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Tumor Intrinsic Resistance Mechanisms to CAR T-cells

Introduction

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated unprecedented clinical efficacy in the treatment of hematologic malignancies; however, primary resistance and disease relapse remain significant barriers to durable therapeutic responses. While loss or downregulation of the target antigen is a well-recognized mechanism of immune escape, increasing evidence indicates that tumor cells can retain antigen expression and effectively engage CAR T-cells yet remain resistant to cell-mediated cytotoxicity. This phenomenon suggests that tumor-intrinsic resistance mechanisms can uncouple CAR recognition and activation from target-cell elimination.

Several pathways have been implicated in this process, including dysregulation of apoptotic signaling through overexpression of anti-apoptotic proteins such as BCL-2, BCL-xL, and MCL-1, defects in death receptor signaling pathways involving FAS and caspase-8, and alterations in interferon-responsive pathways mediated by JAK1 and JAK2. Furthermore, expression of immunosuppressive factors, including programmed death-ligand 1 (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO1), and transforming growth factor-β (TGF-β), can impair CAR T-cell effector function despite successful antigen recognition.

Overcoming the molecular mechanisms that enable antigen-positive tumor cells to resist CAR T-cell-mediated killing is critical for the development of next-generation cellular immunotherapies.

Common Mechanisms for Tumor Resistance to CAR T-Cell Killing

Defects in apoptotic pathways

Defects in apoptotic signaling pathways represent a major mechanism by which tumor cells evade destruction by CAR T-cells. Following antigen recognition, CAR T-cells induce target cell death primarily through perforin/granzyme-mediated activation of the intrinsic mitochondrial apoptotic pathway and, to a lesser extent, through death receptor signaling. However, many tumors acquire resistance by overexpressing anti-apoptotic members of the B-cell lymphoma 2 (BCL-2) protein family. Elevated expression of BCL-2 inhibits mitochondrial outer membrane permeabilization (MOMP) by sequestering pro-apoptotic proteins such as BAX and BAK, thereby preventing cytochrome c release and downstream caspase activation. Similarly, overexpression of BCL-xL (BCL2L1) suppresses apoptosis by binding and neutralizing BH3-only proteins and inhibiting BAX/BAK oligomerization, resulting in reduced sensitivity to CAR T-cell derived cytotoxic signals. Increased levels of myeloid cell leukemia 1 (MCL-1) provide an additional survival mechanism by stabilizing mitochondrial integrity and antagonizing pro-apoptotic effectors, particularly in response to granzyme B-induced cellular stress. Collectively, dysregulated expression of BCL-2, BCL-xL, and MCL-1 elevates the apoptotic threshold of tumor cells, enabling survival despite effective CAR T-cell engagement and activation, and has emerged as an important contributor to therapeutic resistance and disease relapse.

Resistance to death receptor signaling

Defects in death receptor-mediated apoptotic signaling represent an important mechanism of resistance to CAR T-cell therapy. In addition to perforin/granzyme-dependent cytotoxicity, activated CAR T-cells can induce tumor cell death through engagement of death receptors, including Fas (CD95) and TRAIL receptors, leading to activation of the extrinsic apoptotic pathway. Loss or downregulation of Fas expression impairs the interaction between tumor cell Fas and Fas ligand (FasL) expressed on activated CAR T cells, thereby preventing formation of the death-inducing signaling complex (DISC) and subsequent apoptotic signaling. Similarly, inactivating mutations, reduced expression, or functional impairment of caspase-8 (CASP8), a key initiator caspase recruited to the DISC, block downstream activation of effector caspases such as caspase-3 and caspase-7, rendering tumor cells resistant to death receptor-induced apoptosis. Defects in TRAIL receptor signaling, including decreased expression or mutation of TRAIL-R1 (DR4) and TRAIL-R2 (DR5), diminish the ability of TRAIL produced by CAR T cells to trigger apoptotic signaling through DISC assembly and caspase activation. Collectively, disruption of Fas, CASP8, and TRAIL receptor pathways compromises extrinsic apoptotic signaling, allowing tumor cells to evade CAR T-cell-mediated cytotoxicity despite effective antigen recognition and immune synapse formation.

Interferon pathway defects

Defects in interferon signaling pathways have emerged as important mechanisms by which tumor cells evade CAR T-cell-mediated cytotoxicity. Activated CAR T-cells secrete interferon-γ (IFN-γ), which enhances antitumor immunity by promoting antigen presentation, increasing sensitivity to apoptotic signals, and inducing the expression of genes involved in growth inhibition and immune recognition. Central to this pathway are Janus kinase 1 (JAK1) and Janus kinase 2 (JAK2), which transduce IFN-γ receptor signaling through activation of the STAT1 transcriptional program. Loss of JAK1 expression disrupts IFN-γ-induced signal transduction, preventing the upregulation of interferon-stimulated genes that enhance tumor cell susceptibility to immune-mediated killing and limiting the expression of molecules involved in antigen processing and presentation. Similarly, loss of JAK2 abolishes downstream IFN-γ signaling and impairs STAT1 activation, resulting in reduced responsiveness to the antiproliferative and pro-apoptotic effects of CAR T-cell derived cytokines. Consequently, tumor cells lacking JAK1 or JAK2 become insensitive to IFN-γ mediated immune pressure, diminishing antigen presentation, reducing sensitivity to cytotoxic effector mechanisms, and promoting resistance to CAR T-cell therapy despite successful antigen recognition and T-cell activation.

CAR T-cell Resistant Tumor Cell Lines

There are many tumor cell lines that can engage CAR T-cells (i.e., express the target antigen and trigger CAR signaling) but are relatively resistant or even refractory to CAR T-cell mediated killing. These models are widely used to study mechanisms of resistance and to develop next-generation CAR T-cell therapies.

The tumor cell lines summarized in Table 1 illustrate that resistance to CAR T-cell mediated cytotoxicity can occur despite sustained expression of the target antigen and effective CAR T-cell engagement. These models encompass both hematologic malignancies and solid tumors and collectively highlight several recurrent mechanisms of tumor-intrinsic resistance, including defects in intrinsic and extrinsic apoptotic pathways, dysregulation of anti-apoptotic BCL-2 family proteins, impaired interferon signaling, and adaptive survival responses. As a result, these models provide valuable tools for investigating mechanisms of primary and acquired resistance and for evaluating next-generation cellular therapies designed to overcome tumor-intrinsic barriers to effective CAR T-cell function.

Table 1. Representative Examples of CAR T-Cell Resistant Tumor Cell Lines

Tumor Cell Line	Antigen	Resistance Mechanisms
JeKo-1	CD19	High expression of anti-apoptotic proteins such as BCL-2 and MCL-1 can reduce susceptibility to CAR T-cell killing.
SU-DHL-4	CD19	Elevated BCL-2 family signaling linked to reduced CAR T-cell cytotoxicity.
A375 (engineered antigen-positive models)	Various CAR targets	Resistance associated with IFN-γ pathway defects, including JAK1/JAK2 loss.
OVCAR3	Mesothelin	Frequently exhibits resistance due to anti-apoptotic signaling and an immunosuppressive phenotype despite CAR T-cell recognition.
PANC-1	Mesothelin, EGFR, HER2	Intrinsically resistant due to high expression of anti-apoptotic proteins (BCL-xL, MCL-1), poor apoptotic priming, and resistance to granzyme B-mediated cell death.
MIA PaCa-2	Mesothelin, Claudin 18.2, EGFR	Demonstrates reduced susceptibility to CAR T-cell killing through activation of survival pathways, defective apoptosis signaling, and immunosuppressive cytokine production.
U87MG	EGFRvIII, IL13Rα2, HER2	Frequently used as a CAR T target-positive model; resistant populations exhibit impaired IFN-γ responsiveness, elevated anti-apoptotic signaling, and adaptive resistance to repeated CAR T-cell exposure.

Clinical Evidence

Three of the clearest examples of clinically relevant tumor-intrinsic resistance to CAR T-cell therapy involve defects in interferon signaling and apoptotic pathways that allow tumor cells to survive despite antigen recognition and T-cell activation. Interestingly, across all three clinical resistance mechanisms CAR T-cell activation is consistently preserved including: cytokine secretion, degranulation, proliferation, and immune synapse formation. The resistance phenotype is therefore tumor-intrinsic, not due to CAR T-cell dysfunction.

CD19 CAR T-cell therapy: Interferon signaling defects (JAK1/JAK2 loss)

One of the best-documented examples of tumor-intrinsic resistance in patients treated with CD19-directed CAR T-cell therapy involves disruption of the IFN-γ signaling pathway. Studies of relapsed B-cell malignancies following CD19 CAR T-cell treatment have identified loss-of-function alterations in JAK1 and JAK2, key mediators of IFN-γ receptor signaling. Tumor cells harboring these defects remain CD19-positive and can still be recognized by CAR T-cells; however, they become insensitive to IFN-γ induced growth arrest, antigen presentation, and apoptotic priming. Functional studies demonstrated that JAK1/JAK2-deficient tumor cells are significantly less susceptible to CAR T-cell mediated killing despite normal CAR T-cell activation. These findings provided some of the first clinical and mechanistic evidence that resistance can arise through tumor-intrinsic signaling defects downstream of antigen recognition rather than through antigen loss alone, establishing interferon pathway dysfunction as a clinically relevant mechanism of CD19 CAR T-cell resistance.

CD19 CAR T-cell therapy: Interferon signaling defects (FAS/FADD/CASP8 death receptor signaling axis)

A well-supported example involving the death receptor pathway comes from studies of patients who relapsed after CD19 CAR T-cell therapy while retaining target antigen expression. Analysis of patient-derived leukemia samples demonstrated that disruption of the FAS/FADD/CASP8 death receptor signaling axis was associated with resistance to CAR T-cell mediated killing despite preserved CD19 expression. Functional genomic studies identified loss of FADD and other components of the Fas signaling pathway as mediators of primary resistance, reducing susceptibility to CAR T-cell induced apoptosis. Importantly, tumor cells harboring these defects remained capable of engaging CAR T cells and stimulating T-cell activation, indicating that resistance occurred downstream of antigen recognition. Furthermore, persistent exposure to apoptosis-resistant tumor cells promoted progressive dysfunction and epigenetic reprogramming of CAR T cells, creating a feed-forward mechanism that further impaired antitumor activity. These findings provided some of the strongest evidence that defects in the death receptor pathway can contribute directly to clinical CAR T-cell failure independent of antigen escape.

HER2 CAR T-cell therapy for solid tumors: Acquired JAK2 downregulation

A particularly compelling example in solid tumors was reported in HER2-targeted T-cell therapies, where tumor cells developed resistance despite continued HER2 expression. Resistant tumor populations exhibited marked downregulation of JAK2, resulting in impaired responsiveness to IFN-γ and reduced expression of interferon-stimulated genes. Importantly, these resistant cells remained capable of engaging HER2-directed CAR T cells and HER2-targeted T-cell engagers but were no longer efficiently eliminated. Restoration of JAK2 expression re-sensitized tumor cells to T-cell-mediated cytotoxicity, demonstrating a direct causal relationship between interferon signaling defects and therapeutic resistance. This work provided strong evidence that defects in the JAK/STAT pathway can drive cross-resistance to multiple T-cell-based immunotherapies and represents one of the clearest examples of a tumor-intrinsic resistance mechanism relevant to CAR T-cell therapy directed against a solid tumor antigen.

Conclusion

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment landscape for several hematologic malignancies, demonstrating the potential of engineered immune cells to induce durable clinical responses. However, a significant proportion of patients either fail to achieve complete remission or ultimately relapse, underscoring the importance of understanding the mechanisms that enable tumor cells to evade CAR T-cell-mediated destruction. While antigen loss and impaired CAR T-cell persistence have received considerable attention, increasing evidence indicates that intrinsic resistance mechanisms within tumor cells can also play a critical role in therapeutic failure.

Studies utilizing CAR T-cell-resistant tumor cell lines have revealed that malignant cells may remain fully capable of engaging and activating CAR T-cells while simultaneously resisting cytotoxic killing. These findings demonstrate that productive immune synapse formation and CAR T-cell activation are not always sufficient to ensure tumor eradication. Defects in apoptotic signaling pathways, alterations in death receptor-mediated cell death, metabolic adaptations, epigenetic reprogramming, and enhanced survival signaling have all been implicated as mechanisms by which tumor cells withstand CAR T-cell attack. Importantly, clinical observations have confirmed that several of these resistance pathways are not limited to experimental models but can contribute directly to treatment failure and disease relapse in patients.

The growing recognition of tumor-intrinsic resistance mechanisms highlights the need to view CAR T-cell therapy as an interaction between both the effector immune cell and the target cancer cell. Future therapeutic advances will likely require combination approaches that simultaneously enhance CAR T-cell function and sensitize tumor cells to immune-mediated killing. Improved understanding of the molecular determinants of resistance, coupled with the development of predictive biomarkers and rational combination therapies, may enable more durable responses and broaden the clinical benefit of CAR T-cell therapy across a wider range of malignancies. Ultimately, overcoming tumor cell resistance represents an important challenge and opportunity for the next generation of cellular immunotherapies.

Image credit: Portions of the figure in this article were generated using ChatGPT (OpenAI) or Google Gemini.

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.

About Marin Biologic Laboratories

Our Recent Publication/Meeting Presentation on Gene Therapy

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.

3. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. Journal of Immunological Methods. 2025. 545-114004.

3. 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.

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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