CD28 Costimulatory Domain-Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function.
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ABSTRACT:
SUBMITTER: Boucher JC
PROVIDER: S-EPMC7864379 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG4) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG4-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.
| S-EPMC8021955 | biostudies-literature
Project description:Transfer of autologous tumor infiltrating lymphocytes (TIL) to patients with refractory melanoma has shown clinical efficacy in a number of trials. However, extending the clinical benefit to patients with other cancers poses a challenge. Inefficient costimulation in the tumor microenvironment can lead to T cell anergy and exhaustion resulting in poor anti-tumor activity. Here, we describe a chimeric costimulatory antigen receptor (CoStAR) comprised of FRα-specific scFv linked to CD28 and CD40 intracellular signaling domains. CoStAR signaling alone does not activate T cells, while the combination of TCR and CoStAR signaling enhances T cell activity resulting in less differentiated T cells, and augmentation of T cell effector functions, including cytokine secretion and cytotoxicity. CoStAR activity resulted in superior T cell proliferation, even in the absence of exogenous IL-2. Using an in vivo transplantable tumor model, CoStAR was shown to improve T cell survival after transfer, enhanced control of tumor growth, and improved host survival. CoStAR could be reliably engineered into TIL from multiple tumor indications and augmented TIL activity against autologous tumor targets both in vitro and in vivo. CoStAR thus represents a general approach to improving TIL therapy with synthetic costimulation.
| S-EPMC10664248 | biostudies-literature
Project description:BackgroundChimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CAR-T cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CAR-T cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering.MethodsTwo second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5+ malignant cell lines, primary CD5+ malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5+ T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo.ResultsBoth BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5+ malignant cells in vitro and prolonged the survival of T-ALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5+ malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK.ConclusionsAnti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cell-associated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.
| S-EPMC6524286 | biostudies-literature
Project description:Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.
| S-EPMC4482147 | biostudies-literature
Project description:Purpose:Hepatocellular cancer (HCC) is the sixth most prevalent cancer and the third leading cause of cancer-related death worldwide. Cellular immunotherapy against glypican 3 (GPC3) has recently been used in the treatment of HCC, following the success of chimeric antigen receptor (CAR)-T therapy in treatment of B cell malignancy. However, CAR-T cells are not "off-the-shelf" and always cause cytokine release syndrome, which can be eliminated by using natural killer (NK) cells as effector cells. Since a costimulatory signal is necessary for the activation, persistence, or cytotoxicity of CAR-T cells, we speculated that the costimulatory signal is also required for CAR-NK cells in HCC treatment. Methods:Five anti-GPC3 CAR plasmids containing different costimulatory domains were constructed. They included Z (only the CD3? domain, no costimulatory domain), CD28.Z (T-cell costimulatory domain CD28), DNAM1/2B4.Z (NK-cell-associated costimulatory domain DNAM1 or 2B4), and DNAM1.2B4.Z (both NK-cell-associated costimulatory domains). Respective CAR-NK-92 cells were generated. The MTT viability assay was performed to evaluate the effect of the different costimulatory domains on CAR-NK-cell proliferation. The effect on persistence was analyzed using an apoptosis assay and flow cytometry. Special cytotoxicity against normal hepatocellular cells and GPC3+ malignant cells was investigated in vitro. The concentration of cytokines (TNF-? and IFN-?) released by CAR-NK-92 cells was also measured by ELISA. Results:NK-cell-associated costimulatory signal was necessary for CAR-NK-92 cells. CAR-NK-92 cells with DNAM1 and/or 2B4 expanded more quickly and persisted with a lower apoptotic ratio, compared to the presence of CD28 or no costimulatory signal. All CAR-NK-92 cells showed special cellular cytotoxicity in vitro. CAR-NK-92 cells with NK-cell-associated costimulatory domains exhibited higher cytotoxic ability compared with those without any costimulatory domain or with T-cell costimulatory domain. CAR-NK-92 cells with both DNAM1 and 2B4 displayed the highest cytotoxicity. The cytokine release assay results were consistent with those of the cytotoxicity assay. Conclusion:We provided the first evidence supporting a strategy using DNAM1 and 2B4 costimulatory domains to generate anti-GPC3 CAR-NK-92 cells, which exhibits enhanced cytotoxicity against hepatocellular cancer cells in vitro.
| S-EPMC7217313 | biostudies-literature
Project description:Adoptive T cell therapy with gene-modified T cells expressing chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and recently has shown dramatic therapeutic success in the treatment of B-cell malignancies. However, challenges to achieve similar response in patients harboring solid tumour are still considerable. To achieve anti-tumor efficacy, these cells must survive, expand and persist after infusion into patients. An important lesson has been derived from clinical trials using CAR technologies: the relevance of the CAR design to enhance signaling and sustain T cell proliferation and survival. Here, we prove that third generation CARs specific for GD2 antigen incorporating CD28.4-1BB costimulatory domains improves T cell immunotherapy in a neuroblastoma (NB) pre-clinical model, as compared to a CAR with the same specificity but including CD28.OX40 costimulation. Indeed, we test the anti-tumor activity of polyclonal T cells genetically modified with third generation CAR.GD2 incorporating either CD28.4-1BB or CD28.OX40 in frame with the safety switch inducible Caspase 9 (iC9). We prove a significant in vitro and in vivo amelioration of the approach by the presence of 4.1BB signaling in terms of: 1) less T cell exhaustion, 2) lower basal T cell activation, 3) higher in vivo tumor control and 4) T cell persistence. In addition, the fine tuning of T cell culture conditions with the use of IL7 and IL15 show to be synergic with the third generation CAR.GD2 design to optimize CAR T immunotherapy for NB. Our results provide a proof-of-concept for the need to optimize not only CAR construct design but also T cell culture conditions in order to boost T cell activity in vivo.
2018-06-27 | PXD005426 | Pride
Project description:PURPOSE:Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN:We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS:We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (K d < 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. CONCLUSIONS:A combinatorial costimulatory design allows the use of very low-affinity binding domains (K d < 1 ?mol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs.
| S-EPMC7477921 | biostudies-literature
Project description:Chimeric antigen receptor (CAR) T cells provide great efficacy in B cell malignancies. However, improved CAR T cell therapies are still needed. Here, we engineered tumor-targeted CAR T cells to constitutively express the immune-stimulatory molecule CD40 ligand (CD40L) and explored efficacy in different mouse leukemia/lymphoma models. We observed that CD40L+ CAR T cells circumvent tumor immune escape via antigen loss through CD40/CD40L-mediated cytotoxicity and induction of a sustained, endogenous immune response. After adoptive cell transfer, the CD40L+ CAR T cells displayed superior antitumor efficacy, licensed antigen-presenting cells, enhanced recruitment of immune effectors, and mobilized endogenous tumor-recognizing T cells. These effects were absent in Cd40-/- mice and provide a rationale for the use of CD40L+ CAR T cells in cancer treatment.
| S-EPMC6428219 | biostudies-literature
Project description:Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR(+) T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4alphabeta) was engineered by fusion of the IL-4 receptor alpha (IL-4Ralpha) ectodomain to the beta(c) subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4alphabeta resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4alphabeta with gamma(c) was implicated in signal delivery. Next, human T-cells were engineered to co-express 4alphabeta with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer.
| S-EPMC2919118 | biostudies-other
Project description:Purpose: The multiple mechanisms used by solid tumors to suppress tumor-specific immune responses are a major barrier to the success of adoptively transferred tumor-specific T cells. As viruses induce potent innate and adaptive immune responses, we hypothesized that the immunogenicity of viruses could be harnessed for the treatment of solid tumors if virus-specific T cells (VST) were modified with tumor-specific chimeric antigen receptors (CAR). We tested this hypothesis using VZV-specific T cells (VZVST) expressing a CAR for GD2, a disialoganglioside expressed on neuroblastoma and certain other tumors, so that the live-attenuated VZV vaccine could be used for in vivo stimulation.Experimental Design: We generated GMP-compliant, GD2.CAR-modified VZVSTs from healthy donors and cancer patients by stimulation of peripheral blood mononuclear cells with overlapping peptide libraries spanning selected VZV antigens, then tested their ability to recognize and kill GD2- and VZV antigen-expressing target cells.Results: Our choice of VZV antigens was validated by the observation that T cells specific for these antigens expanded in vivo after VZV vaccination. VZVSTs secreted cytokines in response to VZV antigens, killed VZV-infected target cells and limited infectious virus spread in autologous fibroblasts. However, while GD2.CAR-modified VZVSTs killed neuroblastoma cell lines on their first encounter, they failed to control tumor cells in subsequent cocultures. Despite this CAR-specific dysfunction, CAR-VZVSTs retained functional specificity for VZV antigens via their TCRs and GD2.CAR function was partially rescued by stimulation through the TCR or exposure to dendritic cell supernatants.Conclusions: Vaccination via the TCR may provide a means to reactivate CAR-T cells rendered dysfunctional by the tumor microenvironment (NCT01953900). Clin Cancer Res; 23(14); 3499-509. ©2017 AACR.
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