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Designing Universal CAR Cells to Overcome Transplantation Rejection  Chimeric Antigen Receptor (CAR) cells are genetically engineered immune cells, most commonly T cells, designed to recognize and attack specific target cells, such as cancer cells. The term “chimeric” refers to the artificial receptor introduced into the immune cell, which combines three main components. The extracellular antigen-binding domain, usually derived from an antibody, recognizes a specific protein on the target cell. The transmembrane domain anchors the receptor in the cell membrane, while the intracellular signaling domains activate the T cell when the receptor binds its target. Adoptive cellular immunotherapies using CAR-engineered lymphocytes have revolutionized the treatment of several hematologic malignancies. Most approved CAR-cell products are autologous, meaning a patient’s own immune cells are collected, genetically modified to express the CAR, expanded ex vivo, and reinfused. While effective, this approach is costly, time-consuming, and often unfeasible in patients whose health is rapidly deteriorating. Allogeneic CAR cells are derived from healthy unrelated donors and infused into patients. Advantages are rapid availability, standardized manufacturing, and reduced cost, offering a more scalable therapy model.However, the introduction of CAR cells derived from an un-related donor into an immune competent patient provokes immune recognition and destruction of the graft through transplant rejection mechanisms. The challenge transplant rejection is a key obstacle to effective allogeneic CAR therapies. Overcoming immune barriers while maintaining therapeutic efficacy has therefore become a central focus area of research, with the hope of developing “universal” allogenic CAR cells that are not rejected when infused into patients.

General Cellular Mechanisms of Allogeneic Transplant Rejection

Rejection of allogeneic transplants, both solid organ and hematologic, result from coordinated immune processes involving both adaptive and innate mechanisms. Recipient T cells play a major role in recognizing mismatched human leukocyte antigen (HLA) molecules. Host CD8⁺ cytotoxic T lymphocytes directly identify allogeneic donor cells and kill them, while CD4⁺ helper T cells provide cytokine support and stimulate B cell antibody production against donor HLA. In parallel, recipient B cells can generate alloantibodies that target mismatched HLA molecules on donor cells, leading to complement activation and antibody-dependent cellular cytotoxicity (ADCC) of the transplanted graft. Innate immune cells also participate actively in rejection. Natural killer (NK) cells detect “missing-self” signals, attacking donor cells that lack appropriate inhibitory ligands. Macrophages can further eliminate donor cells through phagocytosis, often enhanced by the presence of alloantibodies or pro-inflammatory cytokines. Antigen-presenting cells amplify these responses by presenting donor-derived peptides to host T cells and producing inflammatory cytokines such as IFN-γ, IL-12, and IL-6. Together, these cellular mechanisms orchestrate rapid immune clearance of donor cells following allogeneic transplantation.   TABLE 1.
Immune ComponentMechanism of ActionKey Effects/Outcome
Adaptive immune cells; Host CD8⁺ and CD4⁺ T cells Recipient T cells recognize donor HLA, and become activatedActivated host T cells proliferate; CD8⁺ T cells mediate cytotoxicity, and CD4⁺ T cells provide help that coordinates rejection.
Innate immune cells; Host NK cells and macrophagesNK cells detect “missing self” (loss or mismatch of donor HLA) and macrophages are activated by Fc receptors or inflammatory cytokines.NK cells directly lyse donor cells lacking inhibitory HLA ligands; macrophages mediate phagocytosis and release pro-inflammatory cytokines.
Host B cells and alloantibodiesRecipient B cells produce anti-donor HLA antibodies.Alloantibodies opsonize donor cells, activate complement, and trigger antibody-dependent cellular cytotoxicity (ADCC).
Cytokine milieu and antigen-presenting cells (APCs)Host APCs amplify alloimmune responses through antigen presentation, costimulation, and cytokine production.Cytokines recruit and activate effector cells, enhancing donor cell elimination.
 

The Problem of Allogeneic CAR Cell Rejection and the Need for Universal CAR Cells

Allogeneic CAR cells are designed for immediate ‘off the shelf’ therapeutic use, offering clear advantages over patient-derived CAR cells. However, their benefits are limited by immune rejection in the host. This immune clearance diminishes CAR cell expansion, persistence, and overall antitumor efficacy, often necessitating multiple doses or concurrent immunosuppressive therapy. Such outcomes undermine the efficiency and accessibility that off-the-shelf therapies are meant to achieve. Therefore, to maximize efficacy, the field seeks to design “universal” CAR cells that can evade host immunity while maintaining robust tumor-targeting functions.

Mechanism of Allogeneic Rejection of CAR Cells in Transplanted Patients

The rejection of allogeneic CAR cells arises from multiple convergent immune pathways. Host T cells recognize donor HLA molecules as foreign and initiate cytotoxic responses that eliminate infused CAR cells. Both CD8⁺ and CD4⁺ subsets are involved, with the latter providing crucial helper signals that enhance antibody production and sustain cytotoxic activity. B cells further contribute by producing anti-HLA antibodies that mediate complement-dependent lysis or facilitate ADCC through Fc receptor-bearing effector cells. Innate immunity amplifies this rejection process. NK cells respond to the absence of self-HLA molecules, a phenomenon known as “missing-self” recognition, and kill donor cells that lack inhibitory HLA ligands. Macrophages and dendritic cells release pro-inflammatory cytokines that enhance antigen presentation, further stimulating host immune activation. The combined activity of adaptive and innate pathways ensures that unmodified allogeneic CAR cells are rapidly eliminated from circulation unless specifically engineered for immune evasion.   TABLE 2.
Immune ComponentMechanism of ActionKey Effects / Outcome
Host T cells (CD8⁺ and CD4⁺)Donor CAR cells express non-self HLA molecules or minor antigens that are recognized by host T cells. Host CD8⁺ T cells directly kill donor cells expressing foreign HLA, while CD4⁺ T cells provide help for cytotoxic T cell activation and B cell alloantibody production.Direct cytotoxic destruction of donor CAR cells and amplification of adaptive immune rejection through T cell and B cell coordination.
Innate immune and inflammatory cells (myeloid, cytokine-mediated response)Host innate immune activation generates pro-inflammatory cytokines that enhance antigen presentation and recruit effector immune cells.Accelerated clearance of donor CAR cells through amplification of adaptive and innate immune pathways.
NK cell surveillanceNK cells recognize allogeneic CAR cells as “missing-self” targets. Inadequate engagement of inhibitory receptors or expression of stress ligands triggers NK activation.NK-mediated cytotoxic killing of donor CAR cells due to loss of inhibitory signaling.
Antibody-mediated rejectionPre-existing or induced anti-HLA antibodies bind donor CAR cells, initiating complement-mediated lysis and Fc receptor-dependent phagocytosis or antibody-dependent cellular cytotoxicity (ADCC).Antibody-driven elimination of donor CAR cells.

Molecular and Cellular Approaches to Overcome Allogeneic CAR Cell Transplant Rejection

A broad array of molecular and cellular engineering strategies has been developed to overcome allogeneic rejection.
  1. Knockout of donor HLA expression to avoid host T cell recognition
  • b2M knockout. Eliminating β2-microglobulin removes classical HLA, preventing host CD8⁺ T cell recognition. However, HLA loss creates “missing-self” NK susceptibility.
  1. Immune cloaking: expression of inhibitory ligands to prevent NK attack and innate clearance
  • HLA-E overexpression. HLA-E can engage NKG2A inhibitory receptors on NK cells and some T cells, protecting HLA-deficient cells from NK killing. Overexpression of non-polymorphic HLA-E is a widely explored strategy to balance T cell invisibility with NK protection.
  • CD47 (“don’t eat me”) overexpression. CD47 interacts with SIRPα on macrophages to inhibit phagocytosis; over-expressing CD47 by genetic engineering can protect donor cells from myeloid clearance. Preclinical studies show improved survival with CD47 co-expression.
  1. Immune-checkpoint and ligand engineering
  • PD-L1 or other inhibitory checkpoint ligands. Local expression of PD-L1 by donor cells can suppress host T cell activation; however, constitutive expression raises concerns about systemic immunosuppression and tumor immune evasion. Recent studies characterize selective PD-L1 expression or inducible systems to mitigate risk.
  1. Targeted HLA matching and allele-level engineering
  • HLA editing to create “common denominator” alleles or matched alleles. Strategies include selective retention of specific less-polymorphic HLA alleles (e.g., HLA-C with matched allele), or allele editing so that a cell bank can serve a large fraction of the population with defined HLA types — analogous to blood group matching.
  1. Transient or permanent host immunomodulation
  • Lymphodepletion and immunosuppression. Standard preconditioning (fludarabine/cyclophosphamide) reduces host lymphocytes and provides a window for donor expansion. More targeted regimens or short courses of agents (e.g., alemtuzumab, anti-CD52) lower alloresponse but increase infection risk.
  • Adjunctive biologics and cell-depleting antibodies. Anti-CD20, anti-CD38, or B cell-depleting agents can limit humoral responses; anti-NK or anti-myeloid strategies are under exploration.
  1. Use alternative donor cell types with lower alloimmunity
  • CAR-NK cells and CAR-iNKT cells. NK cells and invariant NKT cells pose lower cytotoxic side effect risks and are often less alloreactive; CAR-NK products have demonstrated activity with lower graft toxicity in early trials. NK cells may still be cleared by host immunity, so many of the immune-evasion strategies above are applied to CAR-NK as well.
  1. Inducible safety and control modules
  • Suicide switches and drug-gated receptors. To manage unforeseen toxicity or persistent alloactivity, engineered killswitches (e.g., inducible caspase 9) allow selective elimination of donor cells. These do not prevent rejection but improve safety.
  1. Combinatorial engineering and armoring
  • Multiplex editing/armoring. Many groups combine TCR knockout + B2M KO + HLA-E and CD47 expression + CAR addition in one product to simultaneously avoid T cell recognition, NK killing, and macrophage clearance. This multi-layer engineering aims to create durable, immune-evasive universal cells.

Limitations and Challenges of Current Molecular and Cellular Approaches

Despite significant progress, several limitations remain in overcoming allogeneic CAR cell rejection. Gene-edited donor cells may still retain minor antigens or develop neoepitopes from genome editing that trigger residual immune recognition. Additionally, removing HLA molecules to evade host T cells can provoke NK cell-mediated killing due to “missing-self” recognition, making the balance between T cell evasion and NK inhibition complex and patient-specific. Humoral responses, particularly anti-HLA antibodies, continue to pose a challenge, and strategies to suppress B cell function increase infection risks. Multiplex genome editing introduces safety concerns such as off-target effects, chromosomal rearrangements, and oncogenic mutations, while extensive engineering steps add to manufacturing complexity and cost. Furthermore, genetic modifications may impair cellular proliferation or persistence, as essential signaling pathways can be altered during editing. Overexpression of immunomodulatory proteins like PD-L1 or CD47 carries potential systemic safety issues, including dampening of host antitumor immunity. Finally, inter-patient variability in immune profiles and HLA genotypes means that a truly universal product may not achieve equal efficacy in all individuals. Regulatory hurdles, the need for standardized manufacturing processes, and the long-term monitoring of gene-edited products remain substantial barriers to clinical translation.

Future Considerations

Future research aims to optimize and integrate immune-evasion strategies into durable and safe allogeneic CAR therapies. Rational combinatorial engineering is expected to dominate, combining TCR elimination, HLA modulation, and the introduction of inhibitory ligands such as HLA-E and CD47 within a single construct. The creation of HLA-typed cell banks that partially match broad population haplotypes could further improve persistence without requiring fully individualized manufacturing. Advances in precise genome-editing technologies, such as base and prime editing, promise to enhance the fidelity and safety of multiplexed modifications. Developing inducible or tissue-restricted immune cloaks could provide transient protection that allows engraftment without chronic systemic immunosuppression. Hybrid cell products such as CAR-NK or CAR-iPSC derivatives may further reduce cytotoxic side effects, though optimization of their persistence and expansion remains necessary. Future clinical trials should emphasize deep immune monitoring to identify predictors of host rejection and evaluate the in vivo behavior of engineered cells. The incorporation of robust suicide switches and standardized manufacturing pipelines will also be essential to ensure patient safety and scalability.

Conclusion

The creation of universal, immune-evasive allogeneic CAR cells represents a promising advancement in cellular immunotherapy, potentially enabling faster, more equitable access to treatment. However, achieving durable engraftment and antitumor activity requires overcoming both adaptive and innate immune barriers while maintaining cellular fitness and safety. Current strategies, including multiplex gene editing to remove TCRs and HLAs, expression of immune-inhibitory ligands such as HLA-E and CD47, and the use of alternative effector cell types, are moving from preclinical proof-of-concept into early clinical evaluation. Although these approaches demonstrate encouraging results, challenges remain, including residual immunogenicity, NK cell activation, off target editing risks, and manufacturing complexity. The future of universal CAR therapies will likely involve combinatorial engineering, improved editing technologies, and patient-specific immune profiling to achieve optimal outcomes. If these challenges are met, allogeneic CAR platforms could deliver potent, safe, and widely accessible immune cell therapies that transform the treatment landscape for cancer and beyond. Disclaimer: This blog post is intended solely for educational and scientific informational purposes. Any mention of therapeutic drug names, including FDA-approved medications, is for the purpose of accurate reporting and discussion of biomedical research and does not constitute medical advice, endorsement, or promotion. Readers should not interpret the content as a recommendation for any specific treatment. Always consult a qualified healthcare professional for medical advice or treatment decisions.

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1. Development of VNX-101, an Adeno-Associated Virus with Less Immunogenicity and Efficient Long-Term Expression of a CD19 T-Cell Engager. Molecular Therapy Methods & Clinical Development, published online July 24, 2025.

2. Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217.

3. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1.

4. American Society of Hematology (ASH) Annual Meeting 2024. Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

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