Engineering CAR-T Therapeutics for Enhanced Solid Tumor Targeting

Abstract

Cancer immunotherapy, specifically Chimeric Antigen Receptor (CAR)-T cell therapy, represents a significant breakthrough in treating cancers. Despite its success in hematological cancers, CAR-T exhibits limited efficacy in solid tumors, which account for more than 90% of all cancers. Solid tumors commonly present unique challenges, including antigen heterogeneity and complex tumor microenvironment (TME). To address these, efforts are being made through improvements in CAR design and the development of advanced validation platforms. While efficacy is limited, some solid tumor types, such as neuroblastoma and gastrointestinal cancers, have shown responsiveness to CAR-T therapy in recent clinical trials. In this review, it is first examined both experimental and computational strategies, such as protein engineering coupled with machine learning, developed to enhance T cell specificity. The challenges and methods associated with T cell delivery and in vivo reprogramming in solid tumors is discussed. It is also explored the advancements in engineered organoid systems, which are emerging as high-fidelity in vitro models that closely mimic the complex human TME and serve as a validation platform for CAR discovery. Collectively, these innovative engineering strategies offer the potential to revolutionize the next generation of CAR-T therapy, ultimately paving the way for more effective treatments in solid tumors.

Keywords: Chimeric antigen receptor (CAR); cancer immunotherapy; drug delivery; nanoparticle.

Figure 1

A) Existing methods of chimeric antigen receptor (CAR) T designs. The dual/bi‐CAR (left) is tuned to react to any given tumor surface antigen (similar to an OR gate), or it can be constrained to respond only when both types of antigens are present (AND gate). B) Recent methods to improve CAR T specificity. Synthetic Notch (synNotch) was developed for sequential activation of the CAR T cell, requiring a “priming” antigen to activate the CAR gene coding for a receptor specific to the secondary tumor antigen. Inhibitory CAR (iCAR) adds a safety measure to the existing AND‐gate bi‐CAR system to inhibit CAR T from attacking dual‐antigen‐presenting normal cells. ZipCAR has a leucine zipper to selectively attack a specific type of tumor present in the neighboring environment. Oncolytic viruses target the tumor cells and enable surface expression of specific antigens (e.g., CD19), which in turn activate the downstream CD19‐CAR T cell specifically.

Figure 2

In vitro and in vivo CRISPR‐based screening of CAR T cells phenotype. Both modalities require a design of the single‐guide (sg)RNA library to knock‐out or ‐down the genes of interest. For in vitro screening, the transduced T cells are co‐cultured with the tumor cells or stromal cells for proper activation. Then, the manipulated T cells will be sorted, and bulk RNA sequencing will be performed on phenotypes of interest (e.g., exhaustion, migration) to identify the difference in sgRNA enrichment. In vivo screening is conducted with the packaged sgRNA library in delivery vectors or the sgRNA‐transduced CAR T cells. scRNA‐seq is commonly conducted to identify all subtypes at the single‐cell level present in the tumor microenvironment (TME).

Figure 3

Engineering biomaterials for in vivo T cell reprogramming. A) Lentivirus (LV) specificity to T cells is often altered by mutating the glycoprotein vesicular stomatitis virus G protein (VSV‐G). Virus‐like particles (VLPs) have often been more effective when T cell receptor binding antibodies are conjugated to their surface proteins. B) Directed evolution of adeno‐associated viruses (AAVs) can be conducted on short peptide insertion (PDB ID: 7UD4) or DNA‐shuffled (PDB ID: 1LP3) capsid protein libraries. Different variants (indicated by colored icosahedrons) carry a different fitness (e.g., affinity) to the targeted receptors, leading to selective transduction of T cells. Several rounds of iterations are conducted to enrich the variant(s) of interest, which will then be recovered through deep sequencing. C) Production of lipid nanoparticles (LNPs). Components of the lipid bilayer enter the microfluidic channel in an alcoholic phase and the mRNA CAR gene transcript enters the aqueous phase. The single‐chain variable fragment (scFv) that binds to the receptors is conjugated to the eluted LNPs for increased transfection efficiency. LNPs are purified through size‐selection chromatography and used for T cell reprogramming. D) Extracellular vesicles (EVs) for CAR delivery. CAR gene is delivered to EV producer cells via a vector (e.g., AAV). The EVs that bud out of the producer cells are isolated and used to transfect T cells for expression of CAR. E) Polymers, native T cell antigens, and CAR gene‐containing nanoparticles are mixed in a one‐pot hydrogel complex assembly. An in vivo tumor model post‐resection is injected with the hydrogel at the tumor cavity. The hydrogel forms an “immune cell reservoir” and allows local reprogramming of T cells with controlled release.

Figure 4

Illustration of the differential interactions between cancer organoids and various extracellular matrices (ECMs), including basement membrane extract, interstitial ECM, and synthetic polymer‐based matrix. Each ECM type offers distinct properties such as tunable mechanical properties, patient‐specific characteristics, and complex interactions with immune and stromal cells, which influence the outcomes of high‐throughput screening of anticancer antibody therapies. The differences in immunogenicity and lot‐to‐lot variability are also highlighted, emphasizing the importance of selecting the appropriate ECM type for precise screening applications in CAR therapy development.