CRISPR-Cas9 modified T cells could solve immunotherapy problems

The study team of the Institute for Medical Microbiology, Immunology and Hygiene (from left to right): Prof. Dirk Busch, Thomas Müller and Kilian Schober

Successful T cell engineering with gene scissors

The idea of genetically modifying a patient’s own immune cells and deploying them against infections and tumors has been around since the 1980s. But to this day modified T cells are still not as effective as natural T cells and have been only been of limited clinical value. Using the new CRISPR-Cas9 gene editing tool, a team at the Technical University of Munich (TUM) has now engineered T cells that are very similar to physiological immune cells.

There are two forms of T cell therapy: either a recipient receives cells from a donor, or the recipient’s own T cells are removed, genetically reprogrammed in a laboratory and unleashed against an infection or tumor in the body. While the first method has proven to be successful in clinical models, reprogramming T cells is still beset with problems.

The team led by Professor Dirk Busch, Director of the Institute for Medical Microbiology, Immunology and Hygiene at the TUM, has generated modified T cells for the first time that are very similar to their natural counterparts and could solve some of those problems. To do so, they utilized the new CRISPR-Cas9 gene scissors, which can be used to snip out and replace targeted segments of the genome.

Both the conventional methods and the new method target the key homing instrument of T cells, known as the T cell receptor. The receptor, residing on the cell’s surface, recognizes specific antigens associated with pathogens or tumor cells, which the T cell is then able to attack. Each receptor is made up of two molecular chains that are linked together. The genetic information for the chains can be genetically modified to produce new receptors that are able to recognize any desired antigen. In this way, it is possible to reprogram T cells.

Targeted exchange using the CRISPR-Cas9 gene scissors

The problem with conventional methods is that the genetic information for the new receptors is randomly inserted into the genome. This means that T cells are produced with both new and old receptors or with receptors having one old and one new chain. As a result, the cells do not function as effectively as physiological T cells and are also controlled differently. Moreover, there is a danger that the mixed chains could trigger dangerous side effects (Graft-versus-Host Disease, GvHD).

“Using the CRISPR method, we’ve been able to completely replace the natural receptors with new ones, because we’re able to insert them into the very same location in the genome. In addition, we’ve replaced the information for both chains so that there are no longer any mixed receptors,” explains Kilian Schober, who is a lead author of the new study along with his colleague Thomas Müller.

Near-natural properties

Thomas Müller explains the advantages of the modified T cells: “They’re much more similar to physiological T cells, yet they can be changed flexibly. They’re controlled like physiological cells and have the same structure, but are capable of being genetically modified.“ The scientists have demonstrated in a cell culture model that T cells modified in this way behave nearly exactly like their natural counterparts.

“Another advantage is that the new method allows multiple T cells to be modified simultaneously so that they’re able to recognize different targets and can be used in combination. This is especially interesting for cancer therapy, because tumors are highly heterogeneous,” Dirk Busch adds. In the future, the team plans to investigate the new cells and their properties in preclinical mouse models, an important step in preparing for clinical trials with humans.

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Scientists are a step closer to developing artificial lymph nodes that can spark T-cells to fight disease

T-cells interacting with the transparent gel. Credit: Hawley Pruitt

In a proof-of-principle study in mice, scientists at Johns Hopkins Medicine report the creation of a specialized gel that acts like a lymph node to successfully activate and multiply cancer-fighting immune system T-cells. The work puts scientists a step closer, they say, to injecting such artificial lymph nodes into people and sparking T-cells to fight disease.

In the past few years, a wave of discoveries has advanced new techniques to use T-cells — a type of white blood cell — in cancer treatment. To be successful, the cells must be primed, or taught, to spot and react to molecular flags that dot the surfaces of cancer cells. The job of educating T-cells this way typically happens in lymph nodes, small, bean-shaped glands found all over the body that house T-cells. But in patients with cancer and immune system disorders, that learning process is faulty, or doesn’t happen.

To address such defects, current T-cell booster therapy requires physicians to remove T-cells from the blood of a patient with cancer and inject the cells back into the patient after either genetically engineering or activating the cells in a laboratory so they recognize cancer-linked molecular flags.

One such treatment, called CAR-T therapy, is costly and available only at specialized centers with laboratories capable of the complicated task of engineering T-cells. In addition, it generally takes about six to eight weeks to culture the T-cells in laboratories and, once reintroduced into the body, the cells don’t last long in the patient’s body, so the effects of the treatment can be short-lived.

The new work, reported April 10 in the journal Advanced Materials, is a bid by Johns Hopkins scientists to find a more efficient way of engineering T-cells.

“We believe that a T-cell’s environment is very important. Biology doesn’t occur on plastic dishes; it happens in tissues,” says John Hickey, a Ph.D. candidate in biomedical engineering at the Johns Hopkins University School of Medicine and first author of the study report.

To make the engineered T-cells’ environment more biologically realistic, Hickey — working with his mentors Hai-Quan Mao, Ph.D., associate director of the Johns Hopkins Institute for NanoBioTechnology and Jonathan Schneck, M.D., Ph.D., professor of pathology, medicine and oncology at the Johns Hopkins University School of Medicine — tried using a jelly-like polymer, or hydrogel, as a platform for the T-cells. On the hydrogel, the scientists added two types of signals that stimulate and “teach” T-cells to hone in on foreign targets to destroy.

In their experiments, T-cells activated on hydrogels produced 50 percent more molecules called cytokines, a marker of activation, than T-cells kept on plastic culture dishes.

Because hydrogels can be made to order, the Johns Hopkins scientists created and tested a range of hydrogels, from the very soft feel of a single cell to the more rigid quality of a cell-packed lymph node.

“One of the surprising findings was that T-cells prefer a very soft environment, similar to interactions with individual cells, as opposed to a densely packed tissue,” says Schneck.

More than 80 percent of T-cells on the soft surface multiplied themselves, compared with none of the T-cells on the most firm type of hydrogel.

When the Johns Hopkins team put T-cells onto a soft hydrogel, they found that the T-cells multiplied from just a few cells to about 150,000 cells — plenty to use for cancer therapy — within seven days. By contrast, when the scientists used other conventional methods to stimulate and expand T-cells, they were able to culture only 20,000 cells within seven days.

In the next set of experiments, the scientists injected the T-cells engineered in either the soft hydrogels or the plastic culture dishes into mice implanted with melanoma, a lethal form of skin cancer. Tumors in mice with T-cells cultured on hydrogels remained stable in size, and some of the mice survived beyond 40 days. By contrast, tumors grew in most of the mice injected with T-cells cultured in plastic dishes, and none of these mice lived beyond 30 days.

“As we perfect the hydrogel and replicate the essential feature of the natural environment, including chemical growth factors that attract cancer-fighting T-cells and other signals, we will ultimately be able to design artificial lymph nodes for regenerative immunology-based therapy,” says Schneck, a member of the Johns Hopkins Kimmel Cancer Center.

Learn more: Scientists Advance Creation of ‘Artificial Lymph Node’ to Fight Cancer, Other Diseases

 

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Engineering the Immune System to Fight Melanoma

Loyola University Medical Center has launched the first clinical trial in the Midwest of an experimental melanoma treatment that genetically engineers a patient’s immune system to fight the deadly cancer.

A batch of the immune system’s killer T cells will be removed from the patient and genetically modified in a Loyola lab. Two genes will be inserted into the T cells so that they will recognize tumor cells as abnormal.

The patient will undergo high-dose chemotherapy to kill most of his or her remaining T cells. This will make room for the genetically modified T cells when they are put back in the patient. The modified T cells, it is hoped, will recognize the tumor cells as abnormal and then attack and kill them.

“This clinical trial is a unique attempt to manipulate a person’s own immune system to attack their cancer in a more effective and specific manner,” said Joseph Clark, MD, one of the principal investigators of the trial.

The purpose of the Phase 1 trial is to determine the optimum dose and whether the treatment is safe. Four doses will be tested, with the highest dose consisting of about 5 billion genetically modified T cells. If Phase 1 demonstrates the treatment is safe, investigators will proceed to Phase 2, which will determine whether the treatment is effective.

Melanoma is the sixth-most-common cancer in Americans, and the most common fatal malignancy in young adults. Incidence is rising dramatically. About 1 in 50 people will be diagnosed with melanoma. In the 1960s, it was 1 in 600.

Surgery is highly successful if the cancer is caught early. But if the cancer has spread to other parts of the body, the five-year survival rate is only 15 to 20 percent, according to the American Cancer Society.

“This is a terrible, devastating disease,” Clark said. “It starts on the skin and can spread to just about anywhere in the body.”

The clinical trial is open to patients with metastatic melanoma who are no longer responding to standard therapy. “We need better treatments,” Clark said. “Our clinical trial is designed for patients who have no other options.”

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Research unveils Viagra’s potential in treating skin cancer

A clinical breakthrough might be on the cards as a German study has suggested that the impotence drug may be the most effective skin cancer treatment, reports Forbes.

A clinical breakthrough might be on the cards

 
According to the US publishing house, earlier studies established the effectiveness of Viagra sildenafil in skin cancer treatment. The new study, spearheaded by Dr. Viktor Umansky of the University Medical Center Mannheim, confirms that the erectile dysfunction medicine can help keep the number of T cells intact to negate the inflammatory reaction of malignant cells.

The 7-week study involved two groups of carcinogenic mice one of which was given Viagra while the other was left untreated, Forbes notes. At the end of the study period it was found that the T cell count in the treated sample restored to a normal level, which implied that they were not being subdued by the malignant cells.

The way T cells respond to the inflammatory reaction of skin cancer is an important factor as far as treatment is concerned, Forbes says, adding that the findings from the study have a positive bearing on the future of malignant melanoma treatment.

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New nanoparticle for vaccine delivery developed at MIT

Immune cells, tagged with green fluorescent protein, are surrounded by nanoparticles (red), after the nanoparticles are injected into the skin of a mouse (Image: Peter DeMuth and James Moon)

Vaccines work by exposing the body to an infectious agent in order to prime the immune system to respond quickly when it encounters the pathogen again.

Some vaccines, such as the diphtheria vaccine, consist of a synthetic version of a protein or other molecule normally made by the pathogen, while others, such as the polio and smallpox vaccines, use a dead or disabled form of the virus. However, such an approach cannot be used with HIV because it’s difficult to render the virus harmless. MIT engineers have now developed a new type of nanoparticle that could safely and effectively deliver vaccines for infectious diseases such as HIV and malaria, and could even help scientists develop vaccines against cancer.

When designing a vaccine, scientists either try to provoke T cells, which attack body cells that have been infected with a pathogen, or B cells, which secrete antibodies that target viruses or bacteria present in the blood and other bodily fluids. However, for diseases such as HIV in which the pathogen tends to stay inside cells, a strong response from a type of T cell known as a “killer ” T cell is required.

The best way to provoke these killer T cells into action is to use a killed or disabled virus, but the difficulty in rendering HIV harmless and the danger of using live viruses has led to scientists working on synthetic vaccines for HIV and other viral infections, such as hepatitis B. However, while these synthetic vaccines are safer, they do not elicit a very strong T cell response.

Recently, scientists have tried encasing the vaccines in fatty droplets called liposomes, which could help promote T cell responses by packaging the protein in a virus-like particle. However, these liposomes have poor stability in blood and body fluids and tend to break down quickly inside the body.

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