In-body CAR-T cell generation proves effective, safe in mice in Stanford Medicine-led study

CAR-T cell therapy has transformed the treatment of many blood cancers since it was first approved by the Food and Drug Administration in 2017 for the treatment of acute lymphoblastic leukemia.

But chimeric antigen receptor therapy — in which a patient’s own T cells, a type of immune cell, are removed, genetically engineered and returned to the patient — is onerous and expensive. It requires a series of time- and labor-intensive steps over two to three weeks, and one treatment costs hundreds of thousands of dollars. Patients must also undergo a procedure to deplete the numbers of remaining T cells, which helps the altered T cells expand after they are re-introduced into the body but leaves patients at risk for infection.

Now, a new study led by Stanford Medicine researchers has shown that it’s possible to generate CAR-T cells in laboratory mice with the same technique used for mRNA-based vaccines. And by including two sets of protein-making instructions — one that encodes a protein that binds to tumor cells and another that allows the researchers to track where the modified cells are in the body — they can assess the impact of the therapy in real time.

Unlike standard CAR-T treatment, the mRNA messages can be delivered multiple times in succession, enhancing its duration and amping its curative effects. Tumors in 75% of mice with B cell lymphoma treated with the mRNA messages were eradicated after several doses. Crucially, the approach also does not require a pretreatment to deplete existing immune cells.

“We didn’t see any toxicity, even with a fairly large number of injections,” said professor of radiology Katherine Ferrara, PhD. “It could theoretically be repeated multiple times to enhance the cancer-killing effect. The toxicity of current CAR-T cell treatment is significant, and it can be difficult to achieve a cure with a single infusion.”

Ferrara, who is the chief of the Molecular Imaging Program at Stanford Medicine, is the senior author of the study, which was published June 10 in the Proceedings of the National Academy of Sciences. Postdoctoral scholar Nisi Zhang, PhD, is the lead author of the research. The researchers collaborated with Ronald Levy, MD, professor of oncology and the Robert K. and Helen K. Summy Professor in the design of the mRNA messages and to establish tumor models for cell and animal studies.

“The generation of CAR-T cells inside the body rather than making custom-produced cells from the patient outside the body will make CAR-T cell therapy safer and available to a greater number of patients,” Levy said.

Creating a cancer killer

CAR-T cells are made in the laboratory by tinkering with the genetic instructions in immune cells called T cells that are removed from a patient. In particular, the T cells are tweaked to recognize and bind to a protein called CD19 that is abundant on other immune cells called B cells. Many blood cell cancers, including some types of lymphomas and leukemias, develop due to uncontrolled B cell growth.

Zhang and Ferrara used tiny, fat-soluble bubbles called lipid nanoparticles to package mRNA molecules encoding a receptor protein that binds to CD19 as well as a modified version of another protein that is highly expressed in prostate cancer cells but is rare in other tissue. This second protein allows the researchers to trace the generation and movement of the recipient cells noninvasively using a common medical imaging technique called positron emission tomography, or PET.

Finally, they designed the surface of the nanoparticles to include an antibody that binds to a protein called CD5 that is primarily found on T cells. Once the nanoparticle latches onto the T cell, it is engulfed, the lipid bubble disintegrates and the mRNA molecules are released into the interior of the cell to be made into proteins. (In contrast, mRNA vaccines are not targeted and are taken up non-specifically by muscle and immune cells near the injection site.)

When Zhang added the mRNA-containing nanoparticles to mouse T cells growing in a laboratory dish, she found that 11% of the T cells began making the CD-19 receptor within 24 hours. Furthermore, these newly modified T cells sought out and killed B cells.

When Zhang injected the nanoparticles into mice with a type of B cell lymphoma, she was able to track the generation of the CAR-T cells in the animals — or “in situ” — and see that they traveled to the location of the animals’ tumors.

“Using this method, we can generate sufficient numbers of the CAR-T cells in vivo, and we can see these in situ-generated CAR-T cells are infiltrating the tumors after repeated dosing,” Zhang said.

The in situ method generated about 3 million CAR-T cells per animal, which is similar to the cell numbers infused into patients undergoing conventional CAR-T therapy. 

Eradicating tumors

Importantly, the newly generated CAR-T cells were efficient cancer killers; six out of eight mice with lymphoma were tumor-free 60 days after treatment began, and tumor growth in the remaining two was controlled.

Although in situ CAR-T cell generation has not yet been attempted in humans, it was safe and effective in the mice.

“We did not detect any signs of toxicity or other safety issues even after up to 18 doses.” Zhang said. “And we saw tumor-free survival of 75% of the mice at the end of the study.”

Zhang and Ferrara are hopeful that in situ CAR-T cell generation will make the treatment faster, less expensive, better tolerated and more efficient than the current approach. And tracking the cells in the patients’ body is critical to assess whether and how well the treatment is working.

“These imaging tools can really help evaluate what exactly is happening in patients in real time,” Zhang said. “With our imaging protocol we can evaluate the efficiency of CAR-T cell production, as well as whether they are responding to and infiltrating the tumors.” 

They are also studying whether the in situ approach would increase the effectiveness of CAR-T cell therapy for solid tumors — a goal that has largely eluded researchers since the treatment’s inception.

“The combination of safety and efficacy we’ve seen in the mice is impressive,” Ferrara said. “Furthermore, this imaging technique could easily be translated to humans and would allow us to track off-target effects — say the cells were homing not just to the cancer but to healthy organs or tissue — and quickly change the dose or approach.”

Ferrara is a member of the Stanford Cancer Institute, Bio-X, the Stanford Cardiovascular Institute and the Wu Tsai Neurosciences Institute.

Researchers from the University of Texas Southwestern Medical Center contributed to the work.

The study was funded by the NIH (grants R01CA253316, T32CA118681, P41EB024495 and R01CA134675) and by the Leukemia and Lymphoma Society.

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