Cytotoxic T cells swarm by homotypic chemokine signalling

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

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Chimeric Antigen Receptor T Cells Specific for CLL-1 for Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-113438 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2205-2205 ◽

Cited By ~ 3

Author(s):

Elisa De Togni ◽

Miriam Y Kim ◽

Matt L Cooper ◽

Julie Ritchey ◽

Julie O'Neal ◽

...

Keyword(s):

T Cells ◽

Cell Line ◽

Myeloid Leukemia ◽

Cytotoxic Effects ◽

Car T Cells ◽

Human T Cells ◽

Car T ◽

Acute Myeloid

Abstract Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML. C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML. We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B). We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells. In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. Disclosures Cooper: WUGEN: Consultancy, Equity Ownership.

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Efficacy and Long Term Survival of Genetically Modified T Cells Targeting CD19 in a Syngeneic Immune Competent Transgenic Murine Tumor Model Is Dependent on Prior Lymphodepleting Chemotherapy.

Blood ◽

10.1182/blood.v110.11.2754.2754 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2754-2754

Author(s):

James Lee ◽

Yan Nikhamin ◽

Gavin Imperato ◽

Adam Cohen ◽

Michel Sadelain ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Clinical Setting ◽

Peripheral Blood ◽

Tumor Model ◽

B Cell Malignancies ◽

Car T Cells ◽

Human T Cells ◽

Car T

Abstract T cells may be genetically modified ex vivo to target specific antigens by retroviral transduction of genes encoding chimeric antigen receptors (CARs). We have previously constructed a CAR, termed 19z1, specific for the CD19 antigen expressed on most B cell malignancies. Human T cells modified to express the 19z1 CAR specifically eradicate systemic human CD19+ tumors in SCID-Beige mice. However, these models are limited by the xenogeneic nature of the human T cells and tumor cells and the immune compromised state of the host. Here, we studied the biology of adoptively transferred 19z1+ T cells in a syngeneic immune competent murine model designed to better mimic the clinical setting of patients with B cell malignancies. We utilized transgenic C57BL6 mice which lack expression of mouse CD19 (mCD19−/−) and have a single copy of the human CD19 (hCD19+/−) gene (C57BL6(mCD19−/− hCD19+/−)) kindly provided by Dr. T. Tedder, Duke University. These mice are functionally immune-competent with hCD19 expression restricted to the B cell population. To assess whether syngeneic 19z1+ T cells were capable of eradicating normal hCD19+ B cells, we infused C57BL6(mCD19−/− hCD19+/−) mice with either 19z1+ or control prostate specific membrane antigen-targeted (Pz1+) T cells. As assessed by flow cytometric analysis of peripheral blood, we neither found evidence of hCD19+ B cell aplasias in 19z1+ T cell treated mice nor were able to demonstrate the persistence of infused CAR+ T cells. To investigate whether the lack of 19z1+ T cell efficacy and persistence was due to an absence of homeostatic drive, we next lymphodepleted C57BL6(mCD19−/− hCD19+/−) mice with cyclophosphamide prior to T cell infusion. Mice lymphodepleted prior to 19z1+ T cell infusion demonstrated marked and sustained B cell aplasias when compared to lymphodepleted Pz1+ T cell and non-lymphodepleted T cell treated controls. Furthermore, while no CAR+ T cells were identifiable in the Pz1 and non-lymphodepleted control groups, 19z1+ T cells were consistently present in the peripheral blood of the cyclophosphamide pre-treated, 19z1+ T cell treated mice (3–5% of white blood cells). To assess the anti-tumor efficacy of the 19z1+ T cells, we next established a systemic tumor model utilizing mouse EL4 thymoma cells retrovirally modified to express hCD19 (EL4(hCD19)). C57BL6(mCD19−/− hCD19+/−) mice pre-treated with cyclophosphamide, subsequently infused systemically with EL4(hCD19) tumor, followed by systemic 19z1+ T cell infusion, had a significant survival advantage (80% survival at >120 days) over untreated controls or controls treated with Pz1+ T cells or 19z1+ T cells in the absence of lymphodepletion (0% survival). In conclusion, we have developed a syngeneic immune competent tumor model of hCD19 disease that is highly relevant to the clinical setting. Using this model, we demonstrate the significance of lymphodepletion on the prolonged in vivo persistence and anti-tumor efficacy of 19z1+ T cells. Data derived from this model will be correlated to findings obtained from a recently initiated clinical trial for patients with chronic lymphocytic leukemia, and will significantly impact the design of subsequent trials in the future.

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Increasing AMPK Activity in Human T Cells Enhances Memory Subset Formation without Sacrificing in Vitro Expansion

Blood ◽

10.1182/blood-2020-141605 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 38-39

Author(s):

Erica Lynne Braverman ◽

Andrea Dobbs ◽

Darlene A. Monlish ◽

Craig Byersdorfer

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Therapy ◽

Car T Cell ◽

Car T Cell Therapy ◽

Human T Cells ◽

Car T ◽

Ampk Activity

BACKGROUND: While chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL), treatment failures continue to occur. In studying therapeutic T cell function, it has become clear that achieving a memory-like phenotype is ideal for CAR-T production. This is likely related to the enhanced oxidative metabolic potential of this subset, which allows for improved persistence and enhanced anti-leukemia activity in vivo. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. Finding methods to improve the yield of memory-like cells without sacrificing T cell expansion has been challenging. AMP-activated protein kinase (AMPK) is a key metabolic regulator responsible for promoting mitochondrial biogenesis and oxidative metabolism, and is more active in memory T cells at baseline. It is similarly induced by TCR ligation, making it unlikely that it would significantly detract from proliferation. These properties make activation of AMPK a potential candidate pathway for improving the yield of more functional T cells for CAR-T cell therapy. METHODS: AMPK is a heterotrimeric protein complex consisting of alpha, beta, and gamma domains. Functionally, the alpha subunit contains the kinase domain, which is activated by phosphorylation. The gamma subunit controls the phosphorylation, and therefore the activity, of the alpha domain. To increase AMPK signaling in T cells, we cloned the gamma subunit into a lentiviral plasmid containing the elongation factor 1a (EF1a) promoter and a green fluorescent protein (GFP) tag. An empty vector, containing GFP only, served as a negative control. Human T cells were isolated from three separate donors, transduced with our lentiviral construct, and expanded in vitro in the presence of IL-2. AMPK activity was assessed by phosphorylation of Thr172 on the AMPKα subunit as well as phosphorylation of S555 on downstream target Unc-51-like autophagy activating kinase (ULK1) using western blot densitometry, normalized to the total protein amounts. Memory marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes for in vitro expansion were calculated by adjusting manual cell counts to reflect GFP positivity and CD4+/CD8+ surface staining. RESULTS: The AMPK gamma subunit was efficiently transduced and expressed by human T cells as measured by GFP expression, qRT-PCR, and western blot analysis. Further, AMPK activity increased in GFP+ cells as indicated by the phosphorylation of AMPKα Thr172 (1.93 +/- 0.05 vs 0.6 +/- 0.09, p<0.001) and ULK1 S555 (1.28 +/- 0.11 vs 0.67 +/- 0.08, p<0.01). Cells transduced with AMPK augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (Figure 1). In addition, AMPK-transduced T cells showed a statistically significant increase in mitochondrial density along with notable enhancement of SRC and maximal oxygen consumption rates (Figure 2A,B). Furthermore, the rate of expansion of AMPK-transduced T cells did not differ significantly from Empty-transduced controls, and in fact trended towards increased in both CD4+ and CD8+ cells (Figure 3A). Indeed, the improved rate of expansion in AMPK-transduced CD4+ T cells led to a measurable increase in CD4+ T cell percentages by flow cytometry (Figure 3B). DISCUSSION: Here we present an efficient and direct method to increase AMPK activity in human T cells and demonstrate that increased AMPK activity endows T cells with a variety of characteristics ideal for CAR-T cell therapy. These features include increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

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p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.698565 ◽

2021 ◽

Vol 12 ◽

Author(s):

Valérie Janelle ◽

Mathieu Neault ◽

Marie-Ève Lebel ◽

Dave Maurice De Sousa ◽

Salix Boulet ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cellular Senescence ◽

Ex Vivo ◽

Cell Dysfunction ◽

Human T Cells ◽

Car T ◽

P38mapk Signaling ◽

T Cell Dysfunction

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

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Synthetic Chimeric Antigen Receptors (CARs) Rapidly Induce Exhaustion and Augmented Glycolytic Metabolism In Human T Cells and Implicate Persistent CD28 Signaling As a Driver Of Exhaustion In Human T Cells

Blood ◽

10.1182/blood.v122.21.192.192 ◽

2013 ◽

Vol 122 (21) ◽

pp. 192-192

Author(s):

Adrienne H. Long ◽

Rimas J. Orentas ◽

Crystal L. Mackall

Keyword(s):

T Cells ◽

Antitumor Efficacy ◽

Antigen Receptors ◽

Antitumor Effects ◽

Car T Cells ◽

Human T Cells ◽

Car T ◽

In Vitro Expansion

Abstract Introduction Chimeric antigen receptors (CARs) provide a promising new approach for the adoptive immunotherapy of cancer. Though impressive antitumor activity has been observed with some CAR T cells, other CAR T cells demonstrate poor antitumor efficacy in vivo despite high cytolytic capacity in vitro due to poor expansion and persistence. Whether exhaustion of CAR T cells mirrors exhaustion that occurs naturally in chronically stimulated human T cells has not yet been studied. Here, we report that expression of select CD28 containing CARs in normal human T cells rapidly induces an exhausted state characterized by high PD-1 expression, poor persistence and poor antitumor efficacy, whereas other CARs do not induce this phenotype. Results Human T cells were expanded with anti-CD3/CD28 beads, and then transduced with a second-generation (CD28-CD3ζ) disialoganglioside 2 (GD2) specific CAR or a second-generation (CD28-CD3ζ) CD19 specific CAR. By day 7 of in vitro expansion, GD2 CAR T cells developed a metabolism more highly dependent on glycolysis compared to CD19 CAR T cells or untransduced controls. Neither CAR population was exposed to antigen during this expansion period. Using a Seahorse Extracellular Flux Analyzer, the ratio of glycolysis to oxidative phosphorylation rates (ECAR:OCR ratio) of GD2 CAR T cells was found to be double that of CD19 CAR T cells or controls on day 7. The highly glycolytic metabolism of GD2 CAR T cells was associated with an exhausted phenotype. GD2 CAR T cells expressed higher levels of PD-1, TIM-3 and LAG-3, and transcription repressor BLIMP-1, compared to CD19 CAR T cells or untransduced controls. Additionally, GD2 CAR T cells were poor cytokine producers, generating <10x lower levels of IL2, TNFα and IFNγ than CD19 CAR T cells upon in vitro co-incubation with a GD2+CD19+ osteosarcoma line (143B-CD19), despite maintaining comparable in vitro cytolytic ability. GD2 CAR T cells showed poor in vitro expansion and increased rates of apoptosis compared to controls. GD2 CAR T cells also did not persist and did not mediate antitumor effects against GD2+CD19+ tumors in a murine xenograft model in vivo, whereas CD19 CAR T cells completely eradicated CD19+ tumors and persisted in both the spleen and tumor compartments. To rule out the possibility that diminished cytokine production and in vivo efficacy was related to antigen specific effects, T cells were co-transduced with both the GD2 and CD19 CARs. Though single-transduced CD19 CAR T cells show no signs of an altered metabolism or exhaustion and have strong antitumor efficacy, CD19 CAR T cells co-transduced with the GD2 CAR demonstrate an exhausted phenotype and diminished antitumor efficacy similar to that of single-transduced GD2 CAR T cells. Thus, expression of the GD2 CAR confers a dominant exhausted phenotype in T cells, and prevents otherwise efficacious CARs from mediating strong antitumor effects. We hypothesized that chronic signaling of CD3ζ and CD28 via the GD2 CAR results in exhaustion. Interestingly, however, we did not identify GD2 expression in the culture system. Point mutations in the CAR antigen-binding site, though abrogating GD2 binding, did not prevent the development of exhaustion. Thus, we postulate that constitutive receptor signaling may occur via interactions between the framework regions of the CAR receptors. Importantly however, substitution of 4-1BB for the CD28 domain in the GD2 CAR substantially diminished PD-1 expression, one of the hallmark features of exhausted T cells. Conclusions We report that expression of a CD28 containing GD2 CAR induces both an altered metabolism and an exhausted state in human T cells, resulting in poor in vivo persistence and antitumor efficacy. We hypothesize that tonic signaling through the GD2 CAR induces this phenotype and have identified the CD28 domain as an important component contributing to this phenotype. Rapid induction of exhaustion mediated via a synthetic receptor provides a novel model system to identify mechanistic factors required for this phenotype in human T cells. Work is currently underway to molecularly define the basis for the exhaustion of GD2 CAR T cells and to probe a potential role for altered T cell metabolism as a contributor to T cell exhaustion in human T cells. Disclosures: No relevant conflicts of interest to declare.

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CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth

Frontiers in Immunology ◽

10.3389/fimmu.2019.01149 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 27

Author(s):

Ru Zhou ◽

Mahboubeh Yazdanifar ◽

Lopamudra Das Roy ◽

Lynsey M. Whilding ◽

Artemis Gavrill ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Chimeric Antigen Receptor ◽

Triple Negative ◽

Antigen Receptor ◽

Xenograft Model ◽

Car T Cells ◽

Human T Cells ◽

Car T

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells.

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CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo

Blood ◽

10.1182/blood-2011-03-344275 ◽

2012 ◽

Vol 119 (3) ◽

pp. 696-706 ◽

Cited By ~ 180

Author(s):

De-Gang Song ◽

Qunrui Ye ◽

Mathilde Poussin ◽

Gretchen M. Harms ◽

Mariangela Figini ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Car T Cells ◽

Human T Cell ◽

Human T Cells ◽

Car T ◽

Cd27 Costimulation

AbstractThe costimulatory effects of CD27 on T lymphocyte effector function and memory formation has been confined to evaluations in mouse models, in vitro human cell culture systems, and clinical observations. Here, we tested whether CD27 costimulation actively enhances human T-cell function, expansion, and survival in vitro and in vivo. Human T cells transduced to express an antigen-specific chimeric antigen receptor (CAR-T) containing an intracellular CD3 zeta (CD3ζ) chain signaling module with the CD27 costimulatory motif in tandem exerted increased antigen-stimulated effector functions in vitro, including cytokine secretion and cytotoxicity, compared with CAR-T with CD3ζ alone. After antigen stimulation in vitro, CD27-bearing CAR-T cells also proliferated, up-regulated Bcl-XL protein expression, resisted apoptosis, and underwent increased numerical expansion. The greatest impact of CD27 was noted in vivo, where transferred CAR-T cells with CD27 demonstrated heightened persistence after infusion, facilitating improved regression of human cancer in a xenogeneic allograft model. This tumor regression was similar to that achieved with CD28- or 4-1BB–costimulated CARs, and heightened persistence was similar to 4-1BB but greater than CD28. Thus, CD27 costimulation enhances expansion, effector function, and survival of human CAR-T cells in vitro and augments human T-cell persistence and antitumor activity in vivo.

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Author response: Cytotoxic T cells swarm by homotypic chemokine signalling

10.7554/elife.56554.sa2 ◽

2020 ◽

Author(s):

Jorge Luis Galeano Niño ◽

Sophie V Pageon ◽

Szun S Tay ◽

Feyza Colakoglu ◽

Daryan Kempe ◽

...

Keyword(s):

T Cells ◽

Cytotoxic T Cells ◽

Author Response ◽

Chemokine Signalling

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Potent and Timed Epigenetic Repression of PD-1 in Human T Cells

Blood ◽

10.1182/blood-2018-99-119011 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3493-3493 ◽

Cited By ~ 1

Author(s):

Jocelynn Pearl ◽

Fyodor D Urnov ◽

John A Stamatoyannopoulos

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

B Cell ◽

Single Gene ◽

Hematologic Malignancies ◽

Cell Exhaustion ◽

Human T Cells ◽

Car T ◽

Post Infusion

Abstract T cell exhaustion mediated by checkpoint genes - most prominently PD-1 - is believed to play a major role in limiting the efficacy of chimeric antigen receptor (CAR) T cell therapies of hematologic malignancies. Clinically durable responses to CAR-T therapy of B cell malignancies almost invariably occur in patients who achieve a complete remission by day 28 post infusion. Accumulating data indicate that the impact of T cell exhaustion is most prominent during the early post-infusion interval. PD-1 has recently been found to function as a tumor suppressor in T cells; as such permanent disablement of PD-1 expression via genetic knockout may thus increase long term adverse events from cell therapy. We sought to develop an approach for increasing the potency and efficacy of CAR-T cell therapy of B cell and other hematologic malignancies by timed epigenetic abrogation of PD-1 expression that was limited to the critical early (21-28d) therapeutic interval. To accomplish this we engineered synthetic transcription factors coupled to a modified KRAB repressive domain that densely tiled the transcriptional regulatory regions of PD-1 at up to single-base resolution. To identify TF-repressors that were both potent and specific, we transiently transduced each engineered TF-repressors separately into primary total CD3+, CD4+, or CD8+ human T-cells. These experiments revealed striking position dependence of TF-repressor activity, indicating that spatial and rotational positioning of the repressive domain was critical for function. We identified several TF-repressors that exhibited highly potent repression (>90%), and screened these for gene selectivity using transcriptome-wide deep RNA-seq. These studies yielded a TF-repressor with both high potency (>90%) and true single-gene (PD-1) specificity on a genomic scale. Next we examined the durability of expression. Transduction of TF-repressor mRNA resulted in rapid expression and activity, with PD-1 repression observable beginning at 12 hours and peaking by 48 hours post transduction. Experiments with tagged TF-repressors revealed that substantially all translated protein had dissipated within 72 hours post transduction. Remarkably, however, potent PD-1 repression reliably persisted for >3 weeks post-transduction. Collectively, our studies show the feasibility of generating highly potent and single-gene-selective epigenome editing reagents capable of reprogramming clinically relevant genes over a defined temporal interval. This approach should be broadly applicable to other key regulators of clinical response to produce genome-targeted medicines without incurring the genotoxic consequences of double-stranded DNA breaks. Disclosures No relevant conflicts of interest to declare.

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Modulation of inhibitory signals in CAR T cells leads to improved activity against glioblastoma.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.3031 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 3031-3031

Author(s):

Khaled Sanber ◽

Zeid Nawas ◽

Vita Salsman ◽

Ahmed Gad ◽

Pretty Matthew ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Inhibitory Receptors ◽

Car T Cells ◽

Ribonucleoprotein Complexes ◽

Human T Cells ◽

Car T ◽

Anti Tumor Activity

3031 Background: Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the immunosuppressive tumor microenvironment. Adoptively transferred CAR T cells remain susceptible to inhibition via the engagement of co-inhibitory receptors on their surface such as PD1, BTLA, CTLA4 and LAG3. The subsequent recruitment of Src homology region 2 containing protein tyrosine phosphatase 2 (SHP2) by these receptors to the immune synapse may represent a common mechanism of T cell inhibition, as SHP2 can de-phosphorylate key signaling molecules that mediate T cell activation (including CD28 and CD3ζ). We hypothesized that SHP2 deletion will simultaneously offset the effects of multiple co-inhibitory receptors, thereby improving the anti-tumor activity of CAR T cells. Methods: Electroporation of sgRNA/Cas9 ribonucleoprotein complexes into human T cells was used to knockout (KO) SHP2. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 endodomain) targeting HER2. The phenotype of wild-type (WT) and SHP2KO CAR T cells was evaluated with mass cytometry and flow cytometry. Their anti-tumor function was tested in vitro using the xCELLigence assay (an impedance-based cytotoxicity assay), and in vivo, in an orthotopic xenograft mouse model of GBM. Results: Efficient and reproducible depletion of the SHP2 protein in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect CAR T cell expansion, proliferation or baseline phenotype. However, following co-culture with HER2+ LN229-GBM cells, the CD8+ central memory (CCR7+ CD45RA-) and effector memory (CCR7- CD45RA-) subsets were enriched to a greater extent in the SHP2KO CAR T cells. The pattern of cytokine co-expression varied between donors in a single-cell analysis comparing SHP2KO to WT CAR T cells after encountering LN229 cells. Functionally, SHP2KO CAR T cells derived from the majority of healthy donor and patient peripheral blood eliminated LN229 cells more rapidly in vitro. In an orthotopic mouse model of GBM, SHP2KO CAR T cells showed better early control of established LN229 xenografts and improved survival in comparison to WT CAR T cells. Conclusions: SHP2 deletion in CD28ζ.CAR T cells improves their anti-tumor activity.

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