The biotech start-up Werewolf Therapeutics, founded in 2017, is out to prove they are more than their pithy name. Scientists at Werewolf Therapeutics are gearing up to take their newly developed cancer immunotherapy to the clinic to see if it has teeth.

Cancer immunotherapies strengthen the body’s immune defenses to help stave off tumors. Immunotherapies don’t work for everyone, but in the 15-20% of patients that benefit, the treatment completely eradicates their cancer. Cytokines, inflammatory molecules that empower the immune response, help the body kill cancer cells. But not all cytokines in the body are well-suited for cancer immunotherapy. The cytokine interleukin-2 (IL-2) was one of the first immunotherapies to hit the scene in the 1990s, but now it’s only used as a last resort due to its high toxicity. 

Biotech and pharma companies are looking for ways to make IL-2 therapy more effective and less toxic. Werewolf Therapeutics developed a modified form of IL-2, dubbed WTX-124 that is only activated once it enters the tumor microenvironment. They recently teamed up with Merck to conduct a clinical trial to test the efficacy of their therapy alone and in combination with Merck’s FDA-approved immunotherapy, KEYTRUDA.

The race to develop effective, non-toxic IL-2 therapies is filled with promising contenders, but this predator thinks WTX-124 will take them to the finish line.

“There's reason to believe that [WTX-124] can be quite effective and quite advantageous in cutting down on toxicity,” said Brent Hanks, cancer and pharmacology researcher at Duke University.

IL-2 therapies make a comeback

Scientists gave the first successful IL-2 treatment to a 33-year-old patient with aggressive, treatment-resistant melanoma in 1984 (1). After IL-2 therapy, the patient went into remission and remains cancer-free to this day. Although IL-2 is still a miracle drug for some patients, the side effects associated with its toxicity can be severe and even deadly. Some patients develop capillary leak syndrome in which plasma from blood vessels leaks into the body, causing severe organ damage and dangerously low blood pressure.

“By minimizing toxicity, [clinicians] will be able to dose patients for a longer period of time, and that would allow the agent to be more effective,” said Hanks. 

Making the perfect IL-2 molecule involves more than reducing side effects; it also needs to activate the right immune cells. IL-2 primarily promotes the expansion of T cells. Regulatory T cells dampen the immune response, while effector T cells ramp up the immune response. Unfortunately for cancer researchers, IL-2 binds regulatory T cells more strongly.

The IL-2 receptor on T cells is composed of three subunits: alpha, beta, and gamma. IL-2 tightly binds the alpha subunit, which is often found on regulatory T cells, rather than the beta or gamma subunits, which are the primary receptor components of effector T cells and cytotoxic natural killer (NK) cells that fend off tumor and microbial cells.

Researchers developing IL-2 molecules perform a high-stakes balancing act. They need to modify IL-2 to be less toxic but also increase its ability to activate effector T cells and NK cells, which can cause an unnecessary immune response outside of the targeted tumor.

Research teams at biotech and pharma companies engineer IL-2 to make it the best cancer therapeutic possible. For example, scientists at Sanofi, engineered an IL-2 molecule called, THOR-207, containing an unnatural amino acid developed in-house that prevents it from binding to the alpha subunit of the IL-2 receptor primarily found on the regulatory T cells so that the molecule only binds and activates effector T cells and NK cells. 

At the American Association for Cancer Research (AACR) Annual Meeting this year, Sanofi scientists presented data from their phase I clinical trial. In 45 patients with various types of late-stage cancer, the researchers found that none developed vascular leakage, which is often what causes patients to stop IL-2 therapy. The patients had increased levels of effector T cells and NK cells, and three patients saw their tumors shrink by as much as 80%.

While THOR-207 showed anti-tumor activity alone, Sanofi scientists also tested its efficacy in combination with KEYTRUDA, a programmed cell death protein 1 (PD-1) inhibitor. KEYTRUDA has proven effective in a variety of cancers, often doubling patient survival rate compared to chemotherapy. PD-1 is a receptor on T cells that keeps them in check. If a cell expresses the receptor’s ligand, PD-L1, it tells the T cell not to attack. Cancer cells use this tactic to protect themselves from immune cell mercenaries. By inhibiting PD-1, cancer cells cannot protect themselves from effector T cells.

Most companies developing IL-2 therapies are testing their drugs in combination with PD-1, including Werewolf Therapeutics. The founder of Werewolf Therapeutics, in fact, helped develop KEYTRUDA during his time at Merck.

Researchers at Werewolf Therapeutics are developing an engineered IL-2 molecule that tackles the same problems as countless other biotechnology companies pitting themselves against pharma behemoths like Sanofi.

“There’s so many super cool [IL-2] agonists that have been designed to bind with higher affinity,” said Hanks. “What’s so special about this one in particular?”

Turning IL-2 into a predatory protein

Cynthia Seidel-Dugan, Chief Scientific Officer at Werewolf Therapeutics, thinks the key to their IL-2 molecule, WTX-124, is its selective activity.

“The whole concept is optimizing the half-life so that we can achieve good tumor exposures, but keeping the molecule inactive in the periphery while activating the full biology of the cytokine once it's released in the tumor. We think that differentiates us from the other approaches,” said Seidel-Dugan.

WTX-124 has three primary parts that dictate its therapeutic function: a half-life extension domain, a native IL-2 protein, and an inactivation domain. The inactivation domain prevents IL-2 from binding to the beta and gamma IL-2 receptor subunits, preventing IL-2 activation of effector T cells and NK cells. When the molecule enters the tumor microenvironment, the inactivation domain and half-life extension domain are removed through cleavage by a tumor-specific protease.

“We had extensive discovery efforts to identify linkers that had the exact profile we were interested in,” said Seidel-Dugan. They wanted linkers that were very stable in circulation, not activated in normal tissues, but then efficiently activated once they reached the tumor microenvironment via tumor-selective proteases.

Seidel-Dugan emphasized that the IL-2 molecule released in tumors is native IL-2, rather than an engineered version, something unique to their design.

Randi Isaacs, Chief Medical Officer at Werewolf Therapeutics, added that in addition to the potential for systemic delivery, the increased half-life garnered by the half-life extension domain may offer convenience to patients and clinicians.

“For oncology patients, certainly in terms of combinations with standards of care, because of the half-life extension domain, it can be delivered infrequently. It’s a very acceptable regimen. We expect to deliver the compound every two to three weeks in the clinic, which matches very nicely with combinations and other cytokines, checkpoint inhibitors, immunotherapeutic agents, or other standards of care,” said Isaacs.

Preclinical studies in mice showed WTX-124 could effectively reduce tumor size without the nasty side effects seen with classic IL-2 therapy. They also found increased numbers of effector T cells. Based on these results, they think WTX-124 will work as a monotherapy when they start running clinical trials towards the end of the year or early next year.

Werewolf Therapeutics isn’t just in the IL-2 business though. The team at Werewolf Therapeutics wants to use their selectively active engineered system to develop other effective, non-toxic cytokine therapies. They collectively call these INDUKINE molecules.

Another INDUKINE molecule in development, WTX-330, aims to deliver another interleukin, IL-12 to fight tumors. IL-12 not only activates effector T cells and NK cells without activating regulatory T cells, but it also prevents the formation of new vessels, cutting off the tumor’s food supply and slowing its growth. Although IL-12 works well in mice, it’s too toxic to use in humans (2). 

If WTX-330 proves safe in humans, it could offer an entirely new therapeutic agent. Hanks thinks that the toxicity limiting design of the INDUKINE molecules may benefit IL-12 efficacy more than IL-2. “It’s always tough to tell, and you never know until you run a trial,” said Hanks.

References

1. Rosenberg, S.A. et al. IL-2: The first effective immunotherapy for cancer. J Immunol  192, 5451-8 (2014).
2. Nguyen, K.G. et al. Localized IL-12 for cancer immunotherapy. Front Immunol (2020).