The Costimulatory Role of CD28 Enhances the Function of CAR-NK Cells through the LCK/CD3ζ/ZAP70 Signaling Axis

Research Background and Objective

Chimeric Antigen Receptor (CAR) T-cell therapy has shown significant effects in treating certain advanced cancer patients. However, CAR-T cell therapy has many limitations, such as high cost, complex preparation process, cytokine release syndrome, and neurotoxicity. Therefore, researchers are exploring alternative methods, with Natural Killer (NK) cells becoming a popular candidate for cancer immunotherapy, as they can serve as off-the-shelf cell immunotherapy without causing Graft-Versus-Host Disease (GVHD).

In this study, researchers investigated the impact of different costimulatory molecules in CAR constructs targeting CD70 on the antitumor activity of NK cells. The goal was to identify the most effective costimulatory signaling domain and evaluate its antitumor effectiveness in various xenograft models.

Source of the Paper

The primary authors of this paper include Sunil Acharya, Rafet Basar, and others from the MD Anderson Cancer Center at the University of Texas, University of Houston, and other research institutions. This research was published in the journal “Cancer Discovery.”

Research Process and Methods

Overview of the Research Process

The research team constructed multiple CAR systems targeting CD70, incorporating NK cell-related costimulatory signaling domains (DAP10 and DAP12), T-cell-related costimulatory signaling domains (CD28), and dual costimulatory signaling domains (4-1BB). The research mainly comprised the following steps: a) preparation and in vitro screening of different CAR-structured NK cells; b) analysis of the antitumor activity of these NK cells in vitro and in vivo; c) detailed study of the signaling mechanisms to explore whether the costimulatory role of CD28 enhances the function of CAR-NK cells through the conventional T-cell receptor (TCR) signaling pathway.

Cell Preparation and CAR Construction (Generation and In Vitro Screening)

Researchers conducted an in-depth analysis of CD70 expression in various solid tumors and hematologic malignancies through tissue microarray immunohistochemistry (IHC) and flow cytometry. They confirmed that CD70 is overexpressed in various cancers while being lowly expressed in normal tissues, validating CD70 as a potential target for CAR.

Next, the research team designed multiple CAR constructs based on CD27, linking them with NK cell-related DAP10/DAP12, T-cell-related CD28, and 4-1BB costimulatory signaling domains. These CAR genes were transduced into NK cells derived from umbilical cord blood and screened in vitro. The experiments showed similar transduction efficiency among CAR-NK cells, with no significant differences in in vitro proliferation and cytokine secretion. Different CAR-NK cells exhibited varying degrees of cytotoxicity against various cancer cell lines to assess their effector functions.

Long-term and Repeated Stimulation Experiments and Multifunctionality Analysis

Researchers simulated the complex environment inside tumor patients by conducting long-term in vitro repeated tumor rechallenge experiments. Results indicated that CAR-NK cells with CD28 costimulatory signaling domains (CAR27-28ζ) exhibited stronger long-term cytotoxicity against CD70-positive tumor cells. Moreover, IsoPlexis platform analysis of single-cell multifunctionality showed that CAR27-28ζ NK cells exhibited high multifunctionality in both basal state and after CAR stimulation, mainly secreting effector and chemotactic cytokines.

Using time-of-flight mass cytometry (CyTOF), the study found that CAR-NK cells with CD28 costimulatory signaling domains maintained high levels of killing ability even after repeated tumor cell rechallenges, showing stable expression of key effector cytokines such as perforin and granzyme B.

In Vivo Experiments in Animal Models

In four tumor models (two hematologic malignancy models and two solid tumor models), CAR27-28ζ NK cells demonstrated significant antitumor activity and enhanced survival rates. Especially in the highly invasive CD70+ Raji Burkitt lymphoma mouse model, CAR27-28ζ NK cells exhibited the best antitumor effects.

Mechanism Study of CD28 Costimulatory Signals

To explore the downstream signaling pathways of CD28 costimulatory signals in NK cells, researchers observed that the phosphorylation levels of CD3ζ significantly decreased when the PYAP domain of CD28 was deleted or when ITAM tyrosine residues were mutated. This indicates that the association of LCK with the costimulatory domain of CD28 is critical for signal transduction.

Through a series of immunoprecipitation experiments, researchers confirmed that the binding of ZAP70 to CD3ζ within CAR structures containing CD28 significantly increased. Further validation showed that ZAP70 and CD3ζ form a complete signaling pathway involving LCK.

Research Conclusions and Significance

The study found that integrating CD28 costimulatory signaling domains can significantly enhance the antitumor activity of CD70-targeted CAR-NK cells. Detailed signaling mechanism studies confirmed that CD28 enhances downstream signaling transduction and NK cell effector functions by recruiting LCK and promoting the phosphorylation of CD3ζ.

This study not only provides new insights academically but also offers a theoretical basis for the clinical application of CAR-NK cells in cancer immunotherapy. Based on the findings of this study, Phase I/II clinical trials are currently underway to evaluate the safety and efficacy of CAR-NK cells with CD28 costimulatory domains in patients with CD70-positive hematologic malignancies and solid tumors.

Research Highlights

1. Target Selection: The study validated the potential of CD70 as a “pan-cancer antigen” in multiple cancer types, indicating broad clinical application prospects.
2. Costimulatory Signaling Domain: For the first time, a systematic evaluation of the effects of various costimulatory signaling domains on CAR-NK cells was conducted, revealing the unique advantages of the CD28 costimulatory domain in enhancing NK cell effector functions.
3. Signaling Mechanisms: The study detailed the role of CD28 signals in CAR-NK cells, uncovering the critical role of the LCK/CD3ζ/ZAP70 signaling axis in cell activation and antitumor function.

This research provides important theoretical support and experimental basis for advancing the clinical application of CAR-NK cells, having significant implications in the field of cancer immunotherapy.