Spatial Insights for CAR-Engineered Cells in Solid Tumors: Antibody-Based CAR Detection Method

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Chimeric Antigen Receptor (CAR) cell therapies are a rapidly evolving cancer treatment option that has shown incredible efficacy for hematologic malignancies such as B-cell leukemia and lymphoma. However, translating this success to solid tumors remains challenging—as of April 2025, no CAR-T or CAR-NK cell therapy for solid tumors has received regulatory approval, although multiple candidates are in clinical trials in the US.^1,2,3

This blog explores recent strategies employed to leverage CAR-based cellular therapies in solid tumor immunotherapies, and describes a new antibody for spatial interrogation of CAR-engineered cells that express a single-chain variable fragment (scFv)-based CAR containing a Whitlow/218 linker: The Whitlow/218 Linker (F2G3S) Rabbit mAb #47414, now validated for use in immunohistochemistry (IHC) assays.

A Solid Chance: Overcoming Challenges in Solid Tumor Immunotherapy

At the highest level, solid tumors are a fundamentally different biological landscape than hematologic malignancies. Unlike leukemias and lymphomas, where tumor cells circulate freely in the bloodstream, solid tumors constitute a highly complex cellular neighborhood that is specifically organized to suppress immune infiltration, prevent activation, and protect tumor cells from immune cell-mediated attack.

Additionally, while lineage-restricted surface antigens such as CD19 and BCMA serve as well-defined targets for CAR-based cellular therapies for hematological malignancies, the heterogeneity of tumor antigen expression in solid tumors dictates that multiple antigens for each tumor type must be strategically selected for therapeutic targeting. Not only is it difficult to identify appropriate target antigens, but tumor antigen expression can vary among patients with the same cancer type, meaning that the panel of antigens targeted for one patient might not be a suitable approach for another. Compounding this challenge is the fact that many target antigens expressed in solid tumors are also expressed in healthy tissue, leading to the potential for on-target, off-tumor toxicities—a critical issue that can result in serious safety risks for patients.^1,4

Explore CST videos and posters to learn how to characterize your CAR-engineered cells easier and faster.

To address these and other obstacles, researchers are exploring a number of different approaches. One strategy that has shown promise is to leverage CAR systems based on AND logic-gating, in which T cells engineered to express two unique CARs that target different antigens are only activated when both CARs engage the target antigens. This dual antigen recognition can help increase specificity and reduce the likelihood of damage to healthy tissues. 

Strategies to protect CAR-T cells once they enter the TME are also being explored, including engineering CAR-T cells to express dominant negative receptors in order to resist immune-suppressive cytokines like TGF-β.

Promising CAR cell therapies for solid tumors that incorporate strategies to enhance specificity and resist immunosuppression include: 

• GD2-targeted CARs for diffuse midline gliomas (DMG), with early trials showing tumor shrinkage and neurological improvement in children⁶
• CEA-targeted CARs for treating gastrointestinal and lung cancers¹
• Claudin18.2-specific CARs for gastric and pancreatic tumors, though clinical data remain preliminary¹

Spatial Analysis of CAR-T Cells in Solid Tumors

As researchers continue to refine CAR cell engineering strategies, one critical question remains: Are these cells actually reaching their targets within the tumor, and are they persisting long enough to mount an effective attack? To answer this, researchers need tools that allow them to visualize and quantify CAR-engineered cells in their spatial context within the tumor microenvironment.

Several methods are currently used to detect engineered CAR-T cells in tissues:

Anti-idiotype antibodies

Anti-idiotype antibodies have long been the gold standard for CAR detection. However, these antibodies are typically custom-made to identify a specific CAR construct, and are time-intensive and expensive to develop. Additionally, any modifications to the CAR structure—such as optimizing its binding properties—may alter the epitope recognized by the anti-idiotype antibody, rendering it ineffective.

RNA in situ hybridization (RNA ISH)

Another commonly used method is RNA in situ hybridization (RNAish), which detects the presence of the CAR mRNA transcripts within tissues. This technique provides valuable spatial information, allowing researchers to see where CAR-T cells are localizing within the tumor. However, it comes with a major drawback: It only confirms that the RNA is present, and not whether it is being translated into a functional CAR protein. The many complex post-translational regulatory mechanisms in the TME therefore leave researchers uncertain about whether the detected cells are actually present and actively fighting the tumor.

Anti-CAR Linker antibodies

Anti-CAR linker antibodies are a new class of reagents for detecting the presence of scFv-based CAR-engineered cells in IHC assays, where the spatial biology of the heterogeneous TME can be preserved. Unlike anti-idiotype antibodies, anti-CAR linker antibodies target the ubiquitous peptide linkers (G4S and Whitlow/218) that are present in a majority of scFv-based CARs and remain effective even when modifications are made to non-linker CAR structures. This means that one detection reagent can be leveraged across multiple programs, even as CARs targeting different antigens are designed and tested. Additionally, unlike RNA-based methods, which only indicate the presence of genetic material, the Whitlow/218 linker rabbit antibody directly detects CAR protein, confirming whether engineered cells are actively expressing CAR molecules at the cell surface.

IHC analysis of FFPE spleen tissue of Raji B-Cell lymphoma in an immunocompromised mouse (NSG strain) using the recombinant monoclonal antibody Whitlow/218 Linker (F2G3S) Rabbit mAb #47414. Treated with primary human CD19 CAR-T cells (left) or with PBS control (right).

The Whitlow/218 Linker (F2G3S) rabbit antibody provides a highly specific, sensitive, and reproducible method for CAR detection in formalin-fixed paraffin-embedded tissues (FFPE) tissues, and allows researchers to accurately assess whether their engineered T cells are successfully infiltrating tumors and persisting in the TME.

How can I detect G4S linker CARs in tissue? CST scientists are actively working to validate an anti-G4S linker antibody for use in IHC. Check back here for the latest, or reach out to us for more information.

The Future of Solid Tumor Immunotherapy

CAR therapies for solid tumors show promising potential, but hurdles need to be overcome. Logic-gated systems, designs resistant to immune suppressive mechanisms, and novel target identification are helping researchers explore new avenues for success. Tools like the Whitlow/218 linker antibody can help researchers quickly interrogate CAR presence, localization, and persistence, enabling the more rapid development of novel treatment strategies. 

Interrogate the TME with CST Antibodies CST offers an expansive IHC-validated portfolio of unconjugated and conjugated antibodies for interrogating the TME using antibodies to key phenotypic and functional biomarkers including PD1, CD25, TIM3, CD4, LAG3, CD8, and many more. The SignalStar® multiplex IHC technology is ideal for visualizing up to eight biomarkers in the same panel. Explore CAR-engineered cell characterization solutions from CST.

While curative treatments are not yet a reality, the convergence of spatial biology and adaptive cell engineering offers a roadmap for transforming solid tumor immunotherapy using CAR-engineered cells.

その他のリソース

• To see how researchers are using the CST anti-CAR linker antibodies, read the blog: Research Round-Up: CST Anti-CAR Linker Antibodies in the Literature.
• For a downloadable resource for CAR-engineered cell characterization solutions, including T-cell activation assays and conjugated and unconjugated immunophenotyping and functional antibodies, access the brochure.

参考文献

1. Khan SH, Choi Y, Veena M, Lee JK, Shin DS. Advances in CAR T cell therapy: antigen selection, modifications, and current trials for solid tumors. Front Immunol. 2025;15:1489827. Published 2025 Jan 6. doi:10.3389/fimmu.2024.1489827
2. Yeku O, Li X, Brentjens RJ. Adoptive T-Cell Therapy for Solid Tumors. Am Soc Clin Oncol Educ Book. 2017;37:193-204. doi:10.1200/EDBK_180328
3. Peng L, Sferruzza G, Yang L, Zhou L, Chen S. CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors. Cell Mol Immunol. 2024;21(10):1089-1108. doi:10.1038/s41423-024-01207-0
4. Guzman G, Reed MR, Bielamowicz K, Koss B, Rodriguez A. CAR-T Therapies in Solid Tumors: Opportunities and Challenges. Curr Oncol Rep. 2023;25(5):479-489. doi:10.1007/s11912-023-01380-x
5. Punekar SR, Kirtane K, Stein MN, et al. EVEREST-1: A seamless phase 1/2 study of A2B530, a carcinoembryonic antigen (CEA) logic-gated Tmod CAR T-cell therapy, in patients with solid tumors associated with CEA expression also exhibiting human leukocyte antigen (HLA)-A*02 loss of heterozygosity (LOH). J Clin Oncol. 2024;42(16_suppl):TPS2698
6. Monje M, Mahdi J, Majzner R, et al. Intravenous and intracranial GD2-CAR T cells for H3K27M+ diffuse midline gliomas [published correction appears in Nature. 2024 Dec;636(8043):E6. doi: 10.1038/s41586-024-08452-3.]. Nature. 2025;637(8046):708-715. doi:10.1038/s41586-024-08171-9