Boosting CAR T Cell Potency: Membrane-Tethered SLP-76 Tackles Antigen-Low Tumor Resistance

Overcoming a Critical Hurdle in CAR T Cell Therapy

Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment, particularly for hematological malignancies. However, a significant limitation has emerged: many CAR T cells struggle to recognize and eliminate tumor cells expressing low levels of target antigen. This deficiency creates an escape route for cancer cells through antigen downregulation, a phenomenon observed in clinical trials targeting CD22, CD19, and BCMA. Recent research published in Nature Cancer reveals an innovative solution—engineering T cells with a membrane-tethered version of the signaling protein SLP-76 to overcome this fundamental barrier.

The Signaling Deficit in CAR T Cells

Unlike natural T-cell receptors (TCRs), CARs exhibit impaired proximal signaling despite recognizing the same antigens. Phosphoproteomic analyses comparing CAR and TCR signaling networks identified significant deficits in phosphorylation of key signaling molecules. CAR architectures with higher activation thresholds particularly demonstrated reduced phosphorylation throughout the downstream proximal signaling network., according to industry experts

Comparative studies between CD19 CARs with CD28 versus CD8 hinge-transmembrane domains revealed striking differences. The CD28 configuration showed enriched phosphorylation of proximal signaling molecules (including LCK, SLP-76, LAT), distal signaling molecules, and transcription factors. Multiple members of the LAT/SLP-76 signalosome were significantly enriched in CD28 H/TM CAR T cells, along with proteins linking proximal signaling to cytoskeletal rearrangement. This signaling advantage translates to improved recognition of antigen-low targets, mirroring the superior antigen sensitivity naturally present in TCRs., according to industry experts

The Quest for Enhanced Proximal Signaling

Researchers initially explored whether simply overexpressing key proximal signaling molecules could improve CAR T cell function against antigen-low targets. They tested five critical signaling proteins—LCK, ZAP-70, LAT, SLP-76, and PLCγ1—in CD19 CAR T cells. While ZAP-70 and SLP-76 overexpression enhanced interleukin-2 production in response to antigen-high targets, neither enabled recognition of antigen-low tumor cells. This suggested that mere abundance wasn’t sufficient—proper localization and engagement within the signaling complex was crucial., according to according to reports

A Membrane-Tethered Breakthrough

The research team developed an ingenious solution: engineering a membrane-tethered version of SLP-76 (MT-SLP-76). Since CARs struggle to recruit proximal signaling molecules to the immune synapse, physically anchoring SLP-76 to the membrane could facilitate its engagement and enhance signaling activity.

The results were striking. Co-expression of MT-SLP-76 with a CD19-4-1BBζ CAR substantially enhanced IL-2 production against both CD19-high and CD19-low target cells, outperforming overexpression of native SLP-76 or ZAP-70. Most importantly, MT-SLP-76 significantly improved killing of CD19-low cells while maintaining efficacy against antigen-high targets., according to recent studies

When tested across a spectrum of CD19 antigen densities (from 600 to 249,700 molecules per cell), MT-SLP-76 shifted the antigen density response curve, lowering the threshold for cytokine secretion. The approach demonstrated broad applicability, enhancing sensitivity for CD22- and HER2-targeting CAR T cells against both antigen-low and antigen-high targets., according to related coverage

Validating Clinical Relevance in Disease Models

The researchers tested MT-SLP-76 in clinically relevant models where antigen-low resistance poses significant challenges:, according to industry news

• CD22-low leukemia: In a model where CD22 CAR T cells typically fail to control antigen-low disease (1,300 molecules per cell), MT-SLP-76-overexpressing CD22 CAR T cells mediated sustained tumor eradication, while standard CD22 CAR T cells provided only modest control. MT-SLP-76 rescued CAR T cell expansion in vivo, significantly increasing cell numbers in bone marrow and spleens.
• CD19 ultra-low leukemia: Against CD19 ultra-low targets (600 molecules per cell), only MT-SLP-76 enhanced in vivo activity of CD19-4-1BBζ CAR, while cytosolic SLP-76 or ZAP-70 overexpression had minimal effect.
• BCMA-low multiple myeloma: In OPM-2 cells with low BCMA expression (1,200 molecules per cell), MT-SLP-76 substantially improved antitumor activity in vivo, demonstrating applicability across disease types.

Safety and Persistence Considerations

A critical concern with enhancing signaling strength is potential overactivation against antigen-high targets, which could theoretically reduce T cell persistence or cause toxicity. Comprehensive testing revealed that MT-SLP-76:

• Enhanced antitumor activity and survival in stress test models without compromising persistence
• Maintained equivalent persistence and memory phenotype compared to CAR T cells alone
• Demonstrated appropriate contraction after reaching higher peak numbers, indicating maintained regulatory control

Therapeutic Window Considerations

While enhanced sensitivity benefits targets like CD19 and CD22 with limited normal tissue expression, it could narrow the therapeutic window for antigens with low-level expression on vital tissues. Researchers explored this using a ROR1 CAR model known to cause on-target, off-tumor toxicity. The findings highlight the importance of careful antigen selection when implementing sensitivity-enhancing strategies., as additional insights

Clinical Implications and Future Directions

This research identifies a critical bottleneck in CAR T cell signaling and delivers a readily translatable approach that can be paired with existing CAR designs. The membrane-tethering strategy represents a paradigm shift—rather than simply overexpressing signaling components, optimizing their spatial organization within the cell. This approach could potentially benefit patients who develop resistance through antigen downregulation and expand the applicability of CAR T therapy to solid tumors where antigen density is often limited.

The findings underscore that enhancing proximal signaling through strategic protein localization, rather than mere overexpression, can overcome fundamental limitations in synthetic receptor systems while maintaining safety profiles. As the field advances, such engineering approaches will likely play crucial roles in developing next-generation cellular therapies with improved efficacy against heterogeneous tumors.

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