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In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function
Published on: February 27, 2019
Tandem CAR-T cells targeting mesothelin and MUC16 overcome tumor heterogeneity by targeting one antigen at a time
Diego Salas-Benito1,2,3, Filippo Birocchi1,2,3, Sangwoo Park1,2,3
1Cellular Immunotherapy Program, Massachusetts General Hospital, Boston, Massachusetts, USA.
Background:
Tumor heterogeneity and antigen escape are mechanisms of resistance to chimeric antigen receptor (CAR)-T cell therapy, especially in solid tumors. Targeting multiple antigens with a unique CAR construct could be a strategy for a better tumor control than monospecific CAR-T cells on heterogeneous models. To overcome tumor heterogeneity, we targeted mesothelin (meso) and Mucin 16 (MUC16), two antigens commonly expressed in solid tumors, using a tandem CAR design.
Methods:
We designed a series of tandem CAR constructs based on various anti-meso (SS1) and anti-MUC16 ectodomain (MUC16ecto) (4H11) single-chain variable fragment (scFv) arrangements and G4S linker lengths. Then we determined the best tandem CAR design based on binding of soluble antigens, steric hindrance, avidity and functionality against cell lines expressing one or both antigens in vitro. Finally, we compared the tandem CAR to monospecific CAR-T cells in mixed tumor models in vitro (two-dimensional and three-dimensional models) and in vivo.
Results:
We show that the scFv arrangement and linker length impacted antigen binding and CAR expression in T cells. Tandem CAR configuration (TanCAR1) (with SS1 scFv located distally and one G4S repeat as the linker between scFvs) had the best binding and activation profile in vitro and outperformed SS1 and 4H11 monospecific CAR-T cells in mixed tumor models in vitro and in vivo, showing an antigen-driven killing of tumor cells based on antigen density. Moreover, acoustic force microscopy, using tumor cells with different levels of antigen expression, revealed that TanCAR1-T cells likely bind to one antigen at a time rather than simultaneously.
Conclusions:
This is the first time using a tandem CAR design targeting meso and MUC16, and demonstrating a benefit on tumor control over monospecific CAR-T cells. Tandem CAR-T cells targeting meso and MUC16ecto could be employed as a strategy to overcome tumor cell heterogeneity in ovarian and pancreatic tumors, and may help to design therapeutic approaches relying on its one-antigen-at-a-time binding properties and on its antigen-driven killing of tumor cells based on antigen density.
Insights
This study developed a novel tandem CAR-T cell therapy targeting mesothelin and MUC16 to combat tumor heterogeneity. The new tandem CAR-T cells demonstrated superior tumor control compared to single-target CAR-T cells in preclinical models.
Area of Science:
- Immunotherapy
- Oncology
- Biotechnology
Background:
- Tumor heterogeneity and antigen escape limit chimeric antigen receptor (CAR)-T cell therapy efficacy, particularly in solid tumors.
- Targeting multiple antigens simultaneously with a novel CAR construct offers a potential strategy to enhance tumor control against heterogeneous cancer cells.
Purpose of the Study:
- To design and evaluate a tandem CAR construct targeting mesothelin (meso) and Mucin 16 (MUC16) for improved solid tumor treatment.
- To compare the efficacy of the novel tandem CAR-T cells against monospecific CAR-T cells in preclinical models of tumor heterogeneity.
Main Methods:
- Designed and optimized tandem CAR constructs with varying anti-meso (SS1) and anti-MUC16 (4H11) scFv arrangements and linker lengths.
- Assessed CAR construct binding, avidity, and functionality in vitro using cell lines expressing one or both antigens.
- Compared the performance of the lead tandem CAR construct (TanCAR1) against monospecific CAR-T cells in 2D, 3D, and in vivo mixed tumor models.
Main Results:
- The tandem CAR configuration (TanCAR1) exhibited optimal binding and activation profiles, outperforming monospecific CAR-T cells in mixed tumor models.
- TanCAR1-T cells demonstrated antigen-driven tumor cell killing based on antigen density, with evidence suggesting binding to one antigen at a time.
- scFv arrangement and linker length significantly influenced CAR expression and antigen binding in T cells.
Conclusions:
- This study presents the first tandem CAR targeting mesothelin and MUC16, showing enhanced tumor control over monospecific CAR-T cells.
- Tandem CAR-T cells targeting mesothelin and MUC16ecto represent a promising strategy to overcome tumor heterogeneity in ovarian and pancreatic cancers.
- The unique binding properties of TanCAR1-T cells, involving antigen-driven killing based on density, offer a novel therapeutic approach.
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