For almost a quarter-century, doctors have used antiretroviral therapy (ART) to control HIV infection. The basic idea of ART—a combination of several drugs—is to interfere with different stages of the virus’ life cycle, preventing HIV from infecting new cells and replicating within them. ART works remarkably well at keeping the virus in check indefinitely.
“That’s all well and good, but long-term use of ART causes substantial side effects—and if you stop the therapy, the virus quickly rebounds,” says Harris Goldstein, M.D., professor of pediatrics and of microbiology & immunology and the Charles Michael Chair in Autoimmune Diseases at Einstein, and director of the Einstein-Rockefeller-CUNY Center for AIDS Research.
The problem, he explains, is that when HIV is under siege, it can lie dormant in T cells, a type of immune cell that it infects, for decades. (T cells normally play a critical role in orchestrating the immune response and killing pathogen-infected cells. But HIV diabolically infects its most important cellular adversaries, crippling their immune activity and using them as virus incubators and sanctuaries.) Unless HIV is actively replicating, its tell-tale viral proteins don’t appear on the surface of T cells, so the latent virus in its host cell remains invisible to the immune system or to pharmacologic attack.
But invisible does not mean invulnerable. In 2007, doctors in Berlin cured an American of HIV infection by wiping out his bone marrow and then giving him a stem-cell transplant from a donor with a rare mutation affecting CCR5, a receptor found on the surface of T cells. HIV needs CCR5 to bind to and enter a T cell, so T cells with mutated CCR5 are highly resistant to HIV infection.
When the patient’s ART was stopped, the latent HIV reactivated, “assuming” it was safe to emerge from hiding. But the viruses were quickly dispatched by the man’s newly established pool of HIV-resistant T cells. The “Berlin patient” remains virus-free to this day.
A second HIV-infected patient was later cured using the same approach. But the treatment is not suitable for widespread use, mainly because it’s difficult to find compatible stem-cell donors, much less ones with CCR5 mutations. What’s more, such transplants are costly, laborious, and can be fatal.
Borrowing From Cancer Therapy
“The Berlin patient’s success got us and other investigators thinking, ‘What if we could duplicate that strategy of treating with HIV-resistant T cells, but using CAR-T cells instead of a stem cell transplant from a CCR5 mutant donor?’” says Dr. Goldstein.
![Arming T-Cells to Cure HIV: duoCAR-T cells have been genetically engineered to identify and kill HIV-infected T cells like the one at right]()
Arming T Cells to Cure HIV: duoCAR-T cells, like the one at left, have been genetically engineered to identify and kill HIV-infected T cells like the one at right. Two different chimeric antigen receptor (CAR) proteins (yellow and orange globes) extend from the duoCar-T cell’s membrane. Each protein recognizes a different part of gp120 (green balls), the HIV glycoprotein expressed on the surface of HIV-infected T cells. After binding to gp120, the CAR proteins use their signaling pathways (yellow and orange cylinders) to activate the duoCar-T cell to attack the HIV-infected T cell. The duoCAR-T cell first secretes the protein perforin (blue tubes), which opens pores in the infected cell’s membrane; then it sends the deadly enzyme granzyme B (purple balls) through the pores and into the infected cell to kill it.
CAR-T cells were first developed for use in cancer. They are T cells genetically reprogrammed to seek out and kill tumor cells rather than the disease-causing microbes they normally attack, like dogs trained to sniff out explosives instead of bones. In CAR-T therapy, T cells are first harvested from a patient’s blood and treated with a harmless lentivirus; the virus introduces new genes into the T cells that enable the cells to express a synthetic surface receptor. This receptor, called a chimeric antigen receptor (the CAR in CAR-T), was designed specifically to express binders, which can be antibodies or other molecules that specifically recognize proteins expressed by tumor cells. In the final step, the reprogrammed tumor-specific T cells are expanded in number in the lab and then reinfused into the patient, where they seek out and eliminate tumor cells.
Adapting CAR-T therapy for confronting HIV infection was done by two of Dr. Goldstein’s colleagues: Boro Dropulić, Ph.D., a gene therapy expert at Lentigen, a Miltenyi Biotec Company; and Dimiter S. Dimitrov, Ph.D., an expert in antibody and protein design at the University of Pittsburgh.
Their engineered CAR-T cells express binders designed to home in on gp120, a glycoprotein that is part of HIV’s outer layer and that enables HIV to bind to CD4 surface receptor proteins expressed by T cells and thereby infect those T cells. After infection, gp120 appears on the surface of the infected T cells as virus is being produced and budding from the cells—making the infected T cells susceptible to killing by the novel CAR-T cells, which target two highly conserved (rarely mutated) parts of gp120.
“We call our CAR-T cells duoCAR-Ts,” says Dr. Dropulić. “They deliver a one-two punch by expressing two different binders on two different receptor proteins to target infected T cells for killing. One binder is an engineered piece of CD4, the receptor on T cells to which HIV’s gp120 attaches so it can enter and infect cells. Attachment of this binder to a portion of gp120 fools gp120 into thinking it’s about to infect a T cell. As a result, gp120 opens up—revealing a previously hidden part of gp120 that CAR-T’s second binder—an antibody to this hidden region—can now recognize and bind to.”
The Berlin patient’s success got us and other investigators thinking, ‘What if we could duplicate that strategy of treating with HIV-resistant T cells, but using CAR-T cells instead of a stem cell transplant from a CCR5 mutant donor?’
Harris Goldstein, M.D.
When the CAR-T cells are infused into the patient, ART is purposely withdrawn so that latently infected T cells will be flushed out of hiding by activation and expression of gp120, which—at least in theory—now exposes those HIV-infected T cells to CAR-T cell attack.
To evaluate the HIV-specific CAR-T cells created by Drs. Dropulić and Dimitrov, Dr. Goldstein used cell culture assays and a novel humanized mouse model of HIV that he developed. He found that these CAR-T cells reduced cellular HIV infection by 99 percent in cell cultures and by 97 percent in mice. The results were reported in a paper published August 7 in Science Translational Medicine also co-authored by current and former members of the Goldstein lab, Ariola Bardhi, Ph.D., Alex Ray, Ph.D., Nina Flerin, Ph.D., and Ph.D. student Mengyan Li.
“Now we have T cells that can eliminate HIV infection,” says Dr. Goldstein, “and the beauty of it is this: the CAR-T cells themselves are resistant to HIV infection, since they bind to and block multiple sites on gp120 that the virus needs to enter and infect cells, potentially mimicking the immune system that successfully cured the ‘Berlin patient.’”
Sustained Remission
“We call this sustained remission a functional cure,” says Dr. Goldstein. “HIV has not been eradicated, since the mice still have latently infected human cells. But if there are small bursts of HIV replication, the CAR-T cells—which are long-lasting—will be there, ready to control the infection. It’s like a building with a sprinkler system. The minute a small fire starts, the sprinklers turn on and put the fire out before it can spread.”
The treatment should be able to avoid a common side effect that can occur with cancer CAR-T cell therapy: cytokine response syndrome. When CAR-T cells recognize tumor cells, they recruit helper cells that release cytokines, molecules that can ramp up the immune response.
“If you make too many cytokines, the cytokine ‘storm’ can overactivate the immune system, causing fever, chills, even shock and death,” Dr. Goldstein says. “CAR-T cells target millions of cells during cancer therapy, increasing the likelihood of a cytokine storm. But in our system, we’re targeting only HIV-infected cells, which are present in far lower numbers.”
The team is now working to make the CAR-T cells even more potent. If all goes well, clinical trials could begin as early as next year.
Same Goal, Different Strategy
As Dr. Goldstein and his colleagues pursue CAR-T therapy, he and another Einstein scientist will be investigating a different therapeutic strategy for achieving the same goal: functionally curing HIV infection by amplifying the capacity of a patient’s own T cells to eliminate latent HIV lurking within T cells.
![Steven Almo, Ph.D.]()
Steven Almo, Ph.D.
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For the past two years, Dr. Goldstein and Steven Almo, Ph.D., professor and chair of biochemistry at Einstein, have been developing and evaluating a novel class of synthetic proteins that stimulate the immune system’s CD8+ (“killer”) T cells to specifically attack HIV-infected T cells. That research, spearheaded by Ms. Li in the Goldstein lab and Scott Garforth, Ph.D., in the Almo lab, is now supported by the National Institute of Allergy and Infectious Diseases, which in April awarded the two researchers a five-year, $4.2 million grant.
The synthetic immunostimulatory proteins, which were developed by Dr. Almo, are dubbed synTac (short for Synapse for T-Cell Activation). One synTac arm guides it to killer T cells that have been programmed to attack HIV-infected T cells, and a second synTac arm provides a signal that activates the killer T cells. This signal greatly increases the number and potency of the killer T cells, enabling them to eliminate virus-infected cells.
“We have already found that our novel proteins can trigger the specially programmed killer T cells to significantly expand in number,” says Dr. Goldstein. They will now use their humanized mouse model to study whether those amplified T cells can target and eliminate latent HIV-infected T cells that have been intentionally reactivated to make them “visible” to killer T cells and thereby prevent the recurrence of infection.
But HIV isn’t their only target: By using synTac designed to bind to killer T cells specific for other viruses, Drs. Goldstein and Almo can induce them to attack cells infected by viruses other than HIV. They have already designed their immunostimulatory proteins to stimulate killer T cells that suppress infection by cytomegalovirus, a common type of herpes virus that can infect and kill immunosuppressed patients. They have similar plans to use these synTac immunostimulatory proteins to amplify killer T cells that attack cells infected with Epstein-Barr virus, which causes mononucleosis and can also kill immunosuppressed patients.
Posted on: Wednesday, August 07, 2019