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Dual Antigen CAR T-cell Therapy Shows Benefit in Medical College of Wisconsin Study

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NEW YORK – CAR T cells that target both CD19 and CD20 antigens on the surface of B cells in hematologic malignancies may be a promising strategy to avoid relapse and resistance, a recently published Phase I study has shown.

The findings, demonstrating the safety and preliminary efficacy of a bispecific CAR T-cell therapy, are encouraging news for patients with diffuse large B-cell lymphoma (DLBCL), since CD19 antigen loss is a common resistance mechanism in these patients following treatment with CAR T cells that target just CD19: up to 70 percent of them experience relapse following treatment with anti-CD19 CAR T cells.

But the study, published in Nature Medicine earlier this month, is also noteworthy in that it demonstrated the feasibility of point-of-care CAR T-cell manufacturing, which avoids shipping frozen cells to faraway trial sites. During the course of the trial, over two dozen unique bispecific CAR T-cell products were manufactured at the Medical College of Wisconsin's cell therapy lab using CliniMACS Prodigy, a closed-system, tabletop device.

Prior to this trial, the Medical College of Wisconsin purchased the device from Miltenyi Biotech, but the manufacturer was not involved in using it to make CAR T-cell products. This is different from many other cell therapy trials, which tend to be performed at major academic centers with pharmaceutical industry partners or using drugmakers' large and expensive cell manufacturing facilities.  

The study was conducted by a team of Medical College of Wisconsin researchers led by Nirav Shah. Twenty-six patients with relapsed or refractory B-cell non-Hodgkin lymphoma or chronic lymphocytic leukemia were enrolled in the trial, all of whom were confirmed to have either CD19 or CD20 positive disease, as assessed by immunohistochemistry or flow cytometry on a biopsy sample. After harvesting the patients' T cells through apheresis, the cells were manufactured into bispecific "LV20.19" CAR T cells using the CliniMACS Prodigy system. Over the course of 14 days, the targeted dose of the product, 2.5 million cells per kilogram, was successfully produced from the cells of 22 of the 26 patients. The remaining four patients were treated with as many cells as were manufacturable.

At day 28 following treatment, the overall response rate was 82 percent, and 14 patients — or 64 percent — had achieved a complete remission. Four patients achieved a partial response, and the median overall survival in the entire cohort was 20.3 months. Among the 22 patients who were able to receive the full dose, the overall response rate was 88 percent and the complete remission rate was 75 percent. The treatment was safe and tolerable, moreover, with low rates of high-grade cytokine release syndrome and neurotoxicity.

"A lot of people fail single-targeted CD19 CAR," Shah said. "If a [dual-targeting CAR] can decrease the number of people that ultimately relapse, that's an advancement for patients and could be a better first [option] than the single-targeted [approach]."

"Early on, after the first patients were treated with CD19 CAR T cells, it was noticed that people were relapsing," he explained. "And what was found was that in a subset of the patients who relapsed, the tumor cells no longer had CD19 on them. The tumor cells were evading the CAR T cells by eliminating CD19 as a target on the cancer cells themselves."

Shah and colleagues conducted preclinical research with collaborators at Lentigen Technologies, a subsidiary of Miltenyi Biotech that is focused in part on studying bi-specific vectors. This research showed that when dual-targeted CAR T cells were exposed to the tumor cells, there was less downregulation of the CD19 antigen.

That preclinical work gave Shah and his team confidence to test out the theory in humans, even though at the time, the approach was completely new, and researchers hadn't given patients dual-targeted anti-CD19, anti-CD20 CAR T cells before.

Even though the recent Phase I trial yielded encouraging response rates and a good safety profile, Shah noted that there were still patients in the study who did not respond or who responded initially, then experienced relapse. For four of the five patients in the study who had previously received anti-CD19 CAR T-cell therapy, including the US Food and Drug Administration-approved axicabtagene ciloleucel (Gilead's Yescarta) and tisagenlecleucel (Novartis' Kymriah), the researchers were unable to manufacture the full targeted dose. These patients were therefore infused with as many cells as researchers could manufacture, but all four experienced disease progression by day 28.  

In contrast, Shah pointed out that the researchers were able to manufacture the targeted dose of the bispecific CAR T cells in all of the patients who were CAR-naïve. It's not entirely clear why patients previously treated with a CD19 CAR T-cell therapy ended up relapsing, though Shah suspects it may be due to the lower dose of T cells they received or due to their prior treatment exposure. "That's an area that we are actively investigating," he said. "We presented those findings because it's important for the entire field to be aware of that experience in patients who had prior CAR exposure."

Given these findings, the next Phase II clinical trial that Shah and his colleagues are planning for the bispecific CAR T-cell therapy will restrict enrollment to CAR-naïve patients.

Next steps, funding plans

In the wake of the positive Phase I results, his team is planning a larger, multi-center Phase II trial of the bispecific CAR T-cell therapy specifically in B-cell non-Hodgkin Lymphoma patients. Though the COVID-19 pandemic resulted in some slight delays, the trial is slated for launch in early 2021.

Unlike the Phase I trial, which was conducted entirely on-site at the Medical College of Wisconsin, the Phase II trial will take place at multiple locations, and Miltenyi Biotech will manufacture the product.

"Now that [Miltenyi] has the data that we published, they are investing in this product," Shah said, adding that the company will sponsor the Phase II trial, whereas the Phase I trial was funded by the Medical College of Wisconsin through philanthropic donations.

Effects of cryopreservation

In their paper, Shah and colleagues pointed out an important detail: because the T-cell manufacturing for the Phase I trial was all done on-site, most of the patients enrolled were able to receive the bispecific CAR T-cell product almost immediately after its production.

"The advantage of our process was that, right when the CAR T-cells were ready, they would go into the patient within a few hours," he said. "There was no freezing involved."

For 15 patients in the trial who received the fresh product as soon as it was ready, the overall response rate at day 28 was 93 percent. In contrast, among seven patients who, due to clinical complications, needed to delay their infusion slightly and had their products cryopreserved in the interim, the overall response rate at day 28 was only 57 percent.

This finding, Shah and colleagues wrote in their paper, differed from previous studies that found little to no difference in patient responses to CAR T-cell therapy with fresh versus cryopreserved product. Given this discrepancy, Shah said, the effect of cryopreservation on CAR T-cell efficacy is something that he and his team want to explore further.

In the upcoming Phase II trial, researchers will preferentially treat patients with fresh cells rather than cryopreserved product. The multi-site trial will require central manufacturing of CAR T cells and their transport to the treatment sites, Shah noted, but the goal will be to quickly ship and deliver the cells to patients in a time frame that avoids the need for cryopreservation. Still, there may be instances in the Phase II trial where some patients need to delay treatment and freeze cells. This will allow researchers to compare outcomes between those who received treatment with and without cryopreservation.

The findings of this exploratory analysis may eventually have implications for large-scale manufacturing. As it stands, many patients who undergo treatment with FDA-approved cell therapies receive products that have been frozen, since shipping fresh cells to distant locations often necessitates cryopreservation.

While Shah is encouraged by the Phase I findings and eager to evaluate the efficacy of the bispecific anti-CD19, anti-CD20 CAR T-cell therapy in the larger Phase II trial, he was careful to emphasize that the results are early, adding that it is too soon to say whether this dual-targeting approach could someday replace currently marketed CD19 CAR T-cell therapies.

"Everything comes as an iteration," he said. "Every drug we have in cancer is a refinement of an earlier version. In that same way, we're hoping that the doublet CAR can do that for patients."

Already, the field of oncology has benefitted greatly from CD19 CAR T-cell therapies, he noted. "Patients' lives have been saved and we are so grateful to have the technology available," Shah said. "But can this [bispecific CAR T-cell therapy] improve on what's currently available? We will see."