. Results: Following a rat immunisation programme 38 oligloclonal hybridoma clones were single cell sorted and subjected to 5'RACE. Of 13 novel anti-ROR1 antibodies isolated, 10 retained specific binding when cloned into a heavy-linker-light single chain variable fragment (scFv) format. These scFvs in combination with a second generation CAR architecture comprising CD3zeta and 4-1BB demonstrated specific toxicity against ROR1 positive cell lines after T-cell transduction using lentiviral vectors. We found cytotoxicity with ROR1CAR T-cells was dependent on target cell ROR1 density. In order to ensure our screening assays allowed us to select which of the 10 binders was most suitable for targeting primary CLL, we assessed the antigen density of ROR1 and CD19 on CLL cells. Median expression of ROR1 was 2304 molecules/cell (Range 800-4828), compared to CD19, which had a much higher density of 12,583 (Range 5894-23,652). In view of this, subsequent functional assessment was focused on SKW and Jeko1 cell lines with constitutive ROR1 expression at levels similar to CLL cells, as opposed to those transduced to express supra-physiological levels. Our initial optimisation focused on modifying the CAR extracellular spacer region. We demonstrated a reciprocal relationship between cytotoxicity and the distance between T-cells and target cells. This was assessed by using clones that bound either the membrane-distal immunoglobulin domain or a more membrane-proximal frizzled domain of ROR1. The use of an optimum spacer enhanced cytotoxicity of all scFv constructs but yielded two lead candidates: Clones A & F. These showed consistently superior cytotoxicity against target cell lines compared to the other isolated clones. In addition epitope mapping revealed binding sites unique from the previously described rabbit R12 and murine 4A5 anti-ROR1 CAR T-cells. One of the advantages of targeting ROR1 as opposed to CD19 is sparing the normal B-cell compartment from CAR mediated eradication. However this comes with the consequent risk of B-cell mediated immune responses against rat-derived scFvs. To minimise immunogenicity we undertook a humanisation programme and grafted the complementary determining regions (CDR) of the heavy and light chains of Clone A and F into 5 acceptor human germline VH and VL sequences, generating 25 potential scFvs for each. Binding assessment showed seventeen successfully humanised binders for Clone A and three for Clone F. Of these, 5/17 and 3/3 showed activity in a CAR format against target cells. A final selection was made based on specific cytotoxicity, enhanced cytokine secretion (Interleukin-2 and Interferon gamma) and proliferation compared to the parental clones resulting in 2 unique constructs targeting different extracellular domains of ROR1. In addition, we have demonstrated cytotoxicity against a panel of ROR1 positive solid cancer cell lines to demonstrate their wider applicability. Conclusion: ROR1CAR T-cells have the potential to be an effective therapeutic not just for CLL but also Acute Lymphoblastic Leukaemia, Mantle Cell Lymphoma and solid malignancies. We have described the first humanised ROR1 CARs, which target novel epitopes and have proved effective in relevant pre-clinical assays. Although other ROR1 CARs have been described, we believe the unique properties of these constructs merits further investigation and comparison in the preclinical and clinical setting. Disclosures Gohil: UCL Business: Patents & Royalties: ROR1 based immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Paredes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Pule:Roche: Honoraria; UCL Business: Patents & Royalties; Autolus Ltd: Employment, Equity Ownership, Research Funding; Amgen: Honoraria. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies.

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CD19/CD22 Dual-Targeted Chimeric Antigen Receptor T-Cell Therapy for Relapsed/Refractory Aggressive B-Cell Lymphoma: a Safety and Efficacy Study

Blood ◽

10.1182/blood-2020-143239 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-34

Author(s):

Yongxian Hu ◽

Yanlei Zhang ◽

Houli Zhao ◽

Yiyun Wang ◽

Arnon Nagler ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Target Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T ◽

Aggressive B Cell Lymphoma

Introduction Chimeric antigen receptor (CAR)-T-cell therapy has revolutionized the treatment of relapsed/refractory (R/R) B-cell hematological malignancies, primarily acute lymphoblastic leukemia (ALL), and B-cell non-Hodgkin lymphoma (NHL). CD19 CAR-T cells have been extensively studied and have been shown to yield complete remission (CR) rates of about 90% in R/R ALL, but substantially lower (50%) rates in R/R NHL. Moreover, persistence is usually limited, and antigen escape-mediated relapse is a major limitation. Dual CAR-T cells targeting both CD19 and CD22 may address these limitations. Patients and methods We developed a bispecific CAR-T cells that could concomitantly recognize CD19- and CD22-expressing targets by incorporating both CD19 and CD22 single-chain variables in a single CAR construct (Figure 1A). We designed a prospective study to assess the safety and efficacy profiles of the dual CAR-T therapy in patients with R/R aggressive B-cell lymphoma. Results The preclinical cytotoxicity evaluation of the CD19/CD22 dual-targeted CAR-T cells was performed in comparison with mono-specific CD19-BB-002 and CD22-BB-002 CAR-T cells in HeLa cells that were engineered to express CD19, CD22, or both antigens. The dual-antigen specific CAR-T cells performed equally well when compared with the mono-specific CAR-T cells when there was only a single antigen present on the target cells; better performance was observed when both antigens were present on target cells (Figure 1B). In addition, the dual-antigen specific CAR-T cells induced equal amounts of interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-γ, when compared with the two mono-specific CAR-T cells (Figure 1C). Furthermore, the CD19 CAR-T cells induced more IL-2 and tumor necrosis factor (TNF)-α than the CD22 CAR-T cells and dual-antigen CAR-T cells. However, in the presence of both CD19 and CD22 antigens, the dual-specific CAR-T cell tended to produce more granzyme B, which may explain the higher degree of cytotoxicity when compared with the two mono-specific CAR-T cells (Figure 1D). Twenty-four patients were screened. Of the 16 eligible patients 14 (87.5%) achieved objective response (RR), with 10 (62.5%) achieving complete response (CR). The 2-year overall survival (OS) and progression-free survival (PFS) rates were 77.3% and 40.2%, respectively (Figure 2A). Achieving CR (HR: 0.017, 95% CI: 0.000-0.935; P=0.046) and number of prior lines of chemotherapy (n=2) (HR:135.784, 95% CI: 1.069-17248.110, P=0.047) were found as independent prognostic factors associated with favorable PFS. The 2-year OS and PFS of the CR patients were higher than those of the non-CR patients (100% versus 41.7%, P=0.015; 66.7% versus 0%, P < 0.001), respectively (Figure 2B). The 2-year PFS in patients received 2 prior lines of chemotherapy was higher as compared to those that received more than 2 lines of chemotherapy (68.6% versus 16.7%, P=0.049) whereas the OS in the 2 groups did not differ significantly (83.3% and 71.1%, P=0.613) (Figure 2C). Severe grade 3 cytokine release syndrome (CRS) was observed in only one patient, while 4 had grade one and 11 had grade 2, respectively. No patient developed neurotoxicity. Conclusions Immunotherapy with a novel CD19/CD22 dual targeted CAR-T cells yields a potent and durable anti-lymphoma response with no neurotoxicity or severe CRS. Bispecific CD19/CD22 CAR-T cells represent a safe and potent anti-lymphoma cellular based targeted immunotherapy. Figure 1 Disclosures No relevant conflicts of interest to declare.

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SILAC phosphoproteomics reveals unique signaling circuits in CAR-T cells and the inhibition of B cell-activating phosphorylation in target cells

10.1101/2021.09.10.459784 ◽

2021 ◽

Author(s):

Alijah A. Griffith ◽

Kenneth P. Callahan ◽

Nathan Gordo King ◽

Qian Xiao ◽

Xiaolei Su ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Receptor ◽

Target Cells ◽

Tcr Signaling ◽

Car T Cells ◽

Car T Cell ◽

Car T

AbstractChimeric antigen receptor (CAR) is a single-pass transmembrane receptor designed to specifically target and eliminate cancers. While CARs prove highly efficacious against B cell malignancies, the intracellular signaling events which promote CAR T cell activity remain elusive. To gain further insight into both CAR T cell signaling and the potential signaling response of cells targeted by CAR, we analyzed phosphopeptides captured by two separate phopshoenrichment strategies from third generation CD19-CAR T cells cocultured with SILAC labeled Raji B cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we report that CD19-CAR T cells upregulated several key phosphorylation events also observed in canonical T cell receptor (TCR) signaling while Raji B cells exhibited a significant decrease in B cell receptor-signaling related phosphorylation events in response to coculture. Our data suggest that CD19-CAR stimulation activates a mixture of unique CD19-CAR-specific signaling pathways and canonical TCR signaling while global phosphorylation in Raji B cells is reduced after association with the CD19-CAR T cells.

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CSPG4-Specific CAR T Cells for High-Risk Childhood B Cell Precursor Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms20112764 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2764 ◽

Cited By ~ 6

Author(s):

Dennis C. Harrer ◽

Gerold Schuler ◽

Jan Dörrie ◽

Niels Schaft

Keyword(s):

T Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Leukemia Cell Line ◽

Target Cells ◽

Activation Markers ◽

Car T Cells ◽

Car T ◽

B Cell Leukemia

The advent of CD19-specific chimeric antigen receptor (CAR) T cells has proven to be a powerful asset in the arsenal of cancer immunotherapy of acute lymphoblastic leukemia and certain B cell lymphomas. However, a sizable portion of patients treated with CD19-CAR T cells relapse with CD19-negative cancer cells, necessitating the quest for back-up antigens. Chondroitin sulfate proteoglycan 4 (CSPG4) expression has been reported on leukemic blasts bearing the ill-fated MLL 11q23 rearrangement. We aimed at exploring the use of CSPG4-specific CAR T cells against mixed-lineage leukemia (MLL)-rearranged leukemic blasts, using the precursor B cell leukemia cell line KOPN8 (MLL–MLLT1 translocation) as a model. First, we confirmed CSPG4 expression on KOPN8 cells. Bulk T cells electroporated with mRNA encoding a CSPG4-specific CAR upregulated activation markers and secreted the Th1 cytokines TNF and IFNγ in an antigen-specific manner upon co-culture with KOPN8 cells. More importantly, CSPG4-specific CAR T cells evinced specific degranulation towards KOPN8 cells and specifically lysed KOPN8 target cells in chromium lysis experiments. CSPG4 is a well-established CAR target in cutaneous melanoma. Here, we provide proof-of-principle data for the use of CSPG4-specific CAR T cells against MLL-translocated leukemias.

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