The multi-specific VH-based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

Abstract Background Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. Methods CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. Results CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. Conclusions CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

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A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2019.2859 ◽

2019 ◽

Vol 15 (11) ◽

pp. 2229-2239 ◽

Cited By ~ 3

Author(s):

Zhuoran Tang ◽

Fengzhen Mo ◽

Aiqun Liu ◽

Siliang Duan ◽

Xiaomei Yang ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cytotoxic T Lymphocyte ◽

Ex Vivo ◽

T Cell Proliferation ◽

Tumor Cell Apoptosis ◽

Xenograft Models

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

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Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo

Blood ◽

10.1182/blood.v124.21.1427.1427 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1427-1427

Author(s):

Suzanne L Tomchuck ◽

Jin He ◽

Ross W. Perko ◽

Scarlett Evans ◽

Amy McKenna ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Fold Increase ◽

T Cell Proliferation ◽

Gamma Delta T Cells ◽

Memory Cells ◽

Gamma Delta ◽

Gamma Delta T Cell

Abstract Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function. Disclosures No relevant conflicts of interest to declare.

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Sirolimus Inhibits Proliferation but Enhances Suppressive Activity of Rhesus Macaque Natural CD4 Regulatory T Cells

Blood ◽

10.1182/blood.v118.21.1008.1008 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1008-1008

Author(s):

Karnail Singh ◽

Natalia Kozyr ◽

Linda Stempora ◽

Allan D Kirk ◽

Christian P Larsen ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Ex Vivo ◽

Cd8 T Cell ◽

T Cell Proliferation ◽

Suppressive Activity ◽

Suppressive Capacity

Abstract Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p<0.01) A mechanistic evaluation of the increase potency observed with sirolimus pulsed Tregs (SPTs) has revealed several key differences that distinguish these cells from the less-potent unpulsed Tregs: SPTs were found to undergo fewer rounds of proliferation in an MLR when compared with unpulsed Tregs (14% proliferation in SPTs versus 37% proliferation in un-pulsed Tregs, p= 0.015), suggesting that the suppressive capability of Tregs may be inversely related to their proliferative capacity. SPTs were also shown to have significantly increased expression of CD25 (p=0.04) and total CTLA4 (p= 0.009) compared to unpulsed Tregs, implicating signaling through both of these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue by which to achieve enhanced Treg-based suppression of alloimmunity, in a manner that is amenable to large-scale ex-vivo expansion and to combinatorial therapy with novel, costimulation-blockade-based immunosuppression strategies. Disclosures: No relevant conflicts of interest to declare.

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Steroids Abrogate BiTE® Antibody Construct-Mediated Cytotoxicity in Primary AML Cells

Blood ◽

10.1182/blood.v128.22.3940.3940 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3940-3940

Author(s):

Christina Krupka ◽

Bettina Lindl ◽

Thomas Köhnke ◽

Julia Platzer ◽

Lavinia Pachzelt ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cell Function ◽

Ex Vivo ◽

Antibody Therapy ◽

Antigen Expression ◽

T Cell Proliferation ◽

Negative Effect ◽

Equity Ownership

Abstract Antibody based immunotherapy represents a promising strategy to eliminate chemoresistant cells in acute myeloid leukemia (AML). Clinical experience in acute lymphoblastic leukemia (ALL) has shown a clear correlation of leukemic burden and the occurrence of a cytokine release syndrome (CRS) during treatment with blinatumomab (CD19/CD3 BiTE®). A cytoreductive phase prior to or in combination with antibody therapy might be beneficial to reduce the severity of adverse events like CRS. The latter is often treated with steroids (dexamethasone, DEX) or less commonly, with the IL-6R antibody tocilizumab (TOC). As T-cell proliferation and function are of crucial importance for BiTE® activity (Zugmaier 2015), the effect of the drugs on effector cell function will dictate clinical response to therapy. In this study, we evaluated the influence of cytoreductive- (azacythidine, AZA; decitabine, DEC), and immunmodulatory (DEX and TOC) drugs on antibody-mediated cytotoxicity and T-cell proliferation. A CD33/CD3 BiTE® antibody construct (AMG 330) served as model T-cell recruiting antibody in this study. To address this question we set up the following experimental approaches: AML cells were cocultured with healthy donor (HD) T cells for up to 14 days ex vivo. T cells were either incubated with the specific drug for 3 days prior to coculture or the drugs were simultaneously added to AML-T cell cultures. Drug concentrations were chosen based on published serum concentrations in AML patients and their ex vivo stability in culture, validated by mass spectrometry. BiTE® mediated cytotoxicity and T-cell proliferation were assessed by flow cytometry. Preincubation of T cells with AZA and DEC impaired antibody mediated cytotoxicity of HL60 cells in a concentration dependent manner (% lysis control (ctrl) vs AZA at 1, 5, 10 µM: 99.9 vs 99.2 vs 52.1 vs 28.7, n=7; ctrl vs DEC at 0.2, 2, 5 µM: 98.4 vs 71.3 vs 60.0 vs 50.0, n=3). Similarly, T-cell proliferation was also markedly decreased (fold change (FC) T cells ctrl vs AZA at 1, 5, 10 µM: 2.9 vs 2.8 vs 1.5 vs 0.7; ctrl vs DEC at 0.2, 2, 5 µM: 3.8 vs 3.0 vs 2.3 vs 1.2). For DEX it was shown that incubation of T cells with steroids prior to cocultures had no negative effect on BiTE® mediated cytotoxicity (Brandl 2007). However, as steroids are often used simultaneously with T-cell recruiting immunotherapies, we tested the influence of DEX in combination with AMG 330. The addition of DEX to primary AML-T cell cultures (75 ng/ml) significantly impaired AMG 330 mediated cytotoxicity (% lysis AMG 330 vs AMG 330+DEX day (d) 6: 95.9 vs 47.5, n=9). This correlated to a markedly reduced T-cell proliferation (FC T cells AMG 330 vs AMG 330+DEX d6: 11.2 vs 1.2, n=9). Correspondingly, secretion of IFNγ was also decreased (n=3). Upon discontinuation of DEX an increase in AMG 330 mediated cytotoxicity was observed. Nevertheless, cytotoxicity was still considerably lower compared to control cultures (%lysis AMG 330 vs AMG 330+DEX d9: 95.6 vs 77.0). In contrast to DEX, TOC (110 µg/ml) had no negative effect on T-cell proliferation (FC T cells d6: AMG 330 vs AMG 330+TOC: 42.3 vs 36.9, n=4). Similarly, secretion of IFNγ was not affected through the simultaneous addition of TOC to primary AMG 330 cultures (pg/ml AMG 330 vs AMG 330+TOC d6: 543.9 vs 345.8 n=2). Importantly, drugs might not only interfere with effector cell function but also modulate target antigen expression. As we have previously demonstrated that antigen expression levels influence BiTE® mediated cytotoxicity (Krupka 2016), we analysed the effect of the drugs on CD33 expression. None of the drugs induced a significant up- or downregulation of CD33 on AML celllines as detected by flow cytometry. Hence our data support the notion that these drugs do not modulate antigen expression dependent lysis kinectics. We conclude, that drugs given prior or concomitant to BiTE® therapy have the potential to reduce T-cell proliferation and cytotoxicity. In particular, we observed a negative impact of AZA and DEC when given prior to AML-T cell cocultures. Importantly, even short exposure to DEX led to a significanly reduced T-cell responsiveness. Our data suggest the careful evaluation of concomitant drugs in T-cell recruiting antibody therapies and support the restrictive use of steroids in patients receiving BiTE® antibody therapy. For management of severe CRS, TOC could be considered as a targeted biologic therapy that preserves BiTE®-dependent T cell function. Disclosures Krupka: AMGEN Research Munich: Research Funding. Kufer:AMGEN Research Munich: Employment, Equity Ownership, Patents & Royalties. Kischel:AMGEN Research Munich: Employment, Equity Ownership, Patents & Royalties. Subklewe:AMGEN Research Munich: Research Funding.

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Pharmacological Inhibition Of Semi-Direct Alloantigen Presentation and The Generation Of Cross-Dressed Antigen Presenting Cells: A Novel approach To Limit Graft Versus Host Disease Following Allogeneic Hematopoetic Stem Cell Transplantation

Blood ◽

10.1182/blood.v122.21.294.294 ◽

2013 ◽

Vol 122 (21) ◽

pp. 294-294

Author(s):

Sravanti Rangaraju ◽

Junghwa Choi ◽

Cynthia R. Giver ◽

Edmund K. Waller

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Proliferation ◽

Mhc Ii ◽

Direct Pathway ◽

Antigen Presenting

Abstract Background Graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplant (allo-HSCT) is caused by CD4+ and CD8+ donor T cells directed against mismatched recipient antigens, presented in the context of donor MHC-II (indirect pathway) and recipient MHC-I (direct pathway). Recently, the presence of 'cross-dressed' CD11c+ antigen presenting cells (APCs) expressing both donor and recipient type MHC-I molecules has been demonstrated in animal organ and HSCT transplant models supporting 'semi-direct' pathway of allo-activation (Wang et al, Blood. 2011).These APCs can efficiently present allo-antigens to both CD4+ and CD8+ T cells and activate immune responses that could lead to allograft rejection or GVHD. Exchange of membrane fragments and associated proteins between cells, termed trogocytosis, generates cross-dressed APCs.We sought to test whether cross-dressed APCs facilitate antigen presentation to donor T cells and initiate GVHD following allo-HSCT. Further, we tested an array of drugs as inhibitors of trogocytosis, to interrupt the semi-direct pathway of allo-antigen presentation. Methods In vivo experiments used a B6(H2Kb) ˆ B10.BR(H2Kk) murine transplant model. Spleens of transplanted mice were analyzed on days 10, 15, 20 post-transplant for presence of cross dressed CD11c+cells, and their expression of CD80, CD86 and MHC-II by flow cytometry. Cross dressed donor CD11c+ FACS sorted cells from recipient spleens were co-cultured with CFSE labeled donor type T-cells for 6 days, and T-cell proliferation was measured as dilution of CFSE by flow cytometry. In vitro experiments used primary MLR consisting of CFSE labeled B6 bone marrow cells co-cultured with PKH26 (membrane dye) labeled B10.BR splenocytes. B6 antigen presenting cells were analyzed by flow cytometry for the presence of CFSE+PKH26+ double positive cells generated by trogocytosis. Pharmacological inhibitors of cytoskeleton function were added to the primary MLR and their effect on trogocytosis as well as T cell proliferation was assessed. Results Cross-dressed donor CD11c+ APCs were generated in vivo following allo-HSCT (Figure 1). Recipient spleens showed that 50%, 28.6% (p=0.01) and 12% (p=0.02) of donor type CD11c+ cells were cross dressed on days 10, 15 and 20 respectively post transplant (n=5). These cross dressed APCs expressed higher levels of co-stimulatory molecules CD80 (p<0.001) and CD86 (p<0.001), and MHC-II compared to non-cross-dressed donor CD11c+ cells (Figure 2). Sorted cross dressed CD11c+ cells from recipient mice were able to induce in vitro proliferation of co-geneic CD8 T-cells, while their non-crossdressed counterparts did not. We demonstrated that cross-dressed CD11c+ cells were generated in vitro, by exchange of plasma membrane fragments and could be inhibited in vitro by low doses of paclitaxel and VIP antagonist (Figure 3), while preserving cell viability. Further more, bone marrow treated with 0.05uM of paclitaxel, caused significantly decreased T cell proliferation in primary MLR compared to non drug treated bone marrow. Discussion The high frequencies of cross-dressed donor CD11c+ APCs following allo-HSCT suggests that semi-direct allo-antigen presentation may play a key role in the initiation of GVHD, while the decreasing trend could reflect replacement of host cells by donor hematopoetic cells. Reducing the generation of cross-dressed APCs by pharmacological inhibition of trogocytosis is a novel approach to reduce GVHD post allo-HSCT, targeting the semi-direct pathway of allo-antigen presentaion. Our data shows that very low doses of paclitaxel, a microtubule inhibitor and VIPHyb, an antagonist of Vasoactive Intestinal Peptide signaling, can reduce semi-direct presentaion of allo-antigen to Tcells and reduce alloreactivity without direct cytotoxic effect. Disclosures: No relevant conflicts of interest to declare.

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PD-1/PD-L1 Blocking Enhances CD33/CD3-Bispecific BiTE® Antibody (AMG 330) Mediated Lysis of Primary AML Cells

Blood ◽

10.1182/blood.v124.21.3738.3738 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3738-3738 ◽

Cited By ~ 1

Author(s):

Christina Krupka ◽

Peter Kufer ◽

Roman Kischel ◽

Gerhard Zugmaier ◽

Thomas Köhnke ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Ex Vivo ◽

Primary Diagnosis ◽

T Cell Proliferation ◽

Employment Equity ◽

Blocking Antibody ◽

Equity Ownership

Abstract Antibody-based immunotherapy represents a promising strategy to target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). In our previous work, the potency of the CD33/CD3-bispecific BiTE® antibody AMG 330 to activate and redirect T-cells to AML cells was evaluated in a long-term culture system using unmanipulated peripheral blood (PB) or bone marrow (BM) samples from AML patients. We were able to show effective elimination of AML cells by AMG 330-activated and -expanded residual autologous T-cells (n=13, Krupka et. al, Blood 2014 123(3):356-65). The goal of the present study was to identify and manipulate factors that interfere with AMG 330-mediated lysis of primary AML cells. In our ex-vivo long-term culture system (n= 32 primary diagnosis, 3 relapse), we observed that successful elimination of primary AML cells was predominantly influenced by the initial effector:target (E:T) ratio. Reduced short-term lysis efficacy was observed for patient samples with low E:T ratios (median lysis: E:T ratio <1:10 23.5% vs >1:10 99.3%) after 4 days of culture. However, after 12 days of culture this effect was less prominent as effector T-cell numbers increased and lysis was observed even in cultures with very low initial E:T ratios (up to an E:T ratio of 1:80). Considering the ubiquitous expression pattern of CD33 within the myeloid compartment, the number of AMG 330 treatment days will be limited due to expected hematotoxicity. Therefore, AMG 330 efficacy might benefit by increasing the E:T ratio within a short time period. Programmed cell death-1 (PD-1) and its primary ligand PD-L1 are immune checkpoints that limit T-cell responses and may contribute to slower lysis kinetics during AMG 330 treatment. Therefore, we assessed PD-1 expression on T-cells and PD-L1 expression on primary AML cells. No constitutive expression of PD-1/PD-L1 on T-cells and corresponding AML cells was found at primary diagnosis (PD-1: n=23; PD-L1: n=193). Upon the addition of AMG 330 to our primary ex-vivo AML cultures, we observed a strong upregulation of PD-1 on activated T-cells, which correlated with the extent of T-cell proliferation (10/10). This was most prominent within the subpopulation of CD4+/CD45RA-/CCR7- effector memory T-cells. Furthermore, in response to AMG 330-mediated T-cell activation, we observed an upregulation of PD-L1 on primary AML cells (16/19). Data obtained from AML cell lines confirmed an upregulation of PD-L1 after co-cultivation with T-cells and AMG 330. This phenomenon was cytokine-mediated as the addition of IFNγ and TNFα also induced PD-L1 expression (n=6). Interestingly, we also observed a PD-L1 upregulation on T-cells upon activation with AMG 330, but to a much lower extent compared to primary AML cells (n=17; mean MFI ratio: T-cells: 4.7; AML cells: 12.1). Blockade of the PD-1/PD-L1 interaction through the addition of an inhibitory antibody induced an increase in T-cell proliferation in ex-vivocultures resulting in enhanced cytotoxicity against primary AML cells (lysis on day 18: with/without PD-1 blocking antibody: 75 vs 44%; fold change T-cell expansion: 6 vs 3). This was accompanied by a significant increase in IFNγ production (with/without PD-1 blocking antibody: 280 pg/ml vs 81 pg/ml). Complimentary experiments using primary AML cells and allogeneic T-cells at defined E:T ratios demonstrated that this effect is most prominent in co-cultures with a low percentage of T-cells within the primary sample (lysis on day 7 with/without PD-L1 blocking antibody: E:T ratio 1:1: 100% vs 94.5%; E:T ratio 1:5: 96.6% vs 82.7%; fold change T-cell expansion: E:T ratio 1:1: 3.1 vs 3.1; E:T ratio 1:5: 23.4 vs 9.8). Intriguingly, upon addition of lenalidomide to our primary AML cultures, we were also able to circumvent the immune inhibitory effect of the PD-1/PD-L1 interaction by a decrease in PD-L1 upregulation on primary AML cells. Our data show that AMG 330-mediated lysis of primary AML cells can be enhanced by inhibiting the PD-1/PD-L1 axis on T-cells and corresponding AML cells. The modulation of the PD-1/PD-L1 axis increased T-cell proliferation and accelerated attaining a beneficial E:T ratio. We hypothesize that PD-L1 upregulation on primary AML cells is a relevant immune escape mechanism employed by AML cells to escape cytokine-mediated immune responses. Prospective clinical trials will be needed to assess the relevance of our finding in AMG 330-treated AML patients. Disclosures Krupka: AMGEN Inc.: Research Funding. Kufer:AMGEN Inc.: Equity Ownership; AMGEN Research (Munich): Employment. Kischel:AMGEN Inc.: Equity Ownership; AMGEN Research (Munich): Employment. Zugmaier:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Sinclair:AMGEN Inc.: Employment, Equity Ownership. Newhall:AMGEN Inc.: Employment, Equity Ownership. Frankel:AMGEN Inc.: Employment, Equity Ownership. Baeuerle:AMGEN Research (Munich): Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership. Subklewe:AMGEN Inc.: Research Funding.

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BET Inhibition As a Targeted Epigenetic Approach to Reverse T Cell Dysfunction in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2021-153921 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3715-3715

Author(s):

Audrey L Smith ◽

Alexandria P Eiken ◽

Sydney A Skupa ◽

Dalia Y Moore ◽

Avyakta Kallam ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Ex Vivo ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

T Cell Proliferation ◽

Cell Dysfunction ◽

T Cell Dysfunction

Abstract Introduction : Chronic Lymphocytic Leukemia (CLL) is characterized by the clonal expansion of mature CD19+/CD5+ lymphocytes in the peripheral blood and secondary lymphoid organs. The accumulation of B-CLL cells yields profound immune defects in the CLL tumor microenvironment (TME), promoting evasion of immune surveillance that contributes to tumor persistence and thus relapsed/refractory disease. The bromodomain and extra-terminal domain (BET) family of proteins are epigenetic readers that bind acetylated histone residues to regulate transcription of numerous genes involved in critical CLL protumor pathways. Of the BET family proteins, BRD4 is overexpressed in CLL and highly enriched at super-enhancers of genes that regulate CLL-TME interactions such as B cell receptor pathway components, chemokine/cytokine receptors, and immune checkpoint molecules. Pan BET inhibitors (BET-i), such as PLX51107 (Plexxikon Inc.) significantly improve survival in aggressive CLL murine models. Here we demonstrate that blocking BRD4 function with PLX51107 (PLX5) can alleviate the inherent immune defects observed in CLL, hence reducing B-CLL induced T cell dysfunction and allowing for robust B-CLL cell elimination. This therapeutic strategy may be vital in overcoming frequent drug resistance and/or bolstering the anti-tumor effect of current CLL therapies. Methods : Primary leukemic B cells were isolated from the peripheral blood of CLL patients and co-cultured with healthy donor T cells to evaluate the effect of PLX5 (0.1-0.5μM) on CLL-induced T cell immunosuppression ex vivo via an array of flow cytometry assays. T cell proliferation was assessed using CFSE after 96 h co-culture with α-CD3/α-CD28 stimulation. Effector cytokine production was evaluated after 48 h co-culture in the presence of PMA/ionomycin (final 6 h) and brefeldin A (final 5 h). Immune inhibitory molecule surface expression was measured following 48 h co-culture with α-CD3/α-CD28 stimulation. To further validate our ex vivo findings, the E μ-TCL1 adoptive transfer model was used. Once disease onset was confirmed in recipient WT B6 mice (>10% CD45+/CD19+/CD5+ peripheral blood lymphocytes), mice were randomized to receive either PLX5 (20 mg/kg) or vehicle (VEH) equivalent daily by oral gavage for 4 weeks. Following treatment, mouse spleens were processed to evaluate exhaustion marker expression, T cell proliferation (CellTrace™ Violet, 72 h a-CD3/α-CD28 stimulation ex-vivo), and T-cell effector function (ex-vivo mitogenic stimulation, 6 h). Results : T cell proliferation indices were reduced following ex vivo co-culture with primary B-CLL cells (mean ± SEM for T cells vs. co-culture, 2.0 ± 0.13 vs. 1.57 ± 0.05; P<0.01). This suppression was significantly alleviated in 0.5μM PLX5-treated co-cultures (1.84 ± 0.08; P<0.01). In a similar fashion, the percentage of polyfunctional TNF-α+/IFN-γ+ CD4+ T cells markedly increased in PLX5-treated co-cultures (VEH vs. 0.5μM PLX5, 10.0% ± 0.76% vs. 15.2% ± 0.92%; P<0.01). Notably, BET inhibition with PLX5 also bolstered T cell inflammatory function (%TNF-α+/IFN-γ+) in the absence of B-CLL cells (VEH vs. PLX5, 12.9% ± 1.0% vs. 15.3% ± 0.69%; P<0.05). Remarkably, the expression of numerous immune inhibitory molecules (e.g., PDL1, PD1, CTLA4, LAG3) was consistently reduced between 1.8- and 3-fold in PLX5-treated co-cultures (0.1μM). In the adoptive transfer E μ-TCL1 model, mice receiving PLX5 displayed reduced expansion of B-CLL cells and increased T cell infiltration in the spleen (Fig. 1A). Splenic CD4+ T cells from PLX5-treated mice had significantly greater proliferative capacity (Fig. 1B) and pro-inflammatory functionality (Fig. 1C). Finally, PLX5 treatment markedly reduced the surface expression of immune inhibitory molecules (e.g., PDL1, LAG3, VISTA) on CD4+ and CD8+ T cells in the spleen (Fig. 1D). Studies to evaluate the effects of PLX5 on malignant B-CLL and T cells within the bone marrow niche and soluble factors in the plasma are ongoing. Collectively, our data indicate that the novel BET-i, PLX5, exerts beneficial immunomodulatory effects on T cells within the CLL TME. Conclusion : Epigenetic-targeted therapies such as BET-i have the potential to alleviate CLL-induced T cell dysfunction while eliminating B-CLL cells and preventing tumor expansion. Future profiling studies are pending to further illuminate how BET proteins regulate immune function in CLL. Figure 1 Figure 1. Disclosures Lunning: AstraZeneca: Consultancy; Legend: Consultancy; Acrotech: Consultancy; ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Myeloid Therapeutics: Consultancy; Beigene: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Morphosys: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Spectrum: Consultancy; Kite, a Gilead Company: Consultancy. Vose: Kite, a Gilead Company: Honoraria, Research Funding. Powell: Plexxikon Inc.: Current Employment.

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Type D SRV‐2 virus‐specific CD8 + and CD4 ‐ CD8 ‐ T cells that regulate virus‐induced T cell proliferation in Celebes macaques

Journal of Medical Primatology ◽

10.1111/j.1600-0684.1993.tb00643.x ◽

1993 ◽

Vol 22 (2-3) ◽

pp. 80-85

Author(s):

A. Malley ◽

N. Pangares ◽

S.K. Mayo ◽

M. Zeleny‐Pooley ◽

J.V. Torres ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

T Cell Proliferation ◽

Type D

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Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.186.10.1787 ◽

1997 ◽

Vol 186 (10) ◽

pp. 1787-1791 ◽

Cited By ~ 10

Author(s):

Pan Zheng ◽

Yang Liu

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Influenza Virus ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Target Cells ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

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