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.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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 Lenalidomide Enhances CAR-T Cell Activity Against Solid Tumor Cells

2020 ◽  
Vol 29 ◽  
pp. 096368972092082 ◽  
Author(s):  
Zhixiong Wang ◽  
Guomin Zhou ◽  
Na Risu ◽  
Jiayu Fu ◽  
Yan Zou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cytokine Secretion ◽  
Target Cells ◽  
Functional Assay ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T-cell immunotherapy still faces many challenges in the treatment of solid tumors, one of which is T-cell dysfunction or exhaustion. Immunomodulator lenalidomide may improve CAR T-cell function. In this study, the effects of lenalidomide on CAR T-cell functions (cytotoxicity, cytokine secretion, and cell proliferation) were investigated. Two different CAR T cells (CD133-specific CAR and HER2-specific CAR) were prepared, and the corresponding target cells including human glioma cell line U251 CD133-OE that overexpress CD133 and human breast cancer cell line MDA-MB-453 were used for functional assay. We found that lenalidomide promoted the killing of U251 CD133-OE by CD133-CAR T cells, the cytokine secretion, and the proliferation of CD133-CAR T cells. Lenalidomide also enhanced the cytotoxicity against MDA-MB-453 and the cytokine secretion of HER2-CAR T cells but did not affect their proliferation significantly. Furthermore, lenalidomide may regulate the function of CAR T cells by inducing the degradation of transcription factors Ikaros and Aiolos.

scholarly journals Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

2021 ◽  
Vol 12 ◽  
Author(s):  
Ulrich Blache ◽  
Ronald Weiss ◽  
Andreas Boldt ◽  
Michael Kapinsky ◽  
André-René Blaudszun ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Cytotoxic Agents ◽  
Clinical Staging ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

scholarly journals CAR2.0 Therapy for the Management of Post-Transplantation Relapse of B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Rui Zhang ◽  
Juan Xiao ◽  
Zhouyang Liu ◽  
Yuan Sun ◽  
Sanfang Tu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (allo-HCT) is a standard treatment for relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). However ~30-40% of patients (pts) still relapse after HCT. We report a cohort of 20 r/rB-ALL pts, who relapsed after HCT, and enrolled in the CAR2.0 study receiving one or two types of CAR-T cells targeting various B-ALL antigens. METHOD: Pts with r/r B-ALL who relapsed after allo-HCT and did not have significant active comorbiditeis, were enrolled in the study. The target antigens were determined based on immunostaining of each pt's leukemia cells, and CAR-T infusions included a single, or a combination of CAR-Ts targeting the following antigens: CD19, CD22, CD123 and CD38. T cells were collected from pts (N=4) or their allogeneic donors (N=16) and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR). Pts received cyclophosphamide/fludarabine lymphodepleting therapy before infusion of 0.2-5.8x106 CAR-T/kg per infusion. In addition to disease response, we carefully monitored the quality of apheresis cells, efficiency of gene transfer, T cell proliferation rate, CAR-T infusion dose, and the CAR-T copy number in peripheral blood. RESULTS: Among the 20 enrolled pts, 11 were &lt;18 years of age, and 7 were BCR- ABL (P190) positive. Before CAR-T treatment, 7 pts had ≤grade 2 active graft-versus-host disease (GVHD), and 13 pts received chemotherapy or targeted therapy after their relapse post HCT. Six pts had extramedullary relapse and 2 of them also had bone marrow relapse. The tumor burden in bone marrow ranged from minimal residual disease (MRD) negative to 66% of blasts, based on flow cytometry before CAR-T therapy. Five pts had &gt;10% blasts in bone marrow, 8 pts had &lt;3% blasts, and 7 pts had MRD negative bone marrow (summarized in the Table below). Based on the GVHD history, chimerism state and the available T-cell sources, 16 pts used allogeneic HCT donor T-cells for CAR-T preparation. All pts were full donor chimeras prior to CAR-T infusion, except one pt who had 41% donor cells in bone marrow. Eleven pts received a single CD19 CAR-T infusion, with a mean dose of 1.6x106 CAR-T/kg, and ten achieved an MRD remission and one had progressive disease (PD) within 60 days by flow cytometry. The remaining 9 pts received 2 CAR-Ts (CD19 plus CD22, CD123 or CD38 CAR-Ts) given on the same day, and resulted in 8 CR and 1 PD within 60 days. After CAR-T infusion, no cytokine release syndrome (CRS) was observed in 8 pts, and 12 pts experienced CRS of grade 1, which was consistent with the previously described low toxicity profile of the 4SCAR design. Acute GVHD ≤ grade 2 developed in 5 pts within one month following CAR-T cell infusion but all responded well to supportive care and/or cyclosporine infusion. The 2 pts who developed PD after CAR-T infusion included the one with 41% donor chimerism and had grade 2 GVHD and active infections before CAR-T infusion. The other pt with PD following CAR-T had severe bone marrow suppression, low leukocyte count, infections and was transfusion dependent before enrollment. This emphasizes the need for controlling comorbidities before infusion of CAR-T cells. In summary, total 18 patients (90%) achieved negative MRD remission within 2 months of therapy with acceptable CRS. Four pts relapsed (after being in remission for 3 months) and 14 pts are in continued remission, 6 of which for &gt; 1 year. None of these 20 pts received a second HCT after CAR-T infusion. GVHD developed in 5/16 (31%) pts after donor source CAR-T cell infusion within one month, but all responded well to treatment. CONCLUSION: This study focuses on CAR-T cell therapy following relapse after HCT. While the expanded study is ongoing, we present results of the first 20 pts. Use of donor-derived or recipient-derived CAR-T products in pts who relapsed after allo-HCT is well tolerated and it may prolong life expectancy of these pts while maintaining good quality of life. Table Disclosures No relevant conflicts of interest to declare.

scholarly journals Hinge and Transmembrane Domains of Chimeric Antigen Receptor Regulate Receptor Expression and Signaling Threshold

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1182 ◽  
Author(s):  
Kento Fujiwara ◽  
Ayaka Tsunei ◽  
Hotaka Kusabuka ◽  
Erika Ogaki ◽  
Masashi Tachibana ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Transmembrane Domains ◽  
Expression Level ◽  
Expression Levels ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Hinge Domain

Chimeric antigen receptor (CAR)-T cells have demonstrated significant clinical potential; however, their strong antitumor activity may cause severe adverse effects. To ensure efficacy and safe CAR-T cell therapy, it is important to understand CAR’s structure–activity relationship. To clarify the role of hinge and transmembrane domains in CAR and CAR-T cell function, we generated different chimeras and analyzed their expression levels and antigen-specific activity on CAR-T cells. First, we created a basic CAR with hinge, transmembrane, and signal transduction domains derived from CD3ζ, then we generated six CAR variants whose hinge or hinge/transmembrane domains originated from CD4, CD8α, and CD28. CAR expression level and stability on the T cell were greatly affected by transmembrane rather than hinge domain. Antigen-specific functions of most CAR-T cells depended on their CAR expression levels. However, CARs with a CD8α- or CD28-derived hinge domain showed significant differences in CAR-T cell function, despite their equal expression levels. These results suggest that CAR signaling intensity into T cells was affected not only by CAR expression level, but also by the hinge domain. Our discoveries indicate that the hinge domain regulates the CAR signaling threshold and the transmembrane domain regulates the amount of CAR signaling via control of CAR expression level.

Effect of Rituximab On the Activity of T Cells Expressing CD20-Specific Chimeric Antigen Receptors

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4222-4222
Author(s):  
Gregory A. Rufener ◽  
Michael C. Jensen ◽  
Shaunda Brouns ◽  
Lihua E. Budde ◽  
David G. Maloney ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Cell Function ◽  
Cytokine Secretion ◽  
Lymphoma Cells ◽  
Car T Cells ◽  
Car T ◽  
Anti Cd20

Abstract Abstract 4222 BACKGROUND: Adoptive cellular therapy using autologous T cells that have been genetically modified to express a chimeric antigen receptor (CAR) has emerged as a promising therapy for lymphoma. Clinical trials for lymphoid malignancies to date have primarily targeted either the CD19 or CD20 antigens. While CD20 has a more established track record as an immunotherapy target, one potential drawback of targeting CD20 with CAR+ T cells is the theoretical possibility that residual levels of circulating anti-CD20 antibodies (Ab) from prior chemoimmunotherapy regimens could partially or completely block CAR-antigen interactions. This could negatively impact the efficacy of CD20-targeted CAR+ T cells. However, previous data from our group and others indicate that CD20 CAR+T cell function is only partially blocked by anti-CD20 Ab, and T cell function in the setting of anti-CD3 × anti-CD20 bispecific Ab is not blocked by rituximab (R) levels of up to 100 μg/ml. Collectively, these data suggest that a very low number of available CD20 binding sites may be sufficient to trigger CAR signaling and T cell activation. METHODS: We tested the effect of different levels of R on in vitro function of polyclonal T cells from healthy donors negatively selected by MACS, activated with anti-CD3/CD28 beads, and transduced with epHIV7 lentiviral vectors encoding 1st or 3rdgeneration (αCD20-ζ or αCD20-CD28–41BB-ζ) anti-CD20 CARs. T cells were re-stimulated 1 week after initial activation by co-culture with antigen presenting cells (APCs) that had been pre-incubated for 30 minutes with varying concentrations of R (ranging from 0 to 800 μg/ml). APCs were K562 cells transduced to express CD80 with or without CD20 (denoted “K80” and “K80-20”), or Ramos lymphoma cells. Proliferation, cytokine secretion, and cytotoxicity were then assessed as discussed below. RESULTS: We first used flow cytometry to test whether varying concentrations of R blocked binding of the Leu16 Ab and, as expected, found a dose-dependent blockade of CD20 on each cell line, with 50 μg/ml and 200 μg/ml causing near-complete blockade of K80-20 and Ramos cells, respectively. However, despite this apparent blockade, proliferation was largely unimpaired in CFSE-labeled 1st or 3rd generation CAR+ T cells cultured with K80-20 or Ramos cells pre-incubated with R concentrations of up to 400 μg/ml. We concurrently measured cytokine secretion of these T cells using Luminex assays and found that IL-2 and IFN-γ secretion decreased with increasing R levels, but 50–85% of baseline levels were still achieved at R concentrations of up to 100 μg/ml. Cytotoxicity against K80-20 and Ramos target cells in standard 51Cr-release assays by 1st and 3rd generation CAR+ T cells was largely preserved at low R concentrations, and 50–75% of cytolytic activity was retained at 100 μg/ml. Nonspecific proliferation, cytokine secretion, and cytotoxicity were excluded in these experiments by using CAR+ T cells incubated with K80 cells lacking CD20 expression, or T cells transduced with an empty vector as negative controls. Mouse xenograft experiments are currently ongoing to test the effect of serum R levels on the in vivo anti-tumor efficacy of CD20-CAR T cells. CONCLUSIONS: These in vitro results suggest that despite apparent blockade of the CD20 antigen, CAR+ T cells targeting CD20 retain significant activity in the presence of R concentrations of up to 100 ug/ml. Patients receiving 2–3 cycles of R-chemotherapy have serum R trough levels in the range of 30–70 μg/ml. We therefore predict that residual serum R levels will not present a significant impediment to CD20-targeted adoptive T cell therapy given after salvage R-chemotherapy. Disclosures: Jensen: ZetaRx: Equity Ownership, Patents & Royalties. Maloney:Roche: Consultancy; Genentech: Consultancy. Off Label Use: Lentiviral vector encoding a CD20-specific chimeric antigen receptor, used to re-direct autologous T cells to recognize B cell lymphoma cells.

scholarly journals P06.02 Enhancing CAR T cell persistence and memory through modulating mitochondrial function

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A42.1-A42
Author(s):  
A Hosseini Rad ◽  
G Min Yi Tan ◽  
A Poudel ◽  
A McLellan
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Viability ◽  
T Cell Activation ◽  
Mitochondrial Function ◽  
Luciferase Reporter ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundCAR T cell therapy for solid tumours has achieved limited success compared to its application to B cell malignancies. One reason for this failure is the low differentiation rate to memory subsets and low persistence of CAR T cells due to activation-induced cell death (AICD) in lymphoid tissue and the tumour microenvironment. In this study, we have expressed the MCL1 gene within CAR T cells to overcome losses by AICD in adoptively transferred T cells. The MCL1 gene expresses two isoforms; the long isoform localises to the outer membrane of mitochondria and inhibits the CD95 signalling death pathway, while the short isoform localises to the inner membrane of mitochondria to enhance mitochondrial oxidation, phosphorylation and fusion. In addition, we have also utilized a microRNA (miR) 429 to promote memory T cell formation through the suppression of genes such as T-cell-restricted intracellular antigen-1 (TIA-1), T cell activation inhibitor, mitochondrial (TCAIM) and mitochondrial fission factor (MFF).Materials and MethodsOverexpression of MCL1 was confirmed at both mRNA and protein level by real time RT-PCR (qPCR) and western blot. Similarly, overexpression of miR-429 was measured by qPCR and specific binding of miR-429 to the 3′ UTR of target genes was confirmed by luciferase reporter assay. Mitochondrial depolarization and cell viability were assessed by TMRE mitochondrial membrane potential assay (flow-cytometry) and resazurin assay. The effect of MCL1 or miR429 overexpression on HER2-CAR T cells was determined by flow cytometry. Soluble leucine-zipper CD95L (https://www.addgene.org/104349/) was expressed and purified from Expi293 cells.ResultsOverexpression of MCL1 in both Jurkat T cells and primary human T cells protected cells against mitochondria depolarization as well as the loss of cell viability in response to CD95L-triggering. Expression of miR429 downregulated TIA1, TCAIM and MFF. A HER2-CAR construct with either MCL1 or miR429 in a lentiviral system was successfully designed and transduced into primary T cells. Mitochondria in transduced T demonstrated enlarged and fusion morphology - a classic feature of memory T cells.ConclusionsOverexpressing MCL1 or miR429 significantly improves mitochondrial function in T cells. This approach will be used to increase persistence of adoptively transferred CAR T cells.Disclosure InformationA. Hosseini Rad: None. G. Min Yi Tan: None. A. Poudel: None. A. McLellan: None.

scholarly journals Novel CD19t T-Antigen Presenting Cells Designed to Re-Activate and Expand CD19 CAR T Cells In Vivo: Early Demonstration of Feasibility and Safety

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4021-4021 ◽  
Author(s):  
Colleen Annesley ◽  
Rebecca Gardner ◽  
Olivia Finney ◽  
Corinne Summers ◽  
Adam J. Lamble ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Pilot Study ◽  
Antigen Presenting Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Antigen Presenting

Abstract Background: Immunotherapy with CD19 specific chimeric antigen receptor (CAR) T cells for relapsed/refractory acute lymphoblastic leukemia (ALL) demonstrated a minimal residual disease (MRD) negative remission rate of 93% on the phase 1 portion of Pediatric Leukemia Adoptive Therapy (PLAT)-02. However, the 1-year event free survival (EFS) was 50.8%, largely due to recurrence of disease which was frequently associated with early loss of CAR T cell persistence. Subjects on PLAT-02 with <15% CD19 antigen burden in the bone marrow prior to lymphodepletion had a decreased magnitude of CAR T cell expansion and were less likely to have durable persistence of CAR T cells. Also, subjects with a rapid contraction of CAR T cells in the blood between Day +10 and Day+14, as measured by flow cytometry, had inferior long term persistence. These findings led to the hypothesis that loss of persistence may be due to decreased antigen stimulation and subsequent quiescence of CAR+ effector T cells. Episodic target exposure using T cell antigen presenting cells (T-APCs) expressing a truncated human CD19 (CD19t) could trigger CD19 CAR T cell proliferation and re-activation in vivo, resulting in more durable CAR T cell persistence and diminished risk of CD19+ relapse. Methods: Eligible subjects for PLAT-03 (NCT03186118), a pilot study of CD19t T-APCs, must have previously enrolled on the phase 2 portion of PLAT-02 and have a stored apheresis product available. Cohorts include subjects that have an identified predictive factor to lose CAR T cell persistence early, or have already experienced early loss of CAR T cell persistence before 6 months and can receive a reinfusion of CD19 CAR T cells prior to planned T-APCs. Patient-derived T-APC products are manufactured with selected and cryopreserved CD4 and CD8 cells from the subject's original apheresis product. The T cells are activated and transduced with a lentiviral vector to express the CD19t transgene. Transduced cells are expanded in culture for 10-14 days, cryopreserved and release-tested. Subjects <25kg receive 10x106 T-APCs/kg and those ≥25kg receive a flat dose of 5x108 T-APCs. Subjects receive at least one, and up to six total doses of T-APCs at monthly intervals. Results: Seven subjects (ages 9-23 years) have enrolled on PLAT-03; 5 enrolled based on low CD19 antigen burden at the time of lymphodepletion, and 2 based on rapid CAR T cell contraction between Days 10 and 14. T-APC products were successfully manufactured in 5/5 subjects, with two products pending manufacture. One of 5 subjects was unable to receive his planned T-APCs, as he lost CAR T cell persistence prior to T-APC infusion and failed to engraft a second infusion of CD19 CAR T cells. Of the 4 subjects that have received at least one dose of T-APCs, there have been no related adverse events (AEs) > grade 2, and no fever or cytokine release syndrome (CRS) has been observed. Grade 1-2 AEs that are at least possibly related to T-APCs have included hypogammaglobulinemia, nausea, vomiting and headache. Two subjects have completed T-APC treatment; one received all 5 available T-APC doses (subject S001), and one experienced loss of CAR T cell persistence after two T-APC doses and therefore was not eligible for further doses. Data is shown for subject S001, a 20 year old man with multiply relapsed B-ALL. He enrolled on PLAT-03 due to a low CD19 antigen burden of 4.79% by flow cytometry (inclusive of both normal and malignant CD19+ cells) just prior to lymphodepletion. He had minimal toxicity (grade 1 CRS) following CD19 CAR T cell infusion, and subsequently received 5 monthly doses of T-APCs. An increase in the quantity of detectable CAR T cells (EGFRt+ cells) was observed following T-APCs as shown below, and the subject has ongoing CAR T cell persistence at 8 months, as evidenced by B cell aplasia. Conclusion: The first-in-human, pilot study PLAT-03 introduces CD19t T-APCs in an attempt to enhance durable CAR T cell persistence and leukemia remission following CD19 CAR T cell immunotherapy. T-APCs have been successfully manufactured from stored apheresis products collected for CAR T cell production. Four subjects have been successfully treated with at least one T-APC infusion to date, without any significant toxicity and with early evidence of efficacy. Accrual of subjects is ongoing to further assess the safety and feasibility of administering CD19t T-APCs, and to examine the impact of T-APCs on CD19 CAR T cell efficacy. Figure. Figure. Disclosures Park: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Sign in / Sign up

Export Citation Format

Share Document