The immune system can recognize and destroy damaged or faulty cells, including cancer cells. And this natural ability can be harnessed and enhanced for therapeutic purposes. For example, immune system elements can be employed in cancer immunotherapies. Although cancer immunotherapies are available for some forms of cancer, and although some patients have been benefiting, it is apparent that the response rates vary widely. They are generally below 50%. In some circumstances, they are considerably worse.

The greatest challenges are in treating solid tumors, which are complex and continually evolving entities. Besides harboring cancer cells, solid tumors incorporate infiltrating and resident host cells, blood vessels, secreted factors, and extracellular matrix. All these components work together to form a kind of ecosystem, one that is known as the tumor microenvironment (TME).

The TME exploits various mechanisms to resist cancer immunotherapy and thereby sustain itself. These mechanisms include regulatory processes, or checkpoint mechanisms, that ordinarily preserve health by restraining the immune response when it threatens to become overzealous. Although checkpoint mechanisms can prevent autoimmunity, they can also suppress the anticancer immune response.

To restore the immune response and make cancer immunotherapies more effective, many scientists are looking for ways to modify the TME so that it becomes less hospitable to cancer cells and less supportive of their invasive and metastatic activities. Specifically, scientists are working to improve checkpoint inhibition, to help immune cells infiltrate the TME, and to prevent the TME from recruiting immunosuppressive cells.

Stopping TGF-β through integrin inhibition

Transforming growth factor-β (TGF-β), an anti-inflammatory and pro-fibrotic protein that plays an important role in wound healing and other processes, can promote an immunosuppressive TME. When TGF-β is overexpressed in the TME, it interferes with the immune response in a variety of ways, such as inhibiting the function of natural killer cells and CD8+ T cells. Moreover, TGF-β is thought to promote resistance to checkpoint inhibitors.

To prevent TGF-β from defeating checkpoint inhibition, researchers are looking for ways to interfere with TGF-β activation, which depends on a family of proteins called integrins. Integrins regulate many cellular processes by facilitating communication between cells and between cells and the extracellular matrix.

The integrins that activate TGF-β include αvβ1, αvβ6, and αvβ8. To date, researchers interested in preventing TGF-β activation have focused on inhibiting αvβ8, which is expressed by immune cells. But Pliant Therapeutics is going a bit further. The company is developing a small-molecule drug that not only targets αvβ8, but also αvβ1.

“On the outside of some tumors, there’s a lot of fibrotic material that makes penetration into the tumors very difficult,” says Timothy Machajewski, PhD, vice president and head of chemistry at Pliant. “If we can block αvβ1, which is expressed in fibroblasts and is producing all of that fibrotic material in the cancer, we can actually increase the access into the tumor.”

A dual αvβ8 and αvβ1 inhibitor would not only help activate T cells, but also help them infiltrate the tumor. This action would be beneficial for treating cold tumors, which have a resistance to checkpoint inhibitor therapies. And that’s just what Pliant found when the company tested its oral drug candidate, PLN-101095.

Treatment with PLN-101095 in combination with a checkpoint inhibitor in mice with EMT6 breast cancer resulted in the infiltration of CD8+ T cells into the tumor. In some cases, the tumor completely cleared in 28 days and didn’t regrow when the same tumor cell line was reintroduced. In addition, according to Pliant, PLN-101095 is effective even in the absence of another therapy.

“When we started this project, we believed this was going to be an add-on that would require a checkpoint inhibitor in combination,” Machajewski relates. “But we’re very excited by the notion that you might see some activity in some tumors that don’t require checkpoint inhibition.”

Pliant Therapeutics illustration
When transforming growth factor-β is activated by the integrins αvβ8 and αvβ1, the tumor microenvironment becomes immunosuppressive. To inhibit these integrins, Pliant Therapeutics is developing a drug candidate called PLN-101095 that promises to facilitate tumor infiltration of immune cells. Preclinical studies have shown that PLN-101095 can increase the expression of PD-L1 on tumor cells, and that combining PLN-101095 with anti-PD-1 therapy can reduce tumor growth.

Overcoming the challenge of selectively targeting HPK1

Scientists are interested in targeting hematopoietic progenitor kinase 1 (HPK1), an important checkpoint protein. It belongs to the MAP4K kinase family and has a major role as a negative intracellular regulator of T- and B-cell responses. Scientists believe that it works similarly to the well-known checkpoint proteins PD-1 and CTLA-4.

“The working hypothesis in the field is that immune cells that recognize tumors may have activated HPK1, which mutes the activity of those immune cells through a negative feedback loop,” remarks Ryan McClure, PhD, senior scientist, discovery research, AbbVie.

As such, researchers have been working to effectively target and inhibit HPK1 to enhance the activation of these immune cells. However, the inhibition of HPK1, in comparison with the inhibition of other kinases, requires a higher specificity.

“There is a high degree of similarity in the MAP4K kinase family,” McClure points out. “Among the six family members, HPK1 is the only kinase that plays a negative regulatory role. A positive role in immune cell function has been reported for the other five family members, and therefore they cannot be inhibited.”

HPK1 chemical probe illustration
AbbVie developed an HPK1 chemical probe, A-745, and tested its selectivity in an in situ kinase profiling study. Primary cells were pretreated with 0.1, 1.0, and 10 mM A-745, and MAP4K kinase peptides were quantified compared to the control mouse splenocytes. A-745 was shown to be highly selective for HPK1. This chart presents the binding affinities as percentages.

Although AbbVie is not currently pursuing the development of an HPK1 inhibitor, the company did create a chemical probe called A-745 that demonstrated excellent selectivity in unbiased cellular kinase-binding assays. The degree of selectivity was similar to the immune cell activation in T cells with deficient or dead HPK1. The company published its data as part of the general effort to facilitate the development of a selective and potent small-molecule HPK1 inhibitor.

Glenmark Pharmaceuticals is already on this path after overcoming initial challenges with its first HPK1 inhibitor, says Pravin Iyer, PhD, the company’s senior vice president and head of new chemical entity research. “The [target product] profile was good overall, just not good enough,” he adds. “[It needed to have] better synergy with checkpoint inhibitors and a better standalone efficacy.”

After a number of improvements, Glenmark’s preclinical candidate, GRC54276, has demonstrated good efficacy as a single agent and in combination with checkpoint inhibitors when tested in mice with colon cancer. A Phase I study is currently underway.

Preventing Tregs from entering the TME

In healthy bodies, regulatory T cells (Tregs) suppress the immune response to prevent autoimmunity. However, when Tregs infiltrate the TME, they can inhibit the antitumor immune response.

Higher infiltration of the TME by Tregs is associated with elevated expression of the chemokines CCL17 and CCL22, which bind to CCR4, the primary chemokine receptor on Tregs. These chemokines are produced by certain immune cells, such as tumor-associated macrophages, as well as some cancer cells.

By analyzing existing data, RAPT Therapeutics identified which tumors are more likely to express high levels of CCL17 and CCL22 and therefore more Tregs. The company refer to these tumors as “charged tumors.”

“Charged tumors are expected to be ‘hot,’ with high levels of effector CD8 T cells, but also with high levels of Tregs that dampen the antitumor immune response,” says Omar Robles, PhD, associate director of drug discovery at RAPT. “Thus, blocking CCR4-mediated Treg recruitment would be more likely to shift the CD8:Treg ratio toward an enhanced antitumor microenvironment.”

RAPT Therapeutics FLX475 illustration
In response to inflammation, tumor cells and other cells in the tumor microenvironment express the chemokines CCL17 and CCL22 at high levels. Left panel: These chemokines attract regulatory T cells, which express the chemokine receptor CCR4, and suppress the antitumor activity of effector T cells. Right panel: The migration of T regulatory cells into the tumor microenvironment could be selectively inhibited by FLX475, a drug candidate that is being developed by RAPT Therapeutics.

When biologics specific for CCR4, such as mogamulizumab, or other targets are used to deplete Tregs and treat cancer, they do so systemically throughout the body, which can result in autoimmunity. To avoid this problem, RAPT developed a therapy that selectively inhibits the migration of Tregs into the TME. According to results from a Phase I trial, the company’s oral therapy, FLX475, did not deplete any immune cells or cause autoimmunity, but instead resulted in clinical activity.

In an ongoing Phase I/II trial that includes patients with highly charged tumors, an early cohort of 13 patients with non-small cell lung cancer had an overall response rate of 31% after receiving a combination of FLX475 and pembrolizumab. (Patients receiving pembrolizumab alone would be expected to have an overall response rate below 20%.) In addition, single-agent activity of FLX475 was demonstrated in patients with natural killer/T-cell lymphoma, including two patients with durable complete metabolic responses.

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