In the field of modern medicine, CAR-T cell therapy is undoubtedly one of the most revolutionary breakthroughs in cancer treatment. This therapy genetically modifies a patient's own T cells, enabling them to precisely identify and attack cancer cells, and has achieved remarkable results in the treatment of certain hematologic malignancies. However, traditional CAR-T therapy has a fundamental limitation—it must use the patient's own T cells (autologous CAR-T), which leads to high production costs, long waiting times, and many patients' inability to provide enough healthy T cells due to their physical condition. Scientists have long dreamed of developing a universal allogeneic CAR-T therapy, that is, using healthy donor T cells for large-scale production, readily available for any patient in need. But this vision faces significant challenges—allogeneic T cells may be recognized as foreign invaders by the patient's immune system and rejected; at the same time, these foreign T cells may also attack the patient's normal tissues, causing fatal graft-versus-host disease (GvHD).
On October 30, 2025, Wensheng Wei and his collaborators from Peking University published a research paper in Cell entitled "Glycan shielding enables TCR-sufficient allogeneic CAR-T therapy." They discovered that by regulating the distribution of glycans on the surface of T cells, this challenge can be creatively solved, opening a new avenue for developing safe and effective allogeneic universal CAR-T therapies.
The core principle of CAR-T cell therapy is to introduce an artificially designed chimeric antigen receptor (CAR) into T cells through genetic engineering. This receptor can recognize specific Tumor Antigens (such as the CD19 protein on the surface of B cells), enabling T cells to precisely target cancer cells. Since the approval of the first CAR-T therapy in 2017, this "living drug" has shown unprecedented efficacy in the treatment of certain leukemias and lymphomas, even achieving long-term remission in some patients who have not responded to traditional therapies.
However, existing CAR-T therapies face three major bottlenecks. First, they must be personalized using the patient's own T cells, a process that typically takes 3-4 weeks from cell collection and in vitro culture to reinfusion, a time many advanced cancer patients cannot afford. Second, some patients, due to prior chemotherapy or other reasons, have insufficient numbers or quality of T cells, making it impossible to produce qualified CAR-T products. Third, the personalized production process is complex and costly (currently around $400,000 for treatment), limiting its widespread application.
Using healthy donor T cells to prepare off-the-shelf allogeneic CAR-T products is an ideal solution to these problems. Theoretically, these products can be mass-produced in advance, cryopreserved, and readily available to patients in need, significantly reducing costs and shortening waiting times. However, allogeneic CAR-T faces two major biological obstacles:
The first is host-versus-graft (HvG) reaction—the patient's immune system recognizes T cells from another person as foreign and attacks them, leading to rapid clearance of CAR-T cells and preventing them from exerting a long-term anti-tumor effect. This rejection reaction primarily originates from residual T cells and natural killer (NK) cells in the patient's body.
Secondly, there is GvHD—the T cell receptor (TCR) on the surface of donor T cells recognizes MHC molecules (a kind of molecular ID card) from the patient's normal tissues, mistakenly attacking organs such as the skin, liver, and intestines, leading to potentially fatal complications. To avoid GvHD, most current allogeneic CAR-T strategies choose to completely delete the TCR gene, but this brings new problems: the TCR is not only the main mediator of the allogeneic reaction, but also a key signaling molecule for maintaining the long-term survival and function of T cells. CAR-T cells lacking TCR have a short survival time in vivo, and their anti-tumor efficacy is greatly reduced.
How can GvHD be avoided while preserving TCR function? How can allogeneic CAR-T cells evade immune attack without impairing their anti-tumor ability? These seemingly contradictory needs constitute the core challenge of allogeneic CAR-T development. Researchers have focused on this key scientific question, and they have found a breakthrough from an unexpected angle—glycobiology.
The cell surface is covered with complex glycans. These glycan chains modify proteins or lipids, forming a dense sugar coating. For a long time, scientists believed that these glycans mainly played a structural support role, but recent studies have found that glycan modifications play a crucial role in immune recognition—they can physically mask protein antigens, affecting the interaction between immune cells.
The research team first considered whether it was possible to create a glycan shielding effect by altering the distribution of glycans on the surface of T cells, thereby regulating the recognition and response between immune cells without changing the expression of the proteins themselves. To find the key genes regulating glycan modifications, they conducted a genome-wide CRISPR screening, a highly efficient method for gene function research that can systematically knock out every gene in the human genome and observe its impact on specific phenotypes.
The researchers designed two sets of sophisticated screening experiments. The first set searched for genes that could reduce the expression of HLA-I molecules (a key marker recognized by host T cells), and the second set searched for gene modifications that could enable T cells to resist NK cell attacks. Surprisingly, both sets of experiments pointed to the same gene—SPPL3 (signal peptidase-like 3). Further research revealed that SPPL3 encodes a protease located in the Golgi apparatus that cleaves various glycosyltransferases and glycosidases (enzymes responsible for Glycan Synthesis and pruning), thereby negatively regulating the overall level of glycosylation on the cell surface. When the SPPL3 gene was knocked out, the level of glycosylation on the T cell surface significantly increased, forming a thicker and denser glycan shield.
Through mass spectrometry analysis, researchers analyzed in detail the changes in glycosylation in T cells after SPPL3 knockout. Increased levels of N-acetyllactamine (LacNAc) and sialic acid, while decreased mannose levels, indicated an increased proportion of complex N-Glycans. These changes led to three key effects:
Fig. 1 SPPL3 ablation enables immune evasion and persistence of allogeneic CAR-T cells. (Wu, et al. 2025)
Based on solid mechanistic research, the team conducted systematic preclinical evaluations. In vitro experiments confirmed that SPPL3 knockout CAR-T cells maintained strong expansion capacity and tumor-killing activity under repeated antigen stimulation. Mouse Models showed that these cells effectively controlled tumor growth and prolonged survival without inducing typical GvHD symptoms.
In September 2023, the team initiated the first human clinical trial (NCT06014073) to evaluate the safety and efficacy of SPPL3/TCR double knockout anti-CD19 CAR-T cells in patients with relapsed/refractory B-cell non-Hodgkin lymphoma. In 9 treated patients, no dose-limiting toxicities or GvHD were observed; the incidence of grade 3 or higher cytokine release syndrome (CRS) was 33%; and all patients achieved objective responses (66.7% complete response, 33.3% partial response).
An even more surprising finding came from the observation of residual TCR-positive cells. Despite strictly controlling the proportion of TCR-positive cells in the infusion product to below 0.3%, the expansion of these cells was still detected in 7 patients. In 4 of these patients, CAR-T cells persisted for more than 2 months and were associated with the clearance of peripheral B cells. Notably, the number of these TCR-positive cells far exceeded the threshold for GvHD induced in allogeneic hematopoietic stem cell transplantation (2 × 10⁵/kg), yet the patients only experienced transient, mild rashes without typical GvHD. This suggests that the glycosuria created by SPPL3 knockout effectively buffers the TCR signal intensity, placing it within a safe window that supports cell survival without inducing pathological damage.
Based on these results, the team further explored a more challenging approach—SPPL3 single-knockout CAR-T therapy that completely preserves the TCR (NCT06323525). In three compassionate use patients, this fully functional allogeneic CAR-T also demonstrated good safety, with no clinical evidence of GvHD. One 33-year-old female patient with diffuse large B-cell lymphoma achieved complete remission, with CAR-T cells persisting in vivo for over 6 months and effectively inhibiting B-cell regeneration; another B-ALL patient achieved a negative response with minimal residual disease (MRD). These early clinical data provide new insights for the development of allogeneic CAR-T.
This systematic work, from basic research to clinical application, has advanced the field of cell therapy at multiple levels:
Of course, this technology still requires large-scale clinical validation. Future research needs to focus on how to optimize the precise regulation of glycan modification, balance immune escape and functional maintenance, address relapse caused by tumor antigen loss, and extend this strategy to the treatment of solid tumors. Furthermore, the long-term effects of SPPL3 knockout on T-cell metabolism and differentiation need further clarification.
It is promising that this glycan shielding strategy may not be limited to the CAR-T field. Theoretically, any cell therapy that needs to evade host immune rejection (such as stem cell transplantation and islet cell transplantation) could potentially benefit from it. In the longer term, a deeper understanding of the relationship between glycobiology and immune recognition may also lead to the development of novel immunomodulatory drugs.
From basic laboratory discoveries to clinical benefits for patients, this research demonstrates a complete pathway for translational medicine. As research progresses, this strategy may become a key to breaking through the bottlenecks of allogeneic cell therapy, allowing more patients to access timely and accessible innovative therapies. In the long journey of medical progress, humanity's understanding of cancer has evolved from an incurable terminal illness to a manageable chronic disease, and now to the prospect of curing certain hematologic malignancies. Each breakthrough embodies the wisdom and courage of scientists. The exploration of allogeneic universal CAR-T therapy is combining personalized medicine with large-scale production. Perhaps in the not-too-distant future, off-the-shelf cell therapies will be as widely available as antibiotics, giving more lives renewed hope.
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