Pharmaceutical companies are investigating new lines of attack against cancer, which is, according to the World Health Organization, the world’s second leading cause of death. In 2018 alone, cancer accounted for 9.6 million deaths across the globe, or about 1 out of every 6 deaths that year. If cancer statistics are to become less daunting, it may be necessary, many pharmaceutical companies believe, to explore alternatives to
traditional anticancer approaches.

Cancer research has focused on the disease’s underlying genetic and epigenetic defects. Today, however, pharmaceutical companies are turning their attention to the tumor microenvironment (TME), a battleground that consists of the elements surrounding cancerous cells. It includes specialist immune cells, vascular cells, and other factors that contribute to cancer progression.

On this battleground, the fog of war may confound the immune system, preventing it from distinguishing friend and foe. Dispelling the fog, then, could help the immune system fight more effectively. For example, the immune system could gain situational awareness via immunotherapy. Although currently available immunotherapies benefit only a minority of cancer patients and often cause side effects, newly developed immunotherapies may perform better—provided they start targeting the TME.

TME-targeting possibilities were discussed at the 10th Annual Protein and Antibody Engineering Summit (PEGS) Europe. This event, which took place in Lisbon, Portugal, last November, included an immunotherapy track entitled, “Targeting the Tumor Microenvironment.” Several presentations from this track are highlighted in this article, including presentations on bicycle peptides; combination therapies; oncolytic viruses; antagonistic antibodies for killing both tumor cells and immunosuppressive T cells; and improved checkpoint inhibition strategies.

First on a bicycle

Monoclonal antibodies (mAbs) are the commonest type of immunotherapy. These are antibodies grown in cloned immune cells, which target a specific antigen expressed by a tumor. mAbs for immunotherapy have many different mechanisms of action. Some mAbs, for example, flag the tumor for destruction by immune cells, whereas others block antigens that help the tumor grow and spread. Conjoined mAbs, meanwhile, bond to toxins or radioactive particles and deliver them directly to the tumor.

Problems posed by mAbs include their large size and long half-life within the body, says Kevin Lee, PhD, CEO of Bicycle Therapeutics. With conjoined mAbs, especially, “you don’t want the drug hanging around because of the toxin,” he continues. When multiple long-duration antibodies are used in a combination immunotherapy, they can lead to the overactivation of the immune system with consequent side effects and a loss of efficacy because “you’ve got the foot on the accelerator of a car all the time.”

Bicycle Therapeutics is pioneering a new class of immunotherapies based on bicycle peptides, synthetic peptides between 9 and 15 amino acids in size that are tied to a small central molecular scaffold. “I think we have something very different, disruptive, and transformational in the field,” Lee asserts.

Bicycle peptides were invented by Sir Gregory P. Winter, PhD, a member of the MRC Laboratory of Molecular Biology in Cambridge, UK, and a winner of the 2018 Nobel Prize for Chemistry. At PEGS Europe, Winter spoke as the director of Bicycle Therapeutics. “Greg spent a long time working on the minimal chemical footprint of an antibody needed to replicate some of its activities,” Lee points out, emphasizing that bicycle peptides are much smaller than antibodies and capable of rapidly penetrating tumors. Bicycle peptides, then, may exert their effects without having to rely on long circulating half-lives in the body.

The company has a lead compound currently in Phase I testing. This consists of a bicycle bonded to a tumor-killing toxin. Whereas antibodies bonded to toxins kill a tumor as though they were “peeling an onion layer by layer,” Lee explains, a diminutive Bicycle Toxin Conjugate (BTC) can “blow up the tumor from the inside” like the well-targeted torpedo that entered and destroyed the Death Star in Star Wars.

After validating its technology, Bicycle Therapeutics hopes to move two additional BTCs into the clinic. BT5528 targets the ephrin type-A tumor antigen, whereas BT8009 targets the Nectin-4 tumor antigen. The company is also developing a bispecific bicycle that activates T cells to destroy cancers.

In addition, Bicycle Therapeutics is working on a STING (stimulator of interferon genes protein) agonist. STING agonists stimulate the release of interferon gamma, a signaling molecule that upregulates the innate immune system. Lee observes that STING agonists are generating a lot of pharmaceutical interest even though current treatments tend to activate multiple immune cells, causing major side effects. He hopes that a bicycle with a cleavage linker will be developed that stays attached to a STING agonist while the bicycle guides the agonist into a tumor, and that releases the agonist only after it is inside.

Benefitting a majority

Combination therapies tailored to patients are a major trend in targeting the TME, says Jamie Campbell, head of custom services, Abcam. “I think single therapies are not going to be the norm. Duplicate or triplicate therapies will be the norm in future, so having good quality reagents is the core of showing those expensive drugs are performing as expected,” he explains. Abcam provides reagents and tools for research, drug discovery, and diagnostics to pharmaceutical companies targeting the tumor microenvironment.

seondary antibody only control
Abcam scientists subjected formalin/paraformaldehyde-fixed paraffin-embedded sections of human tonsil to immunohistochemistry (IHC) analysis. To detect PD-L1, the scientists used Abcam’s PD-L1 RabMAb antibody (clone 73-10, ab228415) at 1/500 dilution. The secondary antibody came from the company’s ready-to-use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Another trend he identifies is customers wanting reagents that act as indicators of whether a patient group will benefit from a specific therapy. “There’s a trend in the industry to show that new drugs will be effective,” he elaborates, “because antibody- and protein-based therapies are more expensive than small molecule approaches.”

A significant part of reason these trends exist is the limited success of the first generation of cancer immunotherapies, that is, checkpoint inhibition and chimeric antigen receptor (CAR) therapies. Initially, these immunotherapies generated a great deal of enthusiasm. For example, in 2013, cancer immunotherapy was awarded Breakthrough of the Year by the journal Science. Interest in cancer immunotherapy remains high, but developers are increasingly aware of challenges including side effects, partial responses, and lack of efficacy in most patients.

Cancer vaccines

“As we have more knowledge about checkpoint inhibition, we understand that it’s not a therapy for everyone. It benefits in the range of 10% of patients, depending on the tumor indication, but there’s still a majority who aren’t taking a major benefit,” said Philipp Mueller, PhD, principal scientist for cancer immunology and immune modulation, Boehringer Ingelheim.

Boehringer Ingelheim is conducting preclinical testing of an oncolytic virus, a vesicular stomatitis virus carrying a modified glycoprotein, that is, a VSV-GP. This VSV-GP selectively targets cancer cells, and the company hopes that it will benefit patients who don’t respond to checkpoint therapies. These patients often have so-called cold tumors, which don’t have T-cell inflammation, and therefore can’t be treated by checkpoint inhibitors.

“Cancer cells have deficits in their innate immune response pathways and are unable to protect themselves from a viral infection,” Mueller explains. The aim of the viral therapy is for the virus to selectively penetrate and attack the tumor cells without affecting healthy tissue, inducing the patient’s immune system to activate against the tumor.

Oncolytic viruses aren’t new, but the hope is that VSV-GP will improve upon existing therapies. “The oncolytic viruses currently available as a product or next-stage development are frequently based on big DNA viruses; the benefit of this small virus is that it’s able to replicate very fast,” says Knut Elbers, PhD, CEO of ViraTherapeutics, all shares of which were bought by Boehringer Ingelheim in September 2018. VSV belongs to the class of rhabdoviruses which, Elbers explains, can replicate in as little as eight hours. He believes this makes it a powerful tool for fast and effective activation of the immune system, making it ideal for use in combination with other therapies.

Working in combination

Denise L. Faustman, MD, PhD, director of immunobiology at Massachusetts General Hospital, is developing an antibody therapy that works to help patients who fail to respond to checkpoint inhibitors. “If you look at data on checkpoint inhibitor failures,” she says, “you see that when those checkpoints fail therapeutically, the dominant protein on the regulatory T cells (Tregs) is tumor necrosis factor receptor 2 (TNFR2).” When TNRF is expressed on Tregs, which are T cells that regulate or suppress other parts of the immune system, the Tregs become super-suppressive.

TNFR2 is found on the surface of a tiny fraction of the most immunosuppressive Tregs in healthy people, but is found in large quantities in the microenvironment of some tumors. “In all cancer targets to date, I’ve never seen one that’s preferentially expressed in the TME and acts both as a T-cell suppressor and an oncogene, and which has low expression in the human body,” she points out.

tumor micro-environment
Tumor necrosis factor receptor 2 (TNFR2) promotes direct tumor proliferation through oncogene expression or through overexpression on tumor-residing regulatory
T cells and myeloid suppressor cells. According to Massachusetts General Hospital researchers led by Denise Faustman, PhD, targeted alterations in the tumor micro-environment may be possible with multifunctional TNFR2 antagonistic antibodies.

“In TNFR2,” she continues, “you’ve got a protein that is a bad boy in the TME in two limbs: It’s over-expressed on Tregs in the TME, making them super-suppressive, and TNFR2 is also a proliferative pathway, thus also expanding the cells preventing effective therapies—that’s what makes it so different.” After 10 years of work, she has an antibody in preclinical testing that preferentially binds TNFR2 on the surface of proliferating Tregs in the TME, killing these cells while leaving the body’s natural immune response intact.

She hopes that her new therapy will be ready for testing in humans in 12–18 months, and that it will prove useful in combination with other treatments. As TNFR2 is expressed in low concentrations on the surface of normal cells, she hopes it will have fewer side effects than existing therapies. “There’s been a lot of progress in targeting checkpoints,” she notes, “but there’s a lot of toxicity, so a better toxicity profile is a beautiful thing.”

Making checkpoints better

Among the most popular checkpoint inhibitors are those targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1). According to the Cancer Research Institute in 2017, there were more than 1500 clinical trials underway involving PD-1/L1. Researchers at Pfizer are developing a mouse model to aid studies into how patients respond to anti-PD1/PD-L1 therapies.

“One challenge any preclinical researcher faces is whether the murine model will be relevant and translatable to human patients in clinic,” indicates Yan Qu, PhD, senior principal scientist at Pfizer. Her aim is to understand why some patients don’t respond to anti-PD1/PD-L1 therapies and immune activity in the TME during drug treatment. She hopes to gain insights that may lead to more efficient drugs combination therapies.

Qu is seeking to understand the signaling pathways and immune signatures within the TME that lead to the therapies failing. “Just like what has been observed in clinic, we have observed that some preclinical murine models mimic human patients’ response to anti-PD1/PD-L1 therapy,” she notes. “Although these mice are an inbred strain (which means they’re genetically identical), their response to PD-1/PD-L1 blockade are heterogenous.” She is hoping to use data from clinical trials to validate the model.

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