1. Holt, Chuck

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PD-1 checkpoint blockade immunotherapy can stimulate cytotoxic T lymphocytes (CTLs) to kill tumor cells. Unfortunately, a large majority of cancer patients' T cells do not respond to anti-PD-1 therapeutics. Seeking to improve response rates, cancer researchers at the Mayo Clinic in Rochester, MN, led by Haidong Dong, MD, PhD, have identified factors that cause the PD-1 blockade to fail.


The researchers used a single-cell RNA-sequence analysis of peripheral CD8+ T cells from patients who responded to PD-1 immunotherapy and others who did not. They discovered that non-responders had less expression of natural killer cell granule protein 7 (NKG7).


Functional assays showed the knock-down of NKG7 compromised cytolytic granule localization, CTL trafficking, and calcium release, resulting in less killing of tumor cells by the T cells, the researchers reported in Cancer Immunology Research (2022; doi: 10.1158/2326-6066.CIR-21-0539). However, the transfection of T cells with NKG7 mRNA sufficiently improved the tumor-killing ability of peripheral T cells isolated from non-responders and increased their response to anti-PD-1 and anti-PD-L1 therapy in vitro, according to the researchers.


An in vivo model of NKG7 mRNA therapy also showed improved tumor-killing ability in murine tumor antigen-specific T cells. What's more, the study demonstrated how the transcription factor ETS1 plays a role in regulating NKG7 expression.


Having identified that the cytolytic efficiency of T cells was reduced by decreasing NKG7, the researchers hypothesized that increasing NKG7 expression would make T cells better at killing tumor cells and also make immune checkpoint inhibitors more effective.


To test their hypothesis, the researchers used peripheral T cells taken from patients with melanoma who were not responding to anti-PD-1 therapeutics and transfected them with NKG7 mRNA. As theorized, NKG7 mRNA significantly increased the killing of melanoma tumor cells in an in vitro assay, and became even better killers when combined with anti-PD-1 or anti-PD-L1 pre-treatment.


The study data not only shows that deficits in a T cell-intrinsic cytolytic factor can lead to failure of PD-1/PD-L1 blockade, but also establishes NKG7 as a new therapeutic target for enhancing T-cell response to cancer immunotherapy, according to the researchers. The results also drive home the importance of T-cell health, said Dong, the study's lead author, who recently shared additional background and insight into the study with Oncology Times.


Special Delivery

Originally trained as a cancer surgeon, Dong joined the Mayo Clinic nearly 25 years ago and since then became a cancer researcher working with his mentor, Lieping Chen, MD, PhD, helping identify the molecule B7-H1, later renamed PD-L1, and establishing a concept of blocking B7-H1/PD-1 pathway for cancer immunotherapy.


"We discovered the molecule and developed the monoclonal antibody to block this," Dong said. "We found that, if the tumor has more PD-L1, it cannot be killed by the T cell because PD-L1 is a shield around the tumor. But if you block PD-L1, the tumor can be killed by the T-cell again."


Dong next worked on developing a cancer vaccine, a pursuit he continues, and also T-cell transfer therapy, before turning his attention to T-cell gene therapy and, most recently, finding out why T cells in some patients with cancer do not respond to checkpoint blockade therapy.


"Everybody assumes that the immune system is healthy and that everybody's T cells should be OK and functional. But, surprisingly, this is not the case," Dong said.


"But the beauty of our immune system is that we have the CTLs inside the tumor and they have the power to find and destroy the cancer," he continued. "Then we found out that the PD-L1 or PD-1 blockade doesn't function well, because either our responder's T cells need support, or they don't have enough support to do their jobs as in non-responders."


To provide the support CTLs need to be healthy and strong enough to kill tumor cells required a way to get the NKG7 molecule to the immune cells, for which Dong and his team then developed a T-cell gene therapy with mRNA serving as a support for CTLs. He likened the process to a truck delivering cargo that includes lots of cytotoxic molecules that can be released from T cells into the interface with tumor cells. "But, the delivery of the cargo within the T cells has some problems-they are slowed down," he said.


"Normally, T cells have sufficient NKG7 molecules that will kill a tumor cell in 10 minutes, and we show it in our paper," Dong explained. "But with the decrease of NKG7, the knock-down of this molecule, it needs to take 1 hour or more to kill the tumor."


The speed is important due to the swiftness with which cancer causes damage, especially in patients with metastases, Dong said. "We are working on a growing or moving target, which is why it is so difficult. Immune cells can do the job, but they cannot always do the job efficiently enough to help patients with progressive cancer."


The RNA-sequencing used to look inside the tumor-reactive T cells revealed the presence of unhealthy T cells that were too weak to carry their part of the load. "We assumed maybe the unhealthy T cell has another checkpoint it used to slow us, like another brake system on the truck," he said. "But we found we had flat tires."


Messenger Service

Messenger RNA was used in the study because it is safe and dependable for delivering something very controlled like the NKG7 molecule to T cells at the right place and time, Dong noted.


"Without this NKG7 molecule, you don't respond to PD-1, and that's exactly what happened in their CTLs," he said, referring to the non-responders in the study. "But when you give back this molecule using messenger RNA, you quickly turn around the function of the T cell and kill the tumor. You have a turning point.


"The data is so striking. And we are now seeing, to a degree, this messenger RNA to be restorative of patients' immunity," Dong added. "We hope to do as many as we can this year as a proof of concept. And then we are going to the Phase I trial."


Meanwhile, the study signals a shift in how physicians approach cancer treatment, from one that is focused on the cancer cell to one that also pays attention to T-cell health.


"Fundamentally, we know how the bad tissue cells are out of control and that the disease is hard to control because cancer cells are not normal," Dong said. "But this study tells us something else, and that is the T cell has to be healthy. It should be robust and resilient. And resilience means you can still be missing a crucial part, but it can be fixed."


Chuck Holt is a contributing writer.


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