Wielding a Powerful Weapon

When it comes to biological systems, the human immune system surely ranks as one of the most remarkably complex and potent—able to detect and kill disease-causing microbes as well as cancer cells. Now, Einstein researchers are working to modulate the immune system as a way to overcome major diseases, a treatment approach known as immunotherapy.

Recent publicity has centered on using immunotherapy to combat cancer by revving up the immune system. According to a New York Times article published last year, the race among major pharmaceutical companies to develop cancer immunotherapies is potentially worth tens of billions of dollars a year in sales. Three such anticancer immunotherapies are now available. But scientists are also working to turn off the immune response to treat autoimmune diseases such as multiple sclerosis, type 1 diabetes and rheumatoid arthritis. Einstein researchers—all members of the Evolution of Immune Therapeutics Working Group—are involved in both immunotherapy approaches.

Tuning In to T Cells

Xingxing Zang, Ph.D.
Xingxing Zang, Ph.D.

Cancer immunotherapy doesn’t usually target tumor cells directly. Instead, efforts focus mainly on manipulating T cells, a type of white blood cell that helps destroy invaders such as viruses and bacteria and can eliminate cancer cells as well.

“Optimal T-cell activity is crucially important,” says Xingxing Zang, Ph.D., an associate professor of microbiology & immunology and of medicine (oncology). “Abnormally low T-cell activity makes people vulnerable to cancer or to chronic infections such as tuberculosis and herpes simplex. On the other hand, overly active T cells can trigger an immune attack on normal tissues, resulting in autoimmune diseases.”

T-cell activity depends on the numerous proteins attached to the T-cell surface. Steven C. Almo, Ph.D., a professor of biochemistry and of physiology & biophysics, uses an automobile analogy to describe how these proteins, known as cell-surface receptors, govern T-cell activity.

“One protein on the T-cell surface can be thought of as the ignition, since it recognizes infected and malignant cells and turns on the T cell,” says Dr. Almo, Einstein’s Wollowick Family Foundation Chair. “And just as a car needs an accelerator to go somewhere, T cells have another set of proteins that, when stimulated, rev up T cells so they can actually kill the disease-causing cells they’ve recognized. But at some point you want to turn off the immune response so that T cells don’t attack healthy tissues. So additional proteins on the T-cell surface act as brakes, working in opposition to the accelerator receptors to bring the whole system back to normal again.”

Unfortunately, tumors have learned to evade the body’s immune response by exploiting T cells’ finely calibrated control system. Tumors express cell-surface proteins that stimulate the very receptors that put the brakes on T cells’ attack, allowing the tumors to remain unscathed. Tumors are known to activate two “braking” receptors in particular: CTLA-4 (cytotoxic T-lymphocyte antigen-4) and PD-1 (programmed cell death protein-1). A considerable amount of immunotherapy research, at Einstein and elsewhere, is aimed at preventing cancers from turning on those T-cell receptors.

Cutting Brake Lines to Boost Immunity

Steven C. Almo, Ph.D.
Steven C. Almo, Ph.D.

“If we could remove the brakes on the immune response by inactivating CTLA-4 or PD-1, that would enable T cells to mount a much more robust immune response against these cells,” explains Dr. Almo. “Conversely, you could treat autoimmune diseases by boosting the activity of those two receptors, since that would tamp down T-cell activity.”

To attain those goals, Dr. Almo uses high-resolution X-ray crystallography—an imaging technique in which an X-ray beam is shot through purified, crystallized proteins. The beam is scattered, or diffracted, in many different directions, allowing scientists to construct a detailed, 3-D model of the crystallized protein’s molecular structure. Measuring the intensities and angles of the diffracted beams reveals the position of each individual atom in the protein.

Dr. Almo and colleagues have used X-ray crystallography to determine the precise shape of key molecular complexes: those formed when PD-L1 and PD-L2 (proteins expressed on the surface of tumor cells) muffle the immune response by binding to PD-1 receptors on T cells. “Based on that structural analysis, our lab is engaged in an exciting project,” says Dr. Almo. “We’ve developed a range of molecular variants of the PD-1 receptor that have much higher affinity for tumors’ PD-L1 and PD-L2 proteins than the naturally occurring PD-1 protein does.”

The goal, says Dr. Almo, is for these PD-1 receptor variants to bind strongly to the PD-L1 and PD-L2 proteins of the tumor, rendering them unable to bind PD-1 receptors on T cells. This would prevent the T cells’ brakes from being activated and greatly bolster the immune response. “We’re now testing these PD-1 variants in mouse models of malignant melanoma and metastatic cancers, with the aim of finding new and more-effective treatments,” he says.

Ideally, Dr. Almo’s research will lead to new immunotherapies—not only against cancers but also for treating autoimmune diseases and infections caused by microbes resistant even to the most powerful antibiotics. This work will soon be occurring in Einstein’s Center for Experimental Therapeutics, intended specifically to speed the flow of therapies from laboratory to bedside.

X-ray crystallography
This X-ray crystallography image shows PD-L2, a protein on the surface of tumor cells, that has bound to a T cell’s PD-1 receptor. To prevent PD-L1 and PD-L2 from muffling T-cell activity, Dr. Almo’s lab is developing soluble PD-1 receptor variants that will bind to those tumor proteins much more strongly than does the naturally occurring PD-1 receptor.

A Pro-Cancer Protein Family

Dr. Zang is another Einstein researcher trying to “release the brakes” on T cells so they can assault cancer cells. One focus of his research is B7 proteins—a family of proteins that bind to T cells and can speed up or slow down T-cell activity.

Dr. Zang discovered an important member of the B7 family, called B7x, that strongly inhibits T-cell activity by binding to an as-yet-unidentified T-cell receptor. The researchers found that B7x is present at high levels in almost all solid human cancers. The higher the level at which B7 is expressed in tumors, the worse the prognosis is for patients.

One of the monoclonal antibodies developed by Dr. Zang’s team, called 1H3, has successfully blocked the B7x protein expressed on tumors so that T cells can attack them.

“B7x may be one of the most important proteins that human cancers use to hobble the immune system’s ability to combat them,” says Dr. Zang. “It both inactivates T cells and promotes myeloid-derived suppressor cells that help suppress the immune response.”

Dr. Zang has developed a screening system to find monoclonal antibodies that bind to B7x proteins, preventing them from sabotaging the immune response against cancer. (Monoclonal antibodies are designed to target specific proteins or other molecules.)

One of the monoclonal antibodies developed by Dr. Zang’s team, called 1H3, has successfully blocked the B7x protein expressed on tumors so that T cells can attack them. “The beauty of this approach,” says Dr. Zang, “is that it enables T cells to develop an immunological ‘memory’ of the cancer cells, in the same way that vaccines prime the immune system to recognize and fight off disease-causing bacteria and viruses.” Einstein recently licensed Dr. Zang’s B7x technology to a pharmaceutical company for further development.

Recently, the Zang team discovered the newest member of the B7 family of proteins (HHLA2) as well as its T-cell receptor (TMIGD2). HHLA2 is not found on most normal human cells but occurs abundantly on many human cancers, including those of the lung, breast, thyroid, pancreas, prostate, colon and skin. The findings could lead to a novel immunotherapy effective against many different types of tumors.

The New Age of Immunotherapies

Three immunotherapies that unleash the immune system against tumors are now available.

Yervoy is a monoclonal antibody that binds to and blocks CTLA-4 receptors on T cells. It received approval from the Food and Drug Administration in 2011 for treating metastatic melanoma, a usually fatal disease. In clinical trials, Yervoy enabled about a quarter of metastatic melanoma patients to survive for two years—a major improvement over older therapies.

Keytruda, another monoclonal antibody for treating metastatic melanoma, was approved in 2014. (Melanomas are more susceptible than other types of tumors to immune system attack.) Keytruda works by binding to and blocking PD-1 receptors on T cells and so far is approved only for patients who have first tried Yervoy. A clinical trial found that 69 percent of melanoma patients treated with Keytruda were alive after one year.

The third immunotherapy, Opdivo, also inhibits PD-1 receptors. This monoclonal antibody was approved last December for treating metastatic melanoma and this March for treating advanced squamous non-small cell lung cancer, which is typically associated with smoking.

There is room for improving these first-generation immunotherapies. Yervoy, for example, can overstimulate the immune system to attack healthy tissues, resulting in serious adverse effects in up to 15 percent of patients. And these drugs are expensive: A complete course of Yervoy costs $120,000, and Keytruda and Opdivo each cost about $150,000 a year.

New Strategy for a Nasty Cancer

Joseph A. Sparano, M.D.
Joseph A. Sparano, M.D.

Joseph A. Sparano, M.D., a professor of medicine (oncology) and of obstetrics & gynecology and women’s health and associate director for clinical research at the Albert Einstein Cancer Center is exploring whether the immune system can be enlisted to fight “triple-negative” breast cancer (tumors lacking receptors for estrogen, progesterone and Her2/neu). Most chemotherapy drugs target one of those receptors, so triple-negative breast cancer can be challenging to treat.

As chief of the section of breast medical oncology at Montefiore, Dr. Sparano participated in a national clinical trial involving patients with triple-negative breast cancer. The study found that patients whose tumors were densely infiltrated with immune cells had much better outcomes than did patients with fewer immune cells. He has recently begun collaborating with Dr. Zang to study how B7 proteins influence the immune response to triple-negative breast cancer.

An Immunity Boost from Radiation

Chandan Guha, M.B.B.S., Ph.D.
Chandan Guha, M.B.B.S., Ph.D.

Einstein scientists have found that radiation therapy—a standard treatment for many solid tumors—can also enhance the immune system’s ability to attack cancer. They’ve shown that exposing a tumor to radiation can increase the tumor’s immunogenicity—the likelihood that it will provoke an immune response.

“Tumor cells contain a lot of defective proteins,” says Chandan Guha, M.B.B.S., Ph.D., a professor and vice chair of radiation oncology at Einstein and Montefiore and professor of pathology and of urology at Einstein. “When radiation kills tumor cells, those abnormal proteins are released and become detectable by the immune system, which can then target living tumor cells containing those same proteins. Over the past 15 years, my colleagues and I have shown that focused delivery of energy in the form of radiation and ultrasound makes tumors more vulnerable to immune attack.”

“When radiation kills tumor cells, those abnormal proteins are released and become detectable by the immune system…”

The larger the dose, the better. “Standard radiation therapy given at intervals causes a small amount of DNA damage each time, much of which gets repaired by tumor cells,” says Dr. Guha. “Delivering large radiation doses in one to five sessions instead of more frequent smaller doses over several weeks causes more extensive DNA damage that can’t be repaired so well. That means more tumor cells die, releasing large amounts of tumor-specific proteins along with a stronger ‘danger’ signal for arousing the immune system.”

Dr. Guha is also evaluating radiation combined with therapeutic cancer vaccines. Several experimental cancer vaccines boost the immune response by delivering tumor-associated proteins directly into certain immune cells. In one recent study, Dr. Guha and colleagues combined radiation with a prostate cancer vaccine that arouses the immune system against cells producing prostate specific antigen (PSA). In a mouse model of prostate cancer, the combination therapy caused established tumors to regress completely in 60 percent of mice compared with regression of 10 percent or fewer tumors from either radiation or the vaccine alone.

When Brakes Can Be Useful

New immunotherapies to prevent T-cell attacks could transform the treatment of autoimmune diseases. But progress may depend on the answer to a question: Why don’t the T cells of healthy people attack the same tissues and organs they target in autoimmune diseases?

Fernando Macian, M.D., Ph.D., an associate professor of pathology, studies the molecular origins of “anergy”—the condition of nonresponsiveness, or tolerance, that prevents T cells from attacking one’s own tissues. He has found that a protein called NFAT (nuclear factor of activated T cells) plays a crucial dual role in immunity—capable of activating T cells as well as making them tolerant.

A protein called NFAT (nuclear factor of activated T-cells) plays a dual role in immunity, capable of activating T cells or making them tolerant.

NFAT is a transcription factor—a protein that binds to specific DNA sequences and orchestrates gene expression. Whether NFAT activates T cells or makes them tolerant, Dr. Macian has found, depends on which program of gene expression it directs. “To boost T cells’ ability to attack cancer cells, you’d want to suppress NFAT-regulated genes that might otherwise induce T-cell tolerance,” says Dr. Macian. “On the other hand, drugs that induce T cells to over-express those ‘tolerance’ genes could help in treating autoimmune diseases.”

Einstein’s Dr. Zang is studying immunotherapy for use against the autoimmune disease type 1 diabetes, in which T cells destroy the insulin-producing cells of the pancreas. Dr. Zang has shown that the protein B7x—notorious for stifling the immune system’s response to cancer—could actually be an ally in treating or preventing type 1 diabetes and other autoimmune diseases.

Dr. Zang and colleagues recently observed that B7x is normally present in areas of the pancreas called the islets of Langerhans. Islets contain the insulin-producing beta cells that are destroyed in type 1 diabetes. In studies involving mice, Dr. Zang showed that the B7x protein plays a role in protecting beta cells from attack: “If a T cell ‘sees’ B7x on the beta cell, the T cell won’t destroy the beta cell,” he says.

Based on those findings, Dr. Zang is pursuing two approaches against type 1 diabetes. He is developing B7x as a soluble drug and is trying to improve islet cell transplants as a therapy for type 1 diabetes.

Islet cell transplants involve infusing islet cells from a deceased organ donor into a patient with diabetes. Despite use of immunosuppressive drugs, the transplants usually stop working after about five years. “We believe the body’s T cells attack the transplanted islets,” Dr. Zang says. To extend the life of the transplanted cells, Dr. Zang and his colleagues are developing genetically modified islet cells that overexpress B7x protein on their surfaces.

“Immunotherapy is not just a promise anymore,” says Dr. Macian. “The field has progressed rapidly over the past few years. FDA-approved drugs are now in use, clinical trials are under way to evaluate new immunotherapies and scientific advances will allow us to create treatments that are more targeted and more powerful.”

Fernando Macian, M.D.,
Fernando Macian, M.D., Ph.D., with graduate student Rachel Ames, left, and postdoctoral fellow Rut Valdor, Ph.D.