Avelumab Induces Natural Killer Cell Activation and Dendritic Cell Crosstalk

PD-L1 monoclonal antibodies are widely used to treat many types of cancer. Yet the response rates vary greatly and there is a dearth of understanding of what causes this variability.

In an enlightening study, researchers at Cleveland Clinic revealed the mechanism of action behind two of these drugs and their impact on innate immune cell activation.

"There has been a perception that many PD-1/PD-L1 inhibitors are somewhat interchangeable," says Raghvendra Srivastava, PhD, Director of the Discovery Laboratory at Cleveland Clinic's Center for Immunotherapy and Precision Immuno-Oncology. “However, despite belonging to the same drug family, the backbone of each of these medications have notable differences. Without understanding the mechanistic performance of one drug versus another, clinicians won’t know which is best for each type of cancer."

Study design

Researchers conducted side-by-side comparisons of two FDA-approved PD-L1 targeting drugs, avelumab and durvalumab, using two- and three-cell coculture models to better understand the mechanism of action of each antibody. The models included human NK cells, dendritic cells (DC) and tumor cells treated with one of the medications.

They selected these medications since the antibodies have unique designs in their Fc portions, which can affect the drugs’ interactions with immune cells. Avelumab has an active Fc portion so it can bind Fc receptors on the surface of different immune cells, including NK cells. Durvalumab has a mutated Fc portion, so it can't bind Fc receptors.

The researchers also performed bulk RNA sequencing of tumor and immune cell cocultures treated with avelumab or durvalumab to determine the biological pathways triggered by each of the medications.

Study findings

The research team discovered the underpinnings of the cellular immune response initiated by avelumab and durvalumab. Previously, the two therapies were thought to largely have the same effect since they’re directed at the same target, PD-L1; however, the study revealed remarkable variations between the two.

It was already known that avelumab can induce NK-cell-mediated antibody-dependent cellular cytotoxicity, but what was notable was the degree of activation. “By directly comparing avelumab against durvalumab, we saw a massive difference in terms of avelumab’s ability to activate NK cells as well as its ability to enhance dendritic cell function and facilitate robust NK crosstalk with dendritic cells, which play a crucial role in initiating an adaptive anti-tumor response,” says Nicole Osborne, Research Technologist with Cleveland Clinic’s Discovery Lab. “We don't see the same activity in durvalumab.”

These dramatic differences were apparent across a wide range of tumor cell types.

In theory, these drugs are designed to directly impact T cells, but the research shows they may affect NK cell activity as well. "Avelumab, and to a certain extent durvalumab, had a tremendous impact on the transcriptome of NK cells,” says Dr. Srivastava.

Understanding NK-cell effector function may help guide clinicians in choosing which PD-L1 is best for different cancer types. For example, in organs where NK cells are abundant, there may be a good rationale for using avelumab as the primary treatment.

For the first time, the team also demonstrated that avelumab isn't just killing tumor cells but is spurring a massive change in genes. Based on the sequencing data, the researchers found that a number of crucial genes that correlate with positive outcomes were activated when exposed to these medications. Most notably, avelumab initiated tremendous upregulation of:

• IFN-gamma, which encodes a cytokine that plays a key role in immune responses
• CD137, which supports NK cell activation
• Chemokines, which are known to aid in DC recruitment to a tumor site
• CD86 expression on DCs in cocultures containing only DCs and NKcells, indicating that avelumab-activated NKcells can boost DC maturation

"In avelumab-treated cultures, we saw huge upregulation of immune-related genes compared to durvalumab-treated cultures,” says Ms. Osborne. “We also saw chemokines that are important in the interaction between natural killer and dendritic cells. That’s a key finding that has not been seen before, suggesting that there is active NK-DC crosstalk happening. This phenomenon may create a cascade effect where one function enhances the other. As the NK cells become more functional, the DC cells mature and can mount a stronger adaptive immune response.”

“Our hope is that the field will recognize the novel aspects of individual immune checkpoint inhibitors and take these into account when prescribing them or using them in a clinical trial,” says Dr. Srivastava.

Cautions

There were also some negative findings that the researchers discovered. First, they found avelumab-activated NK cells killed roughly 7-10% of DCs in vitro. Whenever the DCs are surviving, they did still see activation and maturation of those cells. Regardless, clinicians need to be aware of this issue, particularly if avelumab is ever considered in conjunction with DC-based therapy,

Second, the researchers found that in the absence of immune cells, PD-L1 medications like avelumab and durvalumab can moderately accelerate tumor cell growth. It is possible this phenomenon may have led to hyper-progression of tumors in some instances. "This data suggests that you need to have a strong supply of immune cells in the tumor microenvironment in order for this immunotherapy to work,” says Dr. Srivastava. “Otherwise, you may see tumor progression."

Next steps

The researchers are now comparing all FDA-approved PD-L1/PD-1 targeting drugs. They have already identified several new immune targets by using the two- and three-cell coculture model. There is a strong possibility that there is a benefit to combining other drugs with avelumab.

The team also hopes that their findings will encourage other experts to use triple cell coculture models to better identify differences in ADCC-stimulating drugs.

In addition, there has been recent work in the field to optimize Fc portions of monoclonal antibodies. The study findings may help build on this work, demonstrating the potential that Fc portions may have in further enhancing innate immune function. "Our hope is that our study will provide the rationale for further examination of therapies that differ in their Fc portion, since it is not known how these Fc modifications will impact treatment response and patient outcomes," says Dr. Srivastava. “Hopefully better understanding these mechanisms will get the right medications to the right patients.”