Kevin Friedman

Selective Inhibition of Regulatory T Cells by Targeting the PI3K–Akt Pathway

Abu-Eid, Samara, and colleagues used PI3K–Akt pathway inhibitors to selectively disrupt homeostasis of immunosuppressive Tregs in naïve and tumor-bearing mice, and to enhance vaccine-induced antitumor immune responses, highlighting the therapeutic potential of these inhibitors as Treg-depleting reagents. Despite the strides that immunotherapy has made in mediating tumor regression, the clinical effects are often transient, and therefore more durable responses are still needed. The temporary nature of the therapy-induced immune response can be attributed to tumor immune evasion mechanisms, mainly the effect of suppressive immune cells and, in particular, regulatory T cells (Treg). Although the depletion of Tregs has been shown to be effective in enhancing immune responses, selective depletion of these suppressive cells without affecting other immune cells has not been very successful, and new agents are sought. We found that PI3K–Akt pathway inhibitors selectively inhibit Tregs with minimal effect on conventional T cells (Tconv). Our results clearly show selective in vitro inhibition of activation (as represented by a decrease in downstream signaling) and proliferation of Tregs in comparison with Tconvs when treated with different Akt and PI3K inhibitors. This effect has been observed in both human and murine CD4 T cells. In vivo treatment with these inhibitors resulted in a significant and selective reduction in Tregs in both naïve and tumor-bearing mice. Furthermore, these PI3K–Akt inhibitors led to a significant therapeutic antitumor effect, which was shown to be Treg dependent. Here, we report the use of PI3K–Akt pathway inhibitors as potent agents for the selective depletion of suppressive Tregs. We show that these inhibitors are able to enhance the antitumor immune response and are therefore promising clinical reagents for Treg depletion. Cancer Immunol Res; 2(11); 1080–9. ©2014 AACR.

Akt1 and -2 inhibition diminishes terminal differentiation and enhances central memory CD8+ T-cell proliferation and survival

The CD8+ T-cell response comprises terminally differentiated effector cells and antigen-experienced memory T cells. The latter encompass central (TCM) and effector (TEM) memory cells. TCM cells are superior in their protection against viral and bacterial challenges and mediation of antitumor immunity due to their higher proliferative ability upon antigen re-encounter. Defining a mechanism to enhance TCM cells and delay terminal differentiation of CD8+ T cells is crucial for cancer immune therapy, as it can promote a better tumor immune response. The differentiation of CD8+ memory T cells is thought to be coordinated by the phosphoinositide 3-kinase (PI3K)/Akt pathway. We, therefore, investigated the role of Akt isoforms in the differentiation and proliferation of memory CD8+ T cells. We found that Akt1 and Akt2, but not Akt3, drive the terminal differentiation of CD8+ T cells, and their inhibition enhances the therapeutically superior TCM phenotype. Furthermore, the inhibition of Akt1 and Akt2, but not Akt 3, delays CD8+ T-cell exhaustion and preserves naïve and TCM CD8+ T cells, thus enhancing their proliferative ability and survival and prolonging their cytokine and Granzyme B production ability. Here, we define a mechanism in which proliferative potential, function, and survival of CD8+ T cells are enhanced by maintaining a reservoir of TCM and naïve cells using only Akt1 and Akt2 inhibition. Therefore, our findings strongly suggest the utility of using Akt1 and Akt2 inhibitors to modulate CD8+ T cells, both for adoptive cell transfer and vaccine-based cancer immune therapies.

A BCMA-specific CAR T cell produced with clinically scalable lentiviral and T cell manufacturing processes has potent anti-multiple myeloma activity

Meeting abstracts Chimeric antigen receptors (CARs) operably link T cell activation domains to the antigen recognition properties of an antibody, thereby redirecting T lymphocytes to cells expressing the antigen. B cell maturation antigen (BCMA) is an attractive CAR T cell target to treat patients

Effective Targeting of Multiple B-Cell Maturation Antigen–Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

Abstract 2296: Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models

Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells for treatment requires ex vivo culture to facilitate CAR gene transfer and to achieve a therapeutic dose of the modified cells. Recent data suggests that specific T cell subtypes can provide enhanced anti-tumor efficacy, spurring efforts to optimize the production of therapeutic T cells via the cumbersome physical isolation of central memory T cells or culture in cytokines such as IL-7 and IL-15. Here we explored the potential for a simple culture modification to improve the therapeutic potential of CAR T cells without adding manufacturing complexity. To this end, we produced CAR T cells specific to B cell maturation antigen (BCMA) using standard IL-2 culture conditions supplemented with a PI3K inhibitor, or with IL-7 and IL-15 in place of IL-2. The in vivo activity of CAR T cells was studied in mouse models of human Burkitt9s lymphoma (Daudi) and multiple myeloma (RPMI-8226), both of which express BCMA. In the Daudi model, NSG mice were injected intravenously with 2 × 106 tumor cells and allowed to accumulate a large tumor burden to model late stage disease observed in relapsed and refractory lymphoma. In this advanced disease model, anti-BCMA CAR T cells (4 × 106/mouse) cultured either in IL-2 or IL-7 and IL-15 had little or no effect on tumor growth (p = 0.22 and 0.23, respectively) and all mice succumbed to tumors within two weeks of treatment. In contrast, all animals treated with the same number of anti-BCMA CAR T cells cultured with PI3K inhibition survived and had complete long-term tumor regression (p = 0.003). The same anti-BCMA CAR T cells were studied in a model of multiple myeloma. NSG mice were injected subcutaneously with 107 RPMI-8226 cells and 22 days later received a single administration of anti-BCMA CAR T cells (4 × 105/mouse) cultured under various conditions. In this model, tumor regression occurred regardless of in vitro culture conditions. To model tumor relapse and evaluate CAR T cell durability, surviving animals were re-challenged with RPMI-8226 cells on the opposite flank two weeks after initial tumor clearance. In contrast to other conditions, all animals treated with anti-BCMA CAR T cells cultured with PI3K inhibition were protected against subsequent tumor challenge (p = 0.005). This improved therapeutic activity of anti-BCMA CAR T cells cultured with PI3K inhibition was associated with an increased frequency of CD62L+ CD8+ T cells in the drug product (p with IL-2 may generate an improved anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Citation Format: Shannon Grande, Molly R. Perkins, Amanda Hamel, Holly M. Horton, Fay Eng, Claire J. Rhodes, Tracy E. Garrett, Sara M. Miller, John W. Evans, Howard J. Latimer, Christopher Horvath, Michael Kuczewski, Kevin Friedman, Richard A. Morgan. Inhibition of the PI3K/Akt pathway during CAR T cell production results in enhanced efficacy across multiple in vivo tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2296.

Effective Targeting of Multiple BCMA-Expressing Hematological Malignancies by Anti-BCMA CAR T Cells

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

Abstract 602: A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function

Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder chimeric antigen receptor (CAR) T cell efficacy. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. A dominant negative receptor version (DNR) of TGFBR2 that does not contain signaling domains protects T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs. Here, we report the development of a novel TGF-β signal conversion platform that provides a T cell stimulatory signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of the T cell simulating IL-12 receptors IL-12R-β2 and IL-12R-β1, respectively. Using a single lentiviral vector encoding both chimeric TGF-β receptors (CTBR) and a CAR, we demonstrated that CAR-CTBR T cells were completely protected from TGF-β-mediated SMAD phosphorylation. In addition, CAR-CTBR T cells generated significant amounts of pSTAT4 and pSTAT5 in response to TGF-β exposure, a response that mimics the T cell stimulation effects of IL-12. To further demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on the secretion of IFNγ, a major downstream target of IL-12 signaling. CAR-CTBR cells secreted significantly greater amounts of IFNγ than either control CAR T or CAR-DNR T cells following activation in the presence of TGF-β. Lastly, we utilized a serial restimulation assay to expand CAR, CAR-DNR, and CAR-CTBR T cells in the presence or absence of TGF-β. As expected, TGF-β exposure resulted in a significant inhibition of T cell proliferation in control CAR T cells. By contrast, both CAR-DNR and CAR-CTBR cells were protected from TGF-β-mediated inhibition of expansion. Gene expression analysis following 21 days of weekly antigen-driven expansion revealed specific TGF-β-mediated gene expression changes in CAR-CTBR cells consistent with increased T cell potency, including significant upregulation of IFNγ, IL10, IL18RAP, IL18R1, IL21R and CD62L transcripts. These data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into an IL-12R-like T cell stimulatory signal that has the potential to produce superior CAR T cell responses in vivo. Note: This abstract was not presented at the meeting. Citation Format: Benjamin Boyerinas, Sara Miller, Ryan Murray, Stacie Seidel, Geoffrey Parsons, Kathy Seidl, Kevin Friedman, Richard Morgan. A novel TGF-β/IL-12R signal conversion platform that protects CAR T cells from TGF-β-mediated immune suppression and concurrently amplifies effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2017-602

747. Towards the Clinical Application of BCMA CAR T Cells: The Importance of Reduced Tonic Signaling and Methods to Enhance Memory T Cells

B cell maturation antigen (BCMA) is expressed on most multiple myeloma (MM) and some lymphoma cells, yet normal tissue expression is limited to plasma and some B cells. Here we describe the development of a CAR T cell that targets BCMA. We tested 19 BCMA-specific single chain variable fragments (scFv) linked to CD137 (4-1BB) and CD3zeta T cell signaling domains. The activity of each CAR T cell candidate differed despite targeting the same antigen. One anti-BCMA CAR T cell candidate (bb2121) was selected based on strong surface CAR expression, superior biological activity to multiple BCMA+ cell lines, and low antigen-independent activity. In vitro analysis demonstrated that antigen-independent activity was associated with T cell differentiation characterized by significantly lower CD62L expression (p=0.003). To determine the impact on tumor control, CAR T cells with antigen-independent activity, or bb2121 CAR T cells, were used to treat an NSG mouse model of MM (RPMI-8226). Immunohistochemical analysis found MM-infiltrated bb2121 T cells within 5 days and mice showed complete regressions by 12 days post treatment. In contrast, mice that received CAR T cells with antigen-independent activity had delayed regressions that occurred 20 days post treatment. Robust recognition of as few as 220 BCMA molecules/cell (compared to >10,000 molecules on MM cell lines) permitted reactivity to primary chronic lymphocytic leukemia (CLL) and other CD19+ B cell tumor lines. Indeed comparable tumor regressions were observed after administration of bb2121 or anti-CD19 CAR T cells into an NSG mouse model of disseminated Burkitts lymphoma (Daudi: 1,170 BCMA and 267,000 CD19 molecules). Prior investigators have shown improved therapeutic efficacy by enriching for memory CAR T cells, yet current antibody-based selection methods are expensive and difficult to scale. Unexpectedly, we found bb2121 manufacture in the presence of a PI3-kinase inhibitor enriched for memory-like CAR T cells without a complicated cell sorting procedure. bb2121 CAR T cells cultured with a PI3K inhibitor expressed markers associated with T cell memory including CD62L, CD127, CD197, and CD38. In a first “stress test,” of advanced disseminated lymphoma (Daudi), NSG mice were administered an amount of bb2121 CAR T cells that failed to control tumor outgrowth. Mice administered the same number of bb2121 CAR T cells cultured with a PI3K inhibitor resulted in complete tumor regressions. A defining property of memory T cells is durability despite multiple antigen encounters. In a second “stress test,” bb2121 CAR T cell durability was evaluated after initial MM (RPMI-8226) clearance. Two weeks post-tumor elimination, mice were re-challenged with tumor on the opposite flank without an additional dose of CAR T cells. None of the mice treated with bb2121 CAR T cells prevented tumor outgrowth in this model. In contrast, all mice treated with bb2121 CAR T cells cultured with a PI3K inhibitor were able to control the tumor re-challenge. These data demonstrate that a potent, antigen-dependent, memory-like BCMA CAR T cell produced with an industrially scalable manufacturing process has promise for robust tumor regressions in clinical applications.

AKT inhibition mitigates terminal differentiation and preserves central memory phenotype of CD8 T cells

CD8 T cell response comprises effector and memory T cells. Effector CD8 T cells become terminally differentiated and are eliminated by apoptosis. Memory CD8 T cells encompass central (TCM) and effector memory T cells (TEM). TCM display a higher proliferative ability, express a higher level of CD62L and are superior in their protection against viral and bacterial challenges and mediation of anti-tumor immunity when compared to TEM. The differentiation of CD8 T cell is thought to be coordinated by the PI3K/Akt pathway.