4^th generation CAR-T cells with inducible cytokine expression targeting CD176 to overcome the immunosuppressive tumor milieu in lung cancer

Background

In the development of effective CAR-Ts against solid tumors, both choosing an appropriate tumor-associated target and overcoming the immunosuppressive tumor microenvironment (TME) remain major challenges. The oncofetal carbohydrate CD176 (Thomsen-Friedenreich antigen), hidden in adult benign tissues by sialylation or prolongation with carbohydrates, is unmasked in 90% of carcinomas, predicting low "on-target/off-tumor"-toxicity.

Methods

To target CD176 and overcome the immunosuppressive TME, we developed 4th-generation CD176-specific CAR-Ts, also known as T cells redirected for universal cytokine-mediated killing (TRUCKs). In addition to the constitutive CD176-CAR expression, these cells were engineered with an inducible cassette expressing either IL-12 (iIL12_CD176_TRUCKs), IL-18 (iIL18_CD176_TRUCKs), or EGFP as a control (iEGFP_CD176_TRUCKs). Functionality of CD176_TRUCKs was tested by co-cultivation with CD176⁺ cell lines and human lung cancer tissue. To characterize activation and cytotoxicity of CD176-TRUCKs following target recognition, multicolor flow cytometry, confocal microscopy, multiplex assays and bulk-RNA transcriptomics were performed.

Results

Following co-culture with different CD176⁺ lung carcinoma cell lines, all CD176_TRUCKs increased NF-κB activity, became activated, released effector molecules (e.g. IFN-γ), and mediated effective cytotoxicity. They did not react towards CD176^- control cells. The inducible cytokines IL-12 and IL-18 were released by respective TRUCKs in a target-specific manner and clearly improved particular effector functions in comparison with iEGFP_CD176_TRUCKs. Precision-cut lung sections (PCLS) were generated from explanted human lung adenocarcinoma tissue and shown to express CD176. Using PCLS as ex vivo model, specific cytotoxicity of CD176_TRUCKs against tumor tissue but not against healthy tissue of the same lung was demonstrated.

Conclusion

CD176_TRUCKs equipped with inducible cytokines were shown to be highly functional, suggesting them as promising strategy to overcome the TME. Based on the tumor-selective expression of CD176, CD176_TRUCKs have a high potential to effectively control lung carcinoma while minimizing "on-target/off-tumor"-toxicity.

Offenlegung Interessenkonflikt:

The authors declare no conflict of interest