A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects

BackgroundDespite recent impressive successes in chimeric antigen receptor (CAR)-T cell therapy, there are still considerable clinical challenges. To improve T cell persistence and antitumor effect, which are critical for clinical...

646. Development of a Novel Chimeric Antigen Receptor as a Therapy Against Solid Tumors

Adoptive transfer of anti-CD19 CAR-engineered T cells have shown impressive and often durable clinical responses in patients with refractory and relapsed pre-B-cell acute lymphoblastic leukemia (ALL). However, clinical efficacy of anti-CD19 CAR-T cell therapy for lymphoma and chronic lymphocytic leukemia as well as CAR-T cell therapies that target solid tumors is not yet satisfactory. Optimal T cell activation and proliferation requires multiple signals including T cell receptor engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3). To the best of our knowledge, CAR genes currently tested in the clinic contain a CD3z domain (signal 1) and costimulatory domain(s) (signal 2), but not a domain that can transmit signal 3. Although forced expression of cytokine genes in CAR-T cells can improve their persistence and antitumor effects, constitutive cytokine expression poses a risk for autonomous T cell growth causing cancers. In this study, we have developed a novel CAR design, 28-IL2RB-z (YXXQ), which can activate the JAK-STAT pathway in an antigen-dependent manner, recapitulating cytokine exposure upon TCR engagement. We incorporated a minimal cytoplasmic domain of the IL-2 receptor β (IL2RB) between CD28 and CD3z, which contains a box1 motif for the binding of JAK family protein tyrosine kinases and the tyrosine residue at position 510, an essential site for STAT5 association. To promote STAT3 recruitment, we introduced a YXXQ motif to the C-terminus end of CD3z (28-IL2RB-z (YXXQ)). T cells transduced with the novel CD19 CAR gene induced phosphorylation of both STAT3 and STAT5 proteins only upon CD19 encounter, and exhibited increased expression of JAK-STAT target genes. Moreover, upon stimulation with CD19+ target cells, these T cells, compared to 2nd and 3rd generation CAR-T cells, demonstrated superior proliferative capacity with increased cellular division and reduced activation-induced cell death. Interestingly, CD8+ T cells engineered with this novel CAR maintained a stem cell-like memory (TSCM) phenotype and polyfunctional cytokine secretion capacity even after repeated antigenic stimulation. Treatment of 28-IL2RB-z (YXXQ) CAR-T cells with a STAT3 inhibitor largely abrogated their proliferative advantage and decreased the frequency of TSCM cells, suggesting that STAT3 signaling played a predominant role in the maintenance of the TSCM phenotype. All the 2nd generation, 3rd generation, and our new generation CAR-T cells similarly demonstrated potent in vitro cytotoxicity and IFN-γ secretion upon initial exposure to CD19+ cells. However, our novel CAR-T cells were superior to the other CAR-T cells in both cytolytic activity and cytokine secretion following multiple antigen exposures. NSG mice were intravenously injected with the human CD19+ leukemia cell line, NALM6, and treated with adoptive transfer of each of the 2nd, 3rd, or new generation CAR-transduced T cells. The 28-IL2RB-z (YXXQ) new generation CAR-T cells demonstrated superior persistence compared to other generation CAR-T cells, which resulted in significantly better overall survival. Long-term persisting 28-IL2RB-z (YXXQ) CAR-T cells from treated mice secreted more IFN-γ when restimulated ex vivo, suggesting the maintenance of a functional and less-exhausted phenotype in vivo. Furthermore, the 28-IL2RB-z (YXXQ) CAR-T cells also demonstrated better in vivo persistence and therapeutic effects than the 28-z and 28-BB-z CAR-T cells in NSG mice inoculated with primary human CD19+ pre-B-ALL cells. Importantly, the 28-IL2RB-z (YXXQ) CD19 CAR-T cells did not exhibit any proliferative responses when adoptively transferred into leukemia-free mice, affirming that JAK-STAT signals are activated only when exposed to the antigen. Taken together, these results suggest that our new generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicities in the clinic. Clinical translation of this novel CAR is warranted. This abstract is also being presented as Poster A73. Citation Format: Yuki Kagoya, Shinya Tanaka, Marcus O. Butler, Mark Minden, Naoto Hirano. A novel chimeric antigen receptor containing JAK-STAT signaling domains mediates superior antitumor effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR09.

XII. Therapeutic exploitation of autologous T-cell activation in B-cell lymphoma.

Enhancing CAR T Cell Anti-Tumor Efficacy through Secreted Single Chain Variable Fragment (scFv) Immune Checkpoint Blockade

T cell therapies have had valuable clinical responses in patients with cancer. Chimeric antigen receptor (CAR) T cells are genetically engineered to recognize tumor cells and CAR T cell therapy has had impressive results in the setting of B cell acute lymphoblastic leukemia but has been less effective in treating other types of hematologic and solid tumors. The inhibitory tumor microenvironment (TME), including expression of ligands that bind inhibitory receptors on T cells, e.g. programmed death receptor 1 (PD-1), can dampen CAR T cell responses. To prevent PD-1-mediated dampening of CAR T cell function, we have co-modified CAR T cells to secrete PD-1 blocking single chain variable fragments (scFv). We first designed mouse constructs with which we could investigate the scFv-secreting CAR T cells in the context of a syngeneic immune-competent intact TME. CAR constructs were engineered directed against either human CD19 or MUC-16 (ecto) and an anti-mouse PD-1 scFv. Mouse T cells transduced with these constructs expressed the CAR on the surface and secreted detectable amounts of scFv that bound to mouse PD-1. The scFv-secreting CAR T cells were cytotoxic and produced IFN- γ when co-cultured with PD-L1 expressing tumors in vitro. We utilized a syngeneic mouse model to study scFv secreting CAR T cells in a model with an intact TME. In tumor-bearing mice treated with CAR T cells, scFv-secreting CAR T cells enhanced survival as compared to second generation CAR T cells. The survival benefit achieved with scFv-secreting CAR T cells was comparable to that achieved with systemic infusion of PD-1 blocking antibody, but with localized delivery of PD-1 blockade. Mice treated with scFv-secreting CAR T cells had detectable scFv in vivo in the TME. Lastly, long term surviving mice had detectable CAR T cells in the bone marrow by PCR, demonstrating persistence and suggesting an immunological memory. We next aimed to translate PD-1 blocking scFv CAR T cells to a clinically relevant human model utilizing a novel anti-human PD-1 blocking scFv. Human T cells modified with the CAR constructs express the CAR on the surface and secrete detectable amounts of PD-1 blocking scFv. The scFv binds to human PD-1 and scFv-secreting CAR T cells are cytotoxic to PD-L1 expressing tumors. Expression of PD-1-blocking scFv enhances CAR T cell function against PD- L1 expressing tumors in xenograft models of hematological and solid tumors by enhancing survival in tumor-bearing mice as compared to second generation CAR T cells. Furthermore, scFv-secreting CAR T cells exhibit in vivo bystander T cell enhancement of function, suggesting scFv-secreting CAR T cells can reactivate endogenous TILs in the TME. These data support the novel concept that localized delivery of scFv by CAR T cells can successfully block PD-1 binding to PD-L1 to enhance the overall anti-tumor efficacy of CAR T cell therapy. Citation Format: Sarwish Rafiq, Oladapo Yeku, Hollie Jackson, Terence purdon, Dayenne van Leeuwen, Su Yan, Pei Wang, Jingyi Xiang, Cheng Liu, Venkatraman Seshan, Renier Brentjens. CAR T cells secreting an immune checkpoint blockade scFv have enhanced anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2568.

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Chimeric Antigen Receptor T Cell Therapies Clinical Trials in Pediatric Oncology: A Retrospective Analysis from Clinicaltrials.Gov

Chimeric Antigen Receptor Transgenic, T Cell Receptor/CD3 Negative Monospecific T Cells Generated from Cord Blood CD34 Positive Cells

Improving Anti-Tumor Efficacy of CAR T-Cell Therapy for Acute Myeloid Leukemia (AML): Results from a Multi-Drug Interaction Screening Approach

BackgroundAllogeneic chimeric antigen receptor (CAR)-T cells from healthy donors provide a readily available ‘off-the-shelf’ solution for improved patient accessibility and reduced cost compared to autologous CAR-T therapy. However, this approach...

Acute Kidney Injury After the CAR-T Therapy Tisagenlecleucel

Introduction: Chimeric antigen receptors (CARs) have been used successfully to retarget T cells in patients with hematologic malignancies. Natural killer (NK) cells offer an alternative to T cells for cellular immunotherapy and are suitable for allogeneic use as they are not HLA-restricted and patients who receive NK cell treatment do not develop graft-versus-host disease (GVHD). Therefore, NK cells can provide an attractive alternative for “off-the-shelf” cellular therapy. Here, we investigated multiple approaches to modify and enhance CD19 CAR expression on NK cells. It has been previously reported that costimulatory domains play an important role in proliferation, efficacy, and persistence of CAR-T cells both in vitro and in vivo. To understand how CAR structure effects the functional behavior of NK cells, we assessed the capability of various costimulatory signaling domains, including CD28, OX40, CD28-41BB, etc., all coupled to CD3ζ, to enhance CD19 CAR signaling and cytotoxicity in NK cells. We demonstrated that NK activity and persistence can be elevated by simultaneous expression of chimeric constructs directing the expression of a CD19 CAR and a membrane-bound form of IL-15 (mbIL-15). Methods: NK cells were generated by coculture of peripheral blood mononuclear cells (PBMC) with genetically modified irradiated K562 feeder cells. NK cells were transduced at a MOI of 1-2 with a gamma-retrovirus encoding a CD19 CAR and mbIL-15. NK expansion and CAR expression were evaluated by flow cytometry. In vitro cytotoxicity of transduced NK cells was measured using both flow cytometry and the IncuCyte S3 live cell analysis system. The in vivo activity of engineered NK cells was further assessed in a xenograft tumor model in NSG mice, using a Nalm6 leukemia cell line. Results: CD19 CAR constructs containing various costimulatory domains were all expressed in expanded and transduced NK cells. CAR expression in multiple donor NK cells was typically between 60-90%. In cytotoxicity assays, CD19 CAR constructs containing the costimulatory domains of OX40, CD28, or CD27 generally exhibited the greatest cytotoxic activity against Nalm6 and Raji tumor cell lines in vitro. High expression of these three CD19 CAR constructs was also maintained over the course of 4 weeks in addition to sustained NK proliferation and cell numbers, indicating an increase in NK cell persistence. In comparing these constructs in an in vivo Nalm6 tumor model, all three constructs demonstrated greater antitumor efficacy relative to unmodified NK cells. The CD19 CAR construct containing the OX40 costimulatory domain showed moderately enhanced efficacy than the CD28 or CD27 variants in vivo. Conclusion: Transduction of NK cells with CD19-OX40-CD3ζ CAR and mbIL15 increases their cytotoxic activity and persistence. Based on these data, further development of NK CD19 CAR for clinical use will be pursued. Citation Format: Luxuan G. Buren, Chao Guo, Yanying Fan, Alexander Aronov, Xiumin Wu, Daofeng Liu, Ming-Hong Xie, Sasha Lazetic, Ivan H. Chan, James B. Trager. Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B64.

Inducible MyD88/CD40 Allows AP1903-Dependent Costimulation to Control Proliferation and Survival of Chimeric Antigen Receptor-Modified T Cells