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CD28 Costimulatory Domain-Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function.

ABSTRACT: An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1 Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.

SUBMITTER: Boucher JC

PROVIDER: S-EPMC7864379 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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.

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CD28 Costimulatory Domain-Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function.

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