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Design of diversified chimeric antigen receptors through rational module recombination.

ABSTRACT: Chimeric antigen receptor (CAR)-T cells have shown great promise in cancer therapy. However, the anti-tumor efficiency is limited due to the CAR-induced T cell apoptosis or exhaustion. The intracellular domain of CAR comprised of various signaling modules orchestrates CAR-T cell behaviors. The modularity of CAR signaling domain functions as the "mainboard" to assemble diversified downstream signaling components. Here, we implemented the modular recombination strategy to construct a library of CARs with synthetic co-signaling modules adopted from immunoglobin-like superfamily (IgSF) and tumor necrosis factor receptor superfamily (TNFRSF). We quantitatively characterized the signaling behaviors of these recombinants by both NFAT and NF-κB reporter, and identified a set of new CARs with diverse signaling behaviors. Specifically, the 28(NM)-BB(MC) CAR-T cells exhibited improved cytotoxicity and T cell persistence. The synthetic approach can promote our understanding of the signaling principles of CAR molecule, and provide a powerful tool box for CAR-T cell engineering.

SUBMITTER: Si W

PROVIDER: S-EPMC10123334 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Design of diversified chimeric antigen receptors through rational module recombination.

Si Wen W Fan Ying-Ying YY Qiu Shi-Zhen SZ Li Xin X Wu Er-Yi EY Ju Jian-Qi JQ Huang Wen W Wang Hao-Peng HP Wei Ping P

iScience 20230330 4

Chimeric antigen receptor (CAR)-T cells have shown great promise in cancer therapy. However, the anti-tumor efficiency is limited due to the CAR-induced T cell apoptosis or exhaustion. The intracellular domain of CAR comprised of various signaling modules orchestrates CAR-T cell behaviors. The modularity of CAR signaling domain functions as the "mainboard" to assemble diversified downstream signaling components. Here, we implemented the modular recombination strategy to construct a library of CA ...[more]

PMID: 37102149

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Project description:Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T cell signaling domains. CD30 is a cell surface protein expressed on Hodgkin's lymphoma, some T cell lymphomas, and some B cell lymphomas. CD30 has a restricted expression pattern in normal cells, so CD30 has good potential as a clinical target for CAR T cells. We compared three different anti-CD30 CAR designs incorporating a single-chain variable fragment derived from the 5F11 fully human monoclonal antibody. 5F11-28Z has hinge, transmembrane, and costimulatory domains from CD28 and a CD3ζ T cell activation domain. 5F11-CD828Z has hinge and transmembrane domains from CD8α, a CD28 costimulatory domain, and a CD3ζ T cell activation domain. 5F11-CD8BBZ is identical to 5F11-CD828Z, except for the replacement of the CD28 moiety with a 4-1BB moiety. We found that T cells expressing 5F11-CD8BBZ had lower levels of CD30-specific degranulation and cytokine release compared with CD28-containing CARs. When compared to the CD28-containing CARs, T cells expressing 5F11-CD8BBZ had higher levels of nonspecific functional activity, including degranulation, cytokine release, and proliferation, when stimulated with CD30-negative target cells. We established tumors in nod-scid common gamma-chain deficient (NSG) mice and treated the tumors with T cells expressing different CARs. T cells expressing 5F11-28Z were most effective at eradicating tumors. T cells expressing 5F11-CD828Z had intermediate effectiveness, and T cells expressing 5F11-CD8BBZ were least effective. CD30+ T cells are lost from cultures of T cells containing 5F11-28Z-expressing T cells. This indicated the killing of CD30+ T cells by the 5F11-28Z-expressing T cells. Despite this, the number of T cells in the cultures consistently accumulated to numbers needed for use in a clinical trial. Based on all in vitro and murine experiments comparing the different CARs, we selected 5F11-28Z for further development, and we have initiated a clinical trial testing 5F11-28Z T cells.

Project description:Effective chimeric antigen receptor (CAR)-T cell therapy is dependent on optimal cell culture methods conducive to the activation and expansion of T cells ex vivo, as well as infection with CAR. Media formulations used in CAR-T cell manufacturing have not been optimized for gene delivery, cell expansion, and overall potency. Bioactive components and derivatives that support the generation of functionally-competent T cell progeny with long-lasting persistence are largely undefined. Current media formulations rely on fetal bovine serum (FBS) or human serum (HS), which suffer from a lack of consistency or supply issues. We recognize that components of blood cellular fractions that are absent in serum may have therapeutic value. Here we investigate whether a concentrated growth factor extract, purified from human transfusion grade whole blood fractions, and marketed as PhysiologixTM xeno-free (XF) hGFC (Phx), supports CAR-T cell expansion and function. We show that Phx supports T cell proliferation in clinical and research-grade media. We also show that Phx treatment enhances lentiviral-mediated gene expression across a wide range of multiplicity of infections (MOIs). We compared the ability of anti-GD-2 CAR-T cells expanded ex vivo in medium conditioned with either Phx or HS to clear tumor burden in a human xenograft model of neuroblastoma. We show that T cells expanded in Phx have superior engraftment and potency in vivo, as well as CAR-induced cytolytic activity in vitro. Metabolomic profiling revealed several factors unique to Phx that may have relevance for CAR-T cell preclinical discovery, process development, and manufacturing. In particular, we show that carnosine, a biogenic amine modestly enriched in Phx relative to HS, enhances lentiviral gene delivery in activated T cells. By limiting extracellular acidification, carnosine enhances the metabolic fitness of T cells, shifting their metabolic profile from an acidic, stressed state toward an oxidative, energetic state. These findings are very informative regarding potential derivatives to include in medium customized for gene delivery and overall potency for T cell adoptive immunotherapies.

Dataset Information

Design of diversified chimeric antigen receptors through rational module recombination.

Publications

Design of diversified chimeric antigen receptors through rational module recombination.

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