scholarly journals Beyond the Cell Surface: Targeting Intracellular Negative Regulators to Enhance T cell Anti-Tumor Activity

2019 ◽  
Vol 20 (23) ◽  
pp. 5821 ◽  
Author(s):  
Sitaram ◽  
Uyemura ◽  
Malarkannan ◽  
Riese
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Activity ◽  
Sh2 Domain ◽  
Extracellular Proteins ◽  
Car T Cells ◽  
Car T ◽  
Src Homology ◽  
Anti Tumor Activity

It is well established that extracellular proteins that negatively regulate T cell function, such as Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) and Programmed Cell Death protein 1 (PD-1), can be effectively targeted to enhance cancer immunotherapies and Chimeric Antigen Receptor T cells (CAR-T cells). Intracellular proteins that inhibit T cell receptor (TCR) signal transduction, though less well studied, are also potentially useful therapeutic targets to enhance T cell activity against tumor. Four major classes of enzymes that attenuate TCR signaling include E3 ubiquitin kinases such as the Casitas B-lineage lymphoma proteins (Cbl-b and c-Cbl), and Itchy (Itch), inhibitory tyrosine phosphatases, such as Src homology region 2 domain-containing phosphatases (SHP-1 and SHP-2), inhibitory protein kinases, such as C-terminal Src kinase (Csk), and inhibitory lipid kinases such as Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase (SHIP) and Diacylglycerol kinases (DGKs). This review describes the mechanism of action of eighteen intracellular inhibitory regulatory proteins in T cells within these four classes, and assesses their potential value as clinical targets to enhance the anti-tumor activity of endogenous T cells and CAR-T cells.

scholarly journals Remote control of CAR T cell therapies by thermal targeting

2020 ◽  
Author(s):  
Ian C. Miller ◽  
Lee-Kai Sun ◽  
Adrian M. Harris ◽  
Lena Gamboa ◽  
Ali Zamat ◽  
...  
Keyword(s):  
T Cells ◽  
Remote Control ◽  
T Cell ◽  
Cell Activity ◽  
Therapeutic Window ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity ◽  
Thermal Targeting

ABSTRACTThe limited ability to control anti-tumor activity within tumor sites contributes to poor CAR T cell responses against solid malignancies. Systemic delivery of biologic drugs such as cytokines can augment CAR T cell activity despite off-target toxicity in healthy tissues that narrows their therapeutic window. Here we develop a platform for remote control of CAR T therapies by thermal targeting. To enable CAR T cells to respond to heat, we construct synthetic thermal gene switches that trigger expression of transgenes in response to mild elevations in local temperature (40–42 °C) but not to orthogonal cellular stresses such as hypoxia. We show that short pulses of heat (15–30 min) lead to more than 60-fold increases in gene expression without affecting key T cell functions including proliferation, migration, and cytotoxicity. We demonstrate thermal control of broad classes of immunostimulatory agents including CARs, Bispecific T cell Engagers (BiTEs), and cytokine superagonists to enhance proliferation and cell targeting. In mouse models of adoptive transfer, photothermal targeting of intratumoral CAR T cells to control the production of an IL-15 superagonist significantly enhances anti-tumor activity and overall survival. We envision that thermal targeting could improve the safety and efficacy of next-generation therapies by allowing remote control of CAR T cell activity.

scholarly journals P07.02 Regulation of CD19 CAR T- cell activation based on engineered Nuclear factor of activated T cells artificial transcription factors

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A23-A23
Author(s):  
D Lainšček ◽  
V Mikolič ◽  
Š Malenšek ◽  
A Verbič ◽  
R Jerala
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Activity ◽  
External Control ◽  
Car T Cells ◽  
Car T Cell ◽  
Artificial Transcription Factors ◽  
Car T

BackgroundCD19 CAR T- cells (Chimeric antigen receptor T cells that recognize CD19) present a therapeutic option for various malignant diseases based on their ability to specifically recognize the selected tumour surface markers, triggering immune cell activation and cytokine production that results in killing cancerous cell expressing specific surface markers recognized by the CAR. The main therapeutic effect of CAR is a specific T cell activation of adequate cell number with sequential destruction of tumorous cells in a safe therapeutic manner. In order to increase T cell activation, different activation domains were introduced into CAR. CAR T-cells are highly efficient in tumour cell destruction, but may cause serious side effects that can also result in patient death so their activity needs to be carefully controlled.1 Several attempts were made to influence the CAR T cell proliferation and their activation by adding T cell growth factors, such as IL-2, into patients, however this approach of increasing the number of activating T cells with no external control over their number can again lead to non-optimal therapeutic effects. Different improvements were made by designing synthetic receptors or small molecule-inducible systems etc., which influence regulated expansion and survival of CAR T cells.2Material and MethodsIn order to regulate CD19 CAR-T cell activity, different NFAT2 based artificial transcription factors were prepared. The full length NFAT2, one of the main players in T cell IL2 production, a key cytokine for T cell activation and proliferation was truncated by deletion of its own activation domain. Next, we joined via Gibson assembly tNFAT21-593 coding sequence with domains of different heterodimerization systems that interact upon adding the inductor of heterodimerization. The interaction counterparts were fused to a strong tripartite transcriptional activator domain VPR and/or strong repressor domain KRAB resulting in formation of an engineered NFAT artificial transcription (NFAT-TF) factors with external control. To determine the activity of NFAT-TF HEK293, Jurkat or human T cells were used.ResultsBased on luciferase assay, carried out on NFAT-TF transfected HEK293 cells we first established that upon adding the external inductor of heterodimerization, efficient gene regulation occurs, according to VPR or KRAB domain appropriate functions. Findings were then transferred to Jurkat cells that were electroporated with appropriate DNA constructs, coding for NFAT-TF and CD19 CAR. After Raji:Jurkat co-culture ELISA measurements revealed that IL2 production and therefore CD19 CAR-T cell activity can be controlled by the action of NFAT-TF. The same regulation over the activity and subsequent proliferation status was also observed in retrovirally transduced human T-cells.ConclusionWe developed a regulatory system for therapeutic effect of CD19 CAR-T cells, a unique mechanism to control T cell activation and proliferation based on the engineered NFAT2 artificial transcription factor.ReferencesBonifant CL, et al. Toxicity and management in CAR T-cell therapy. Mol Ther Oncolytics 2016;3:16011.Wu C-Y, et al. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science 2015;80:350.Disclosure InformationD. Lainšček: None. V. Mikolič: None. Š. Malenšek: None. A. Verbič: None. R. Jerala: None.

scholarly journals P08.05 Combined pharmacological targeting of adenosine 2a- and 2b-receptor enhances CAR T cell function

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A26.2-A27
Author(s):  
M Seifert ◽  
M Benmebarek ◽  
B Cadilha ◽  
J Jobst ◽  
J Dörr ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
Cell Function ◽  
Cytokine Secretion ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Cell Effector

BackgroundDespite remarkable response rates mediated by anti-CD19 chimeric antigen receptor (CAR) T cells in selected B cell malignancies, CAR T cell therapy still lacks efficacy in the vast majority of tumors. A substantial limiting factor of CAR T cell function is the immunosuppressive tumor microenvironment. Among other mechanisms, the accumulation of adenosine within the tumor can contribute to disease progression by suppressing anti-tumor immune responses. Adenosine 2a- and 2b-receptor (A2A and A2B)-mediated cAMP build-up suppresses T cell effector functions. In the present study we hypothesize, that combination therapy with the selective A2A/A2B dual antagonist AB928 (etrumadenant) enhances CAR T cell efficacy.Materials and MethodsSecond generation murine (anti-EPCAM) and human (anti-MSLN) CAR constructs, containing intracellular CD28 and CD3ζ domains, were fused via overlap extension PCR cloning. Murine or human T cells were retrovirally transduced to stably express the CAR constructs. A2A/A2B signaling in CAR T cells was analyzed by phospho-specific flow cytometry of CREB (pS133)/ATF-1 (pS63). CAR T cell activation was quantified by flow cytometry and enzyme-linked immunosorbent assay (ELISA) of IFN-γ, IL-2 and TNF-α. CAR T cell proliferation was assessed by flow cytometry. CAR T cell cytotoxicity was assessed by impedance based real-time cell analysis.ResultsAB928 protected murine CAR T cells from cAMP response element-binding protein (CREB) phosphorylation in the presence of stable adenosine analogue 5′-N-ethylcarboxamidoadenosine (NECA). NECA inhibited antigen-dependent CAR T cell cytokine secretion in response to four murine tumor cell lines. CAR T cell-mediated tumor cell lysis as well as proliferation were decreased in the presence of NECA or adenosine. Importantly, AB928 fully restored CAR T cell cytotoxicity, proliferation, and cytokine secretion in a dose dependent manner. Further, AB928 also restored antigen dependent cytokine secretion of human CAR T cells in the presence of NECA.ConclusionsHere we used the A2A/A2B dual antagonist AB928 to overcome adenosine-mediated suppression of CAR T cells. We found that AB928 enhanced important CAR T cell effector functions in the presence of the adenosine analogue, suggesting that combination therapy with AB928 may improve CAR T cell efficacy. This study was limited to in vitro experiments. To confirm the relevance of our findings, this combination therapy must be further investigated in an in vivo setting.Disclosure InformationM. Seifert: None. M. Benmebarek : None. B. Cadilha : None. J. Jobst: None. J. Dörr: None. T. Lorenzini: None. D. Dhoqina: None. J. Zhang: None. J. Zhang: None. U. Schindler: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Amgen Inc., Arcus Biosciences. Other; Significant; Arcus Biosciences. S. Endres: None. S. Kobold: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Arcus Biosciences.

scholarly journals B-CLL Mediated Resistance to CAR T Cell Therapy Via Insufficient Activation Is CAR-Independent

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-44
Author(s):  
McKensie Collins ◽  
Weimin Kong ◽  
Inyoung Jung ◽  
Stefan M Lundh ◽  
J. Joseph Melenhorst
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cytokine Production ◽  
Cell Function ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, analysis of cytokine production by flow cytometry showed that CLL-stimulated CAR T cells did not produce IL-2 following a 6- or 12-hour stimulus, but TNFα was expressed after 12-hours. Similarly, CAR T cell degranulation, a prerequisite for target cell lysis was triggered after CLL recognition. These data again suggested that CLL cells insufficiently stimulate CAR T cell cytokine production, but also showed that cytolytic activity against CLL cells is intact. We further proposed that CLL cells express insufficient levels of co-stimulatory and adhesion molecules to activate CAR T cells. Flow cytometry showed that most CLL cells expressed co-stimulatory and adhesion molecules at low levels; we hypothesized that up-regulating these molecules would enhance CAR T cell targeting of CLL cells. CLL cells were activated with CD40L and IL-4, which increased expression of CD54, CD58, CD80, and CD86. Stimulating CAR T cells with activated CLL cells enhanced CAR T cell proliferation and induced cell conjugate formation, indicating cell activation. Therefore, improving CLL stimulatory capacity can rescue T cell dysfunctions. To assess whether IL-2 addition and CD40 ligation were synergistic, we combined the two assays; however, we saw no additional improvement over IL-2 addition alone, suggesting that the two interventions may act upon the same pathway. Importantly, we also showed that rescue of CAR T cell function via IL-2 addition or CD40 ligation was not CAR-specific, as we observed the functional defects and subsequent rescue with both a ROR1-targeting CAR and the gold standard CD19-targeting CAR. Conclusions: Together, these data show that CAR T cell "defects" in CLL are actually insufficient activation, and improving the stimulatory capacity of CLL cells may enable better clinical responses. Further, this effect is not CAR-specific and these results may therefore be broadly applicable to multiple therapies for this disease. Disclosures Melenhorst: IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Novartis: Other: Speaker, Research Funding; Johnson & Johnson: Consultancy, Other: Speaker; Simcere of America: Consultancy; Poseida Therapeutics: Consultancy.

scholarly journals Treatment with Acalibrutinib, Ibrutinib and CD19 CAR T Cells Restore the Number of Granulocytic Myeloid Derived Supressor Cells in CLL-Bearing Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3032-3032
Author(s):  
Arantxa Romero-Toledo ◽  
Robin Sanderson ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Research Funding ◽  
Cell Function ◽  
Car T Cells ◽  
Car T Cell ◽  
T Cell Function ◽  
Car T ◽  
Secondary Lymphoid Organs

The complex crosstalk between malignant chronic lymphocytic leukemia (CLL) cells and the tumor microenvironment (TME) is not fully understood. CLL is associated with an inflammatory TME and T cells exhibit exhaustion and multiple functional defects, fully recapitulated in Eµ-TCL1 (TCL1) mice and induced in healthy mice by adoptive transfer (AT) of murine CLL cells, making it an ideal model to test novel immunotherapies for this disease. Myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, are immature myeloid cells with the ability to suppress T cell function and promote Treg expansion. In humans, CLL cells can induce conversion of monocytes to MDSCs provoking their accumulation in peripheral blood (PB). MDSCs include two major subsets granulocytic (Gr) and monocytic (M)-MDSC. In mice, Gr-MDSCs are defined as CD11b+Ly6G+Ly6Clo and M-MDSC as CD11b+Ly6G-Ly6Chi. Both murine and human MDSCs express BTK. We observed that in CLL-bearing mice, MDSCs cells are lost in PB as disease progresses. Treatment with both BTK inhibitors (BTKi), ibrutinib (Ibr) and acalabrutinib (Acala), result in shift of T cell function from Th2 towards Th1 polarity and increase MDSC populations in vivo. We aimed to determine whether combination treatment with BTKi and chimeric antigen receptor (CAR) T cells renders recovery of the MDSC population in CLL-bearing mice. To address this question we designed a two-part experiment, aiming to mimic the clinically relevant scenario of pre-treatment of CLL with BTKi to improve CAR T cell function. Part 1 of our experiment consisted of 4 groups (n=12) of 2.5 month old C57/Bl6 mice. Three groups had AT with 30x106 TCL1 splenocytes. A fourth group of WT mice remained CLL-free as a positive control and donors for WT T cells. When PB CLL load reached >10% (day 14) animals were randomized to either Ibr or Acala at 0.15 mg/l in 2% HPBC or no treatment for 21 days. All animals from part 1 were culled at day 35 post-AT and splenic cells were isolated, analyzed and used to manufacture CAR T cells. WT, CLL, Ibr and Acala treated T cells were activated and transduced with a CD19-CD28 CAR to treat mice in part 2. Here, 50 WT mice were given AT with 20x106 TCL1 splenocytes for CLL engraftment. All mice were injected with lymphodepleting cyclophosphamide (100mg/kg IP) one day prior to IV CAR injection. At day 21 post-AT, mice were treated with WT CAR, CLL CAR, IbrCAR, AcalaCAR or untransduced T cells. MDSC sub-populations were monitored weekly in PB and SP were analysed by flow cytometry. As malignant CD19+CD5+ cells expands in PB, the overall myeloid (CD19-CD11b+) cell population was not affected, but MDSCs significantly decreased (p<0.0001). Treatment with Acala, but not Ibr restores total MDSCs. However, MDSC impairment occurs in the Gr- but not M- MDSC population and both Acala and Ibr restores this population (Figure 1a). When we examined the spleen, treatment with both Ibr (p<0.001) and Acala (p<0.001) reduced CD5+CD19+ cells, whereas neither BTKi affected the overall myeloid (CD19-CD11b+) cell population. Gr-MDSCs were restored by both treatments whilst M-MDSCs were only restored after Ibr treatment (p<0.001 in each case). In part 2 of this experiment we observed that treatment with all CAR-T cell groups provokes the clearance of all CD19+CD5+ cells. The overall CD19-CD11b+ population stays the same across all mice groups 35 days after treatment in PB with any group of CAR and untransduced T cells. Overall MDSC population is maintained following all CAR T cells compared to CLL-bearing mice (p<0.0001) and it is the Gr- but not the M- MDSC population which is recovered in PB (Figure 1b). These parts of the experiments can of course be influenced by treatment with cyclophosphamide. We conclude that novel therapies for CLL treatment have an effect not only in CLL cells but also in non-malignant cell components of the TME. In this animal model of CLL, the rapid expansion of CLL cells in PB and secondary lymphoid organs provokes loss of MDSC, particularly the Gr-MDSC subpopulation is affected. Treatment with BTKi and CAR T cells provokes clearance of CLL cells in PB and spleen allowing MDSC recovery; suggesting this may be BTK and ITK independent. We continue to explore secondary lymphoid organs to further characterize the shift of the CLL microenvironment from an immunosuppressive to an immune effective one and its impact on immune function in this model. Disclosures Sanderson: Kite/Gilead: Honoraria. Gribben:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

scholarly journals Next Generation NKG2D-based CAR T-cells (CYAD-02): Co-expression of a Single shRNA Targeting MICA and MICB Improves Cell Persistence and Anti-Tumor Efficacy in vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3931-3931
Author(s):  
Martina Fontaine ◽  
Benjamin Demoulin ◽  
Simon Bornschein ◽  
Susanna Raitano ◽  
Steve Lenger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Next Generation ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Anti Tumor Activity ◽  
Nkg2d Receptor

Background The Natural Killer Group 2D (NKG2D) receptor is a NK cell activating receptor that binds to eight different ligands (NKG2DL) commonly over-expressed in cancer, including MICA and MICB. The product candidate CYAD-01 are chimeric antigen receptor (CAR) T-cells encoding the full length human NKG2D fused to the intracellular domain of CD3ζ. Data from preclinical models have shown that CYAD-01 cells specifically target solid and hematological tumors. Encouraging preliminary results from the Phase I clinical trial THINK, assessing CYAD-01 safety, showed initial signals of objective clinical responses in patients with r/r AML and MDS. The clinical development of CAR T-cells has been limited by several challenges including achieving sufficient numbers of cells for clinical application. We have previously shown that NKG2D ligands are transiently expressed on activated T cells and that robust cell yields are generated through the addition of a blocking antibody and a PI3K inhibitor during cell manufacture. Here, we investigated the ability of an optimized short hairpin RNA (shRNA) technology to modulate NKG2DL expression on CYAD-01 cells and to determine if there is an increase in the anti-tumor activity of NKG2D-based CAR T-cells (termed CYAD-02). Methods Molecular and cellular analyses identified MICA and MICB as the key NKG2DL expressed on activated T-cells and highly likely to participate in driving fratricide. In silico analysis and in vitro screening allowed the identification of a single shRNA targeting the conserved regions of MICA and MICB, thus downregulating both MICA and MICB expression. The selected shRNA was incorporated in the NKG2D-based CAR vector, creating the next-generation NKG2D-based CAR T-cell candidate, CYAD-02. In addition, truncated versions of the NKG2D receptor were generated to explore the mechanisms of action of NKG2D receptor activity in vivo. The in vivo persistence and anti-tumor activity of CYAD-02 cells was evaluated in an aggressive preclinical model of AML. Results Injection of CAR T-cells bearing truncated forms of the NKG2D-CAR in immunosuppressed mice resulted in similar persistence to the control T-cells. In contrast, CYAD-01 cells had reduced persistence, suggesting that the recognition of the NKG2DL by the NKG2D receptor could contribute to this effect. Analysis of cell phenotype upon CAR T-cell activation showed that MICA and MICB were transiently expressed on T-cells during manufacturing. These results collectively suggested that downregulating MICA and MICB expression in CYAD-01 cells could be a mean to increase CAR T-cell persistence in vivo. Candidate shRNA were screened for efficient targeting of both MICA and MICB at the mRNA and protein level. T-cells transduced with a single vector encoding for the NKG2D-based CAR and the selected shRNA targeting MICA and MICB (CYAD-02) demonstrated 3-fold increased expansion during in vitro culture in the absence of the blocking antibody used to increase cell yield during manufacture. When injected into immunosuppressed mice, CYAD-02 cells generated with the Optimab process showed 10-fold higher engraftment one week after injection and potent anti-tumor activity resulting in 2.6-fold increase of mouse survival in an aggressive AML model. Conclusions By using a single vector encoding the NKG2D-based CAR next to a shRNA targeting MICA and MICB and combined with improved cell culture methods, CYAD-02, the next-generation of NKG2D-based CAR T-cells, demonstrated enhanced in vivo persistence and anti-tumor activity. Following FDA acceptance of the IND application, a Phase 1 dose-escalation trial evaluating the safety and clinical activity of CYAD-02 for the treatment of r/r AML and MDS is scheduled to start in early 2020. Disclosures Fontaine: Celyad: Employment. Demoulin:Celyad: Employment. Bornschein:Celyad: Employment. Raitano:Celyad: Employment. Machado:Horizon Discovery: Employment. Moore:Avvinity Therapeutics: Employment, Other: Relationship at the time the work was performed; Horizon Discovery: Employment, Equity Ownership, Other: Relationship at the time the work was performed; Centauri Therapeutics: Consultancy, Other: Current relationship. Sotiropoulou:Celyad: Employment. Gilham:Celyad: Employment.

scholarly journals P12.05 Evaluating the compatibility of tumor treating electric fields with key antitumoral immune functions

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii60-iii60
Author(s):  
G Diamant ◽  
H Simchony ◽  
T Shiloach ◽  
A Globerson-Levin ◽  
L Gasri Plotnitsky ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Function ◽  
Rational Approach ◽  
Immune Functions ◽  
Color Flow ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T

Abstract BACKGROUND TTFields has the ability to induce immunogenic cell death (ICD). As immunotherapy and TTFields have different mechanisms of action (MOA), combining these therapies is a rational approach. Contrarily, TTFields may interfere with immune functions critical for effective T cell function. MATERIAL AND METHODS We cultured T cells from healthy donors’ peripheral blood or from viably dissociated glioblastoma samples under normal or TTFields conditions, with or without superantigen-stimulation. In order to assess T cell responses we used eight-color flow cytometry by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Evaluation of direct cytotoxicity was done by using chimeric antigen receptor (CAR) T cells. RESULTS TTFields did not change T cell activation rates for all evaluated functions with the exception of reduced proliferation - in line with TTFields’ MOA. TTFields substantially reduced the viability of activated proliferating T cells, moderately affected activated nonproliferating T cells and had almost no effect on the viability of non-activated cells. Polyfunctionality analysis of T-cells, associated with effective antitumoral responses, demonstrated that under TTFields, the activated non-proliferating T cells retained polyfunctional capabilities. PD1-expressing TILs, a subset containing most of the tumor antigen-specific TILs, exhibited unaltered viability and functionality under TTFields. CAR T-cells, which utilize the same killing machinery as unmodified T cells, exhibited unaltered cytotoxic capability under TTFields. Immunohistochemical evaluation of GBM samples before TTFields treatment and after recurrence showed that some patients had accommodated large increases in their CD8 and CD4 counts. RNA-Seq performed on GBM samples from 6 standardly-treated and 6 TTFields-treated patients before treatment and after recurrence. The data shows differential increases in TTFields-treated patients to controls, in the expression of immune genes associated with favorable prognosis (e.g. t-bet, NKG2D, ICOS-L, CD70) and concurrent decreases in genes associated with poor prognosis (e.g. IL4, TSLP, various complement genes). CONCLUSION The preclinical data showed that all antitumoral T cell functions examined, but proliferation, were unhindered by TTFields. The clinical data showed that TTFields may shift treated tumors to a state more conducive of antitumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy.

CD19-Redirected Chimeric Antigen Receptor T (CART19) Cells Induce a Cytokine Release Syndrome (CRS) and Induction of Treatable Macrophage Activation Syndrome (MAS) That Can Be Managed by the IL-6 Antagonist Tocilizumab (toc).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2604-2604 ◽  
Author(s):  
Stephan A. Grupp ◽  
David L Porter ◽  
David T Teachey ◽  
David M. Barrett ◽  
Anne Chew ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Tumor Lysis Syndrome ◽  
Antigen Receptor ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

Abstract Abstract 2604 We previously reported on CART19 cells expressing a chimeric antigen receptor (CAR) with intracellular activation and costimulatory domains. Infusion of these cells results in 100 to 100,000× in vivo proliferation, tumor lysis syndrome followed by durable antitumor activity, and prolonged persistence in pts with B cell tumors. Here we report that in vivo proliferation of CART19 cells and potent anti-tumor activity is associated with CRS, leading to hemophagocytic lymphohistiocytosis (HLH), also termed MAS. We propose that MAS/HLH is a unique biomarker that is associated with and may be required for potent anti-tumor activity. Autologous T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3-zeta, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into ALL or CLL pts with persistent disease after 2–8 prior treatments. CART19 anti ALL activity was also modeled in a xenograft mouse model with high level of human ALL/human T cell engraftment and simultaneous detection of CAR T cells and ALL using 2-color bioluminescent imaging. We describe updated results of 10 pts who received CART19 cells elsewhere at ASH (Porter, et al), including 9 pts with CLL and 1 pediatric pt with relapsed refractory ALL. 6/9 evaluable pts had a CR or PR, including 4 sustained CRs. While there was no acute infusional toxicity, all responding pts also developed CRS. All had high fevers, as well as grade 3 or 4 hypotension/hypoxia. CRS preceded peak blood expression of CART19 cells, and then increased in intensity until the CART19 cell peak (D10–31 after infusion). The ALL pt experienced the most significant toxicity, with grade 4 hypotension and respiratory failure. Steroid therapy on D6 resulted in no improvement. On D9, noting high levels of TNFa and IL-6 (peak increases above baseline: IFNg at 6040x; IL-6 at 988x; IL-2R at 56x, IL-2 at 163× and TNFa at 17x), we administered TNFa and IL-6 antagonists entanercept and toc. This resulted in resolution of fever and hypotension within 12hr and a rapid wean from ventilator support to room air. These interventions had no apparent impact on CART19 cell expansion or efficacy: peak of CAR T cells (2539 CAR+ cells/uL; 77% of CD3 cells by flow) occurred on D11, and D23 bone marrow showed CR with negative MRD, compared to her initial on-study marrow which showed 65% blasts. Although she had no history of CNS ALL, spinal fluid showed detectable CART19 cells (21 lymphs/mcL; 78% CAR+). At 4mo post infusion, this pt remains in CR, with 17 CART19 cells/uL in the blood and 31% CAR+ CD3 cells in the marrow. Clinical assessment of subsequent responding patients shows all had evidence of MAS/HLH including dramatic elevations of ferritin and histologic evidence of HLH. Peak ferritin levels range from 44,000 to 605,000, preceding and continuing with peak T cell proliferation. Other consistent findings include rapid onset hepatosplenomegaly unrelated to disease and moderate DIC. Subsequently, 3 CLL patients have also been treated with toc, also with prompt and striking resolution of high fevers, hypotension and hypoxia. 1 received toc on D10 and achieved a CR accompanied by CART19 expansion. 1 had rapid resolution of CRS following toc administration on day 9 and follow up for response is too short. A 3rd CLL pt received toc on D3 for early fevers and had no CART-19 proliferation and no response. To model the timing of cytokine blockade, xenografts using bioluminescent primary pediatric ALL were established and then treated with extra cells from the clinical manufacture. The CART19 cells proliferated and resulted in prolonged survival. Cytokine blockade prior to T cell infusion with toc and/or etanercept abrogated disease control with less in vivo proliferation of infused CART19 cells, confirming the result seen in the one pt given early toc (D3). The optimal time and threshold to trigger cytokine blockade is currently being tested in these models. CART19 T cells can produce massive in-vivo expansion, long-term persistence, and anti-tumor efficacy, but can also induce significant CRS with features suggestive of MAS/HLH that responds rapidly to cytokine blockade. Given prior to initiation of significant CART19 proliferation, blockade of TNFa and/or IL-6 may interfere with proliferation and effector function, but if given at a point where cell proliferation is underway, toc may ameliorate the symptoms that we have observed correlate with robust clinical responses. Disclosures: Off Label Use: tocilizumab for cell therapy toxicity. Levine:University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties; TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kalos:University of Pennsylvania: Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties.

scholarly journals BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved antitumor activity

2021 ◽  
Author(s):  
Taylor L Hickman ◽  
Eugene Choi ◽  
Kathleen R Whiteman ◽  
Sujatha Muralidharan ◽  
Tapasya Pai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Solid Tumor ◽  
Cell Function ◽  
Cell Metabolism ◽  
Car T Cells ◽  
Car T

Purpose: The solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models. Experimental Design: We evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo. Results: Expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions. Conclusions: Together, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).

scholarly journals Suppression of 4.1R enhances the potency of NKG2D-CAR T cells against pancreatic carcinoma via activating ERK signaling pathway

Oncogenesis ◽  
2021 ◽  
Vol 10 (9) ◽  
Author(s):  
Yaoxin Gao ◽  
Haizhen Lin ◽  
Dandan Guo ◽  
Sijia Cheng ◽  
Ying Zhou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Pancreatic Carcinoma ◽  
Signaling Pathway ◽  
Xenograft Model ◽  
Erk Signaling ◽  
Dependent Manner ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity

AbstractPancreatic carcinoma (PC) is one of the most common malignancies. Chimeric antigen receptor (CAR)-modified T cells has achieved remarkable efficacy in the treatment of hematological malignancies. However, lack of tumor-specific targets and the existence of inhibitory factors limit the function of CAR T cells when treating solid tumors. 4.1R has been reported to suppress the anti-tumor activity of T cell responses. In this study, we investigated the anti-tumor activity of 4.1R deletion in natural killer group 2D (NKG2D)-CAR T cells against PC. The CAR T cells were obtained by transfecting T cells with lentiviral vector carrying NKG2D-CAR, NC-NKG2D-CAR, or KD2-NKG2D-CAR. In vitro, NKG2D-CAR T cells showed higher cytotoxicity than Mock T cells. However, compared to NKG2D-CAR T cells, furtherly higher cytotoxicity against PC cells in a dose-dependent manner was found in KD2-NKG2D-CAR T cells. In addition, the proliferation rate and cytotoxic activity of KD2-NKG2D-CAR T cells were significantly higher than those of NKG2D-CAR T cells. Besides, the inhibitory receptors PD-1 and TIM-3 were expressed in lower level on KD2-NKG2D-CAR T cells. In vivo, KD2-NKG2D-CAR T cells suppressed tumor growth more effectively in a xenograft model compared to NKG2D-CAR T cells. Mechanistically, 4.1R regulated CAR T cell function via activating ERK signaling pathway. Therefore, the study provides a new idea to enhance the anti-tumor efficiency of CAR T therapy.

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