Combo treatment harnesses immune system to fight skin cancer

By combining two treatment strategies, both aimed at boosting the immune system’s killer T cells, Johns Hopkins researchers report they lengthened the lives of mice with skin cancer more than by using either strategy on its own. And, they say, because the combination technique is easily tailored to different types of cancer, their findings — if confirmed in humans — have the potential to enhance treatment options for a wide variety of cancer patients.

“To our knowledge, this was the first time a ‘biomimetic,’ artificial, cell-like particle — engineered to mimic an immune process that occurs in nature — was used in combination with more traditional immunotherapy,” says Jonathan Schneck, M.D., Ph.D., professor of pathology, who led the study together with Jordan Green, Ph.D., associate professor of biomedical engineering, both of whom are also members of the Kimmel Cancer Center and the Institute for NanoBioTechnology.

A summary of their study results will be published in the February issue of the journal Biomaterials.

Scientists know the immune system is a double-edged sword. If it’s too weak, people succumb to viruses, bacteria and cancer; if it’s too strong, they get allergies and autoimmune diseases, like diabetes and lupus. To prevent the immune system’s killer T cells from attacking them, the body’s own cells display the protein PD-L1, which “shakes hands” with the protein PD-1 on T cells to signal they are friend, not foe.

Unfortunately, many cancer cells learn this handshake and display PD-L1 to protect themselves. Once scientists and drugmakers figured this out, cancer specialists began giving their patients a recently developed class of immunotherapy drugs including a protein, called anti-PD-1, a so-called checkpoint inhibitor, that blocks PD-1 and prevents the handshake from taking place.

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(Alyssa Kosmides and Randall Meyer, Johns Hopkins Medicine) This immunotherapy technique combines artificial antigen presenting cells (orange) with anti-PD-1 antibodies (yellow) to activate killer T cells (pink) and prevent tumor cells (brown) from damping that response. « Dual Strategy Teaches Mouse Immune Cells to Overcome Cancer’s Evasive Techniques

PD-1 blockers have been shown to extend cancer survival rates up to five years but only work for a limited number of patients: between 15 to 30 percent of patients with certain types of cancer, such as skin, kidney and lung cancer. “We need to do better,” says Schneck, who is also a member of the Institute for Cell Engineering.

For the past several years, Schneck says, he and Green worked on an immune system therapy involving specialized plastic beads that showed promise treating skin cancer, or melanoma, in mice. They asked themselves if a combination of anti-PD1 and their so-called biomimetic beads could indeed do better.

Made from a biodegradable plastic that has been FDA-approved for other applications and outfitted with the right proteins, the tiny beads interact with killer T cells as so-called antigen-presenting cells (APCs), whose job is to “teach” T cells what threats to attack. One of the APC proteins is like an empty claw, ready to clasp enemy proteins. When an untrained T cell engages with an APC’s full claw, that T cell multiplies to swarm the enemy identified by the protein in the claw, Schneck explains.

“By simply bathing artificial APCs in one enemy protein or another, we can prepare them to activate T cells to fight specific cancers or other diseases,” says Green.

To test their idea for a combined therapy, the scientists first “primed” T cells and tumor cells to mimic a natural tumor scenario, but in a laboratory setting. In one tube, the scientists activated mouse T cells with artificial APCs displaying a melanoma protein. In another tube, they mixed mouse melanoma cells with a molecule made by T cells so they would ready their PD-L1 defense. Then the scientists mixed the primed T cells with primed tumor cells in three different ways: with artificial APCs, with anti-PD-1 and with both.

To assess the level of T cell activation, they measured production levels of an immunologic molecule called interferon-gamma. T cells participating in the combined therapy produced a 35 percent increase in interferon-gamma over the artificial APCs alone and a 72 percent increase over anti-PD-1 alone.

The researchers next used artificial APCs loaded with a fluorescent dye to see where the artificial APCs would migrate after being injected into the bloodstream. They injected some mice with just APCs and others with APCs first mixed with T cells.

The following day, they found that most of the artificial APCs had migrated directly to the spleen and liver, which was expected because the liver is a major clearing house for the body, while the spleen is a central part of the immune system. The researchers also found that 60 percent more artificial APCs found their way to the spleen if first mixed with T cells, suggesting that the T cells helped them get to the right spot.

Finally, mice with melanoma were given injections of tumor-specific T cells together with anti-PD-1 alone, artificial APCs alone or anti-PD-1 plus artificial APCs. By tracking blood samples and tumor size, the researchers found that the T cells multiplied at least twice as much in the combination therapy group than with either single treatment. More importantly, they reported, the tumors were about 30 percent smaller in the combination group than in mice that received no treatment. The mice also survived longest in the combination group, with 45 percent still alive at day 20, when all the mice in the other groups were dead.

“This was a great indication that our efforts at immunoengineering, or designing new biotechnology to tune the immune system, can work therapeutically,” says Green. “We are now evaluating this dual strategy utilizing artificial APCs that further mimic the shapes of immune cells, such as with football and pancake shapes based on our previous work, and we expect those to do even better.”

Other authors of the report include Alyssa Kosmides, Randall Meyer, and John Hickey (all of who are INBT training grant students) as well as Kent Aje and Ka Ho Nicholas Cheung of the Johns Hopkins University School of Medicine.

This work was supported in part by grants from the National Institute of Allergy and Infectious Diseases (AI072677, AI44129), the National Cancer Institute (CA108835, R25CA153952, 2T32CA153952-06, F31CA206344), the National Institute of Biomedical Imaging and Bioengineering (R01-EB016721), the Troper Wojcicki Foundation, the Bloomberg~Kimmel Institute for Cancer Immunotherapy at Johns Hopkins, the JHU-Coulter Translational Partnership, the JHU Catalyst and Discovery awards programs, the TEDCO Maryland Innovation Initiative, the Achievement Rewards for College Scientists, the National Science Foundation (DGE-1232825), and sponsored research agreements with Miltenyi Biotec and NexImmune.

Under a licensing agreement between NexImmune and The Johns Hopkins University, Jonathan Schneck is entitled to a share of royalty received by the university on sales of products derived from this article. Jordan Green is on the scientific advisory board for NexImmune. The terms of these arrangements are being managed by The Johns Hopkins University in accordance with its conflict of interest policies.

Read more about the Jordan Green Group here.

Read more about the Jonathan Schneck Lab here.

Read more about NexImmune here.

SOURCE: Johns Hopkins School of Medicine

Nanowires Deliver Biochemical Payloads to One Cell Among Many

Imagine being able to drop a toothpick on the head of one particular person standing among 100,000 people in a sports stadium. It sounds impossible, yet this degree of precision at the cellular level has been demonstrated by researchers affiliated with The Johns Hopkins University Institute for NanoBioTechnology. Their study was published online in June in Nature Nanotechnology.

Arrow points to nanowire placed on cell surface. (Image: Levchenko/Chien labs)

The team used precise electrical fields as “tweezers” to guide and place gold nanowires, each about one-two hundredth the size of a cell, on predetermined spots, each on a single cell. Molecules coating the surfaces of the nanowires then triggered a biochemical cascade of actions only in the cell where the wire touched, without affecting other cells nearby. The researchers say this technique could lead to better ways of studying individual cells or even cell parts, and eventually could produce novel methods of delivering medication.

Indeed, the techniques not relying on this new nanowire-based technology either are not very precise, leading to stimulation of multiple cells, or require complex biochemical alterations of the cells. With the new technique the researchers can, for instance, target cells that have cancer properties (higher cell division rate or abnormal morphology), while sparing their healthy neighbors.

“One of the biggest challenges in cell biology is the ability to manipulate the cell environment in as precise a way as possible,” said principal investigator Andre Levchenko, an associate professor of biomedical engineering in Johns Hopkins’ Whiting School of Engineering. In previous studies, Levchenko has used lab-on-a-chip or microfluidic devices to manipulate cell behavior. But, he said, lab-on-a-chip methods are not as precise as researchers would like them to be. “In microfluidic chips, if you alter the cell environment, it affects all the cells at the same time,” he said.

Such is not the case with the gold nanowires, which are metallic cylinders a few hundred nanometers or smaller in diameter. Just as the unsuspecting sports spectator would feel only a light touch from a toothpick being dropped on the head, the cell reacts only to the molecules released from the nanowire in one very precise place where the wire touches the cell’s surface.

With contributions from Chia-Ling Chien, a professor of physics and astronomy in the Krieger School of Arts and Sciences, and Robert Cammarata, a professor of materials science and engineering in the Whiting School, the team developed nanowires coated with a molecule called tumor necrosis factor-alpha (TNF?), a substance released by pathogen-gobbling macrophages, commonly called white blood cells. Under certain cellular conditions, the presence of TNF? triggers cells to switch on genes that help fight infection, but TNF? also is capable of blocking tumor growth and halting viral replication.

Exposure to too much TNF?, however, causes an organism to go into a potentially lethal state called septic shock, Levchenko said. Fortunately, TNF? stays put once it is released from the wire to the cell surface, and because the effect of TNF? is localized, the tiny bit delivered by the wire is enough to trigger the desired cellular response. Much the same thing happens when TNF? is excreted by a white blood cell.

Additionally, the coating of TNF? gives the nanowire a negative charge, making the wire easier to maneuver via the two perpendicular electrical fields of the “tweezer” device, a technique developed by Donglei Fan as part of her Johns Hopkins doctoral research in materials science and engineering. “The electric tweezers were initially developed to assemble, transport and rotate nanowires in solution,” Cammarata said. “Donglei then showed how to use the tweezers to produce patterned nanowire arrays as well as construct nanomotors and nano-oscillators. This new work with Dr. Levchenko’s group demonstrates just how extremely versatile a technique it is.”

To test the system, the team cultured cervical cancer cells in a dish. Then, using electrical fields perpendicular to one another, they were able to zap the nanowires into a pre-set spot and plop them down in a precise location. “In this way, we can predetermine the path that the wires will travel and deliver a molecular payload to a single cell among many, and even to a specific part of the cell,” Levchenko said.

During the course of this study, the team also established that the desired effect generated by the nanowire-delivered TNF? was similar to that experienced by a cell in a living organism.

The team members envision many possibilities for this method of subcellular molecule delivery. “For example, there are many other ways to trigger the release of the molecule from the wires: photo release, chemical release, temperature release. Furthermore, one could attach many molecules to the nanowires at the same time,” Levchenko said. He added that the nanowires can be made much smaller, but said that for this study the wires were made large enough to see with optical microscopy.

Ultimately, Levchenko sees the nanowires becoming a useful tool for basic research. “With these wires, we are trying to mimic the way that cells talk to each other,” he said. “They could be a wonderful tool that could be used in fundamental or applied research.” Drug delivery applications could be much further off. However, Levchenko said, “If the wires retain their negative charge, electrical fields could be used to manipulate and maneuver their position in the living tissue.”

The lead authors for this Nature Nanotechnology article were Fan, a former postdoctoral fellow in the departments of materials science and engineering and in physics and astronomy; and Zhizhong Yin, a former postdoctoral fellow in the Department of Biomedical Engineering. The co-authors included Raymond Cheong, a doctoral student in the Department of Biomedical Engineering; and Frank Q. Zhu, a former doctoral student in the Department of Physics and Astronomy.

Regarding the faculty members’ participation, Chien led the group that developed the electric tweezers technique and collaborated with Levchenko on its biological applications.

The research was funded by the National Science Foundation and the National Institutes of Health.

Johns Hopkins Institute for NanoBioTechnology