Study: dual‑target off‑the‑shelf CAR‑T cells resist rejection, curb tumor escape in mice

The engineered T cells in the new study carry two artificial receptors instead of one. One homes in on CD19, a common marker on B cells often targeted in blood cancers and some autoimmune diseases. The other targets CD70, a molecule that appears when immune cells become highly activated.

That second receptor is what makes this experimental therapy unusual. In a paper published April 12 in Nature Communications, scientists at Allogene Therapeutics in South San Francisco report that donor‑derived, “off‑the‑shelf” CAR‑T cells equipped with both receptors persisted longer and controlled tumors better in preclinical tests than conventional CD19‑only designs.

By combining those two targets, the team is trying to solve two of the biggest problems in turning CAR‑T cell therapy into a standardized product: rejection of donor cells by the patient’s immune system and the ability of tumors to escape by shedding a single target.

“Co‑expression of this CD70 CAR with a CD19 CAR resulted in sustained CAR T cell persistence in the presence of alloreactive lymphocytes and prolonged antitumor activity in a CD19 antigen escape model,” the authors wrote in the paper’s abstract.

CAR‑T therapies, which reprogram a patient’s T cells to attack cancer, are among the most powerful treatments developed in the past decade. But all approved products so far are autologous, meaning they are manufactured individually from each patient’s own cells. That process is time‑consuming, expensive and not feasible for all patients.

Allogeneic CAR‑T tries to flip that model by using T cells from healthy donors, edited and frozen in advance, so they can be shipped like a drug on a shelf. The challenge is immunologic: the recipient’s immune system often recognizes donor cells as foreign and destroys them, a phenomenon called host‑versus‑graft rejection. There is also a countervailing danger that donor T cells will attack the patient’s tissues, known as graft‑versus‑host disease.

To reduce that risk, the Allogene team engineered T cells by knocking out the TRAC gene, which encodes part of the native T‑cell receptor that can drive graft‑versus‑host reactions. Into that same genomic location, they inserted a bicistronic, or dual‑gene, cassette that encodes both the CD19 and CD70 chimeric antigen receptors, using a targeted editing approach built around a nuclease called ABR‑001 and an adeno‑associated virus (AAV) DNA template.

After editing, the researchers depleted any residual T cells that still expressed a native receptor and cryopreserved the product, modeling how an off‑the‑shelf manufacturing process could work at scale.

The rationale for adding CD70 is twofold. CD70 is transiently upregulated on activated lymphocytes, including the alloreactive T cells that are primed to attack foreign grafts. It is also expressed on some lymphoma cells, including those that may downregulate CD19 to evade therapy. Targeting CD70, the authors argue, allows donor CAR‑T cells to selectively eliminate the host immune cells most likely to reject them, while also giving them a second way to recognize malignant or pathogenic cells.

In mixed lymphocyte reactions — in vitro tests where immune cells from one donor are exposed to HLA‑mismatched cells from another to provoke an alloreactive response — the CD19/CD70 dual‑CAR T cells reduced the proportion and absolute number of CD70‑positive host T cells. At the same time, the engineered donor cells survived better than non‑transduced controls or cells bearing only the CD19 receptor. Those experiments were run across 10 unique donor pairs.

To model tumor “antigen escape,” where cancer cells stop expressing CD19 to avoid being targeted, the researchers created a mixture of Raji lymphoma cells lacking either CD19 or CD70 in equal proportions and implanted them into immunodeficient NSG mice. When treated with CD19‑only CAR‑T cells, the tumors eventually grew out, dominated by cells that no longer carried CD19. In contrast, mice given the dual‑CAR product showed improved tumor control in both dish and animal experiments, with the engineered T cells able to attack cells expressing either of the two targets. In the in vivo Raji studies, each group included 10 mice.

The team also used humanized mouse models to assess how well the dual‑CAR cells might persist in a more complex immune environment. In NSG mice engrafted with human CD34‑positive blood stem cells, a single dose of 6 million dual‑CAR T cells eliminated human B cells by day 7. The engineered cells expanded in the blood to a peak around that time and then contracted, but remained detectable as host B cells started to reappear around day 20. Each group in that experiment included three mice, and the study was replicated.

In another model, NSG mice were infused with 20 million peripheral blood mononuclear cells from people with systemic lupus erythematosus, an autoimmune disease in which B cells produce antibodies that attack the body’s own tissues. Three days later, the animals received 5 million CAR‑T cells. Only mice given the dual CD19/CD70 product showed significant reductions in human T‑cell counts and levels of anti‑double‑stranded DNA autoantibodies, a hallmark of lupus, by day 14. Dual‑CAR cells were still detectable in the spleen at that time point, while CD19‑only CAR‑T cells were not. Groups in these lupus PBMC experiments included three to four mice each.

The paper also notes a safety‑related observation: in one lupus PBMC model, two mice that received CD19‑only CAR‑T cells developed graft‑versus‑host disease and were euthanized before day 14. Mice that received the dual‑CAR cells did not show the same outcome in that experiment, although the authors cautioned that mouse models have well‑known limitations in predicting human toxicity.

Sample sizes throughout the in vivo work, typically three to 10 animals per group, are standard for early preclinical studies but are not powered to capture rare safety events.

The study is not purely academic. All 12 authors are employees of Allogene Therapeutics, and the dual‑CAR design closely tracks with ALLO‑329, an allogeneic CD19/CD70 product the company is already testing in humans. The paper notes that a Phase 1 trial of this strategy in autoimmune diseases is underway, listed on ClinicalTrials.gov as NCT07085104.

That study, called RESOLUTION, is evaluating ALLO‑329 in adults with conditions including systemic lupus erythematosus, lupus nephritis, idiopathic inflammatory myopathies and systemic sclerosis. The registry record, first posted in July 2025 and most recently updated in February 2026, describes it as an early‑stage safety and dose‑finding trial sponsored by Allogene. The Nature Communications authors wrote that the trial “may provide additional evidence for a contribution of the CD70z CAR to allogeneic CAR T cell persistence and efficacy.”

Allogene has prior clinical experience with CD70‑directed CAR‑T cells. Its product ALLO‑316, which targets CD70 alone, has been tested in a Phase 1 trial for renal cell carcinoma. Company presentations have reported objective tumor responses in patients with high CD70 expression, as well as serious adverse events, including on‑study deaths, underscoring that this target is biologically potent and carries risks.

The authors of the new paper acknowledge that humanized NSG mouse systems do not fully mirror human immunity. These mice lack key human cytokines such as interleukin‑7 and interleukin‑15, which influence T‑cell behavior, and their engrafted immune components are incomplete. As a result, the infection risks and long‑term immune consequences of depleting activated CD70‑positive lymphocytes in people remain uncertain.

If the CD70 “anti‑rejection” strategy works as intended in patients, it could lengthen the lifespan of off‑the‑shelf CAR‑T cells without requiring the intense chemotherapy regimens now used to clear space in the immune system before infusion. That could, in turn, lower barriers to using CAR‑T in autoimmune diseases and in cancer patients too frail for aggressive conditioning, while making cell therapies easier to deliver beyond specialized centers.

But the same mechanism that allows donor cells to prune back alloreactive T cells could also dampen beneficial immune responses, including those needed to fight infections or respond to vaccines. With the first clinical trial of a dual CD19/CD70 allogeneic CAR‑T now underway, early human data will be critical in determining whether this carefully engineered compromise between persistence and safety holds outside the lab.