Immunosuppressive efficacy and safety of MSCs bioengineered with chimeric antigen receptor (CAR) in GvHD preclinical models

Mesenchymal stromal cells (MSCs) are a promising therapy candidate for the treatment of graft-versus-host disease (GvHD). Although MSCs are well-tolerated in patients with GvHD, their insufficient immunosuppression at the sites of inflammation remains a challenge.^1,2 To address this limitation, Sirpilla et al. introduced chimeric antigen receptor (CAR) technology to MSCs to enhance their immunosuppressive efficacy at inflammation sites.^1,2

In GvHD, the host epithelial tissues are susceptible to donor immune cell attack through the interaction of T-cell integrins with E-cadherin (Ecad) expressed on epithelia. Thus, Sirpilla et al.¹ hypothesized that an anti-Ecad CAR-MSC (EcCAR-MSC) with Ecad+ (human, mouse, and canine) cross-reactivity and a CD28ζ intracellular signaling domain would induce antigen-specific immunosuppression at these inflammatory target tissue sites.^1,2 The possible mechanism of action of EcCAR-MSCs is shown in Figure 1.

CAR, chimeric antigen receptor; GvHD, graft-versus-host disease; MSC, mesenchymal stromal cells; ScFv, single-chain variable fragment.
*Adapted from Sirpilla, et al.¹ Created with BioRender.com

Here, we summarize the key findings of a study investigating immunosuppressive efficacy and safety of EcCAR-MSCs in GvHD preclinical models, presented was given at the 2023 Tandem Transplantation & Cellular Therapy Meetings of ASTCT and CIBMTR.

Methods^1,2

EcCAR-MSCs were developed by transducing CAR into adipose-derived MSCs via lentiviral vector enhancement. CD28 signal was selected for its ability to activate downstream immunosuppressive factors.

To test T-cell immunosuppression in vitro, MSCs were cocultured for 24 hours with activated donor T cells with or without antigen-specific Ecad stimulation.

The in vivo testing was done using GvHD xenograft models. GvHD was induced in NOD-SCID-γ^-/- mice via intravenous injection of human peripheral blood mononuclear cells. Mice were then treated with intraperitoneal injections of CAR-MSC, untransduced MSCs (UTD-MSC), or vehicle control to monitor weight loss, clinical GvHD score, human T cell suppression, and survival outcomes.

Mechanism of CAR-MSCs

• Mechanisms underlying superior immunosuppression in EcCAR-MSCs upon CAR stimulation were interrogated via RNA sequencing, serum cytokine assays, and immunophenotype analyses.
• To corroborate transcriptional enrichments, serum cytokines were analyzed.

Safety of CAR-MSCs

• The safety of EcCAR-MSCs was investigated in vivo by comparing clearance profiles with UTD-MSCs.
• Additionally, hematologic and organ toxicity studies were carried out in healthy canines through intraperitoneal EcCAR-MSC injection.

Results^1,2

• Cocultures with antigen-stimulated CAR-MSCs led to significant T-cell suppression (p ≤ 0.01) compared with unstimulated CAR-MSCs or UTD-MSCs.
• In GvHD xenograft models, mice treated with CAR-MSCs exhibited significantly reduced weight loss, decreased clinical GvHD score (3 vs 5.2 score; p = 0.0014), increased T-cell suppression (60 vs 450 cells/uL; p = 0.0039), regulator T-cell induction (6.7 vs 2.1% in CD4⁺ cells), and improved overall survival compared with controls.

Mechanism of CAR-MSCs

• Functional CAR-MSCs were supported by mechanistic studies that revealed prominent immunosuppressive gene expression profiles.
• Mice treated with stimulated EcCAR-MSCs vs UTD-MSCs had elevated anti-inflammatory serum cytokines, such as interleukin-10, TNFα, and G-CSF. Immunophenotypic analyses revealed upregulated T-cell inhibitory receptor expression, including programmed death-1 and galectin-9, on stimulated EcCAR-MSCs vs UTD-MSCs (Figure 2).
• CD28-linked transcription factors such as NFκB and JUN were upregulated in Ecad-stimulated CAR-MSCs only.

EcCAR-MSC, E-cadherin targeted chimeric antigen receptor–mesenchymal stromal cells; Gal, galectin; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; PD, programmed death; TNF, tumor necrosis factor; UTD-MSC, untransduced mesenchymal stromal cells.
*Adapted from Sirpilla, et al.^1,2

Safety of CAR-MSCs

• Compared to UTD-MSC, CAR-MSCs showed similar stem and clearance profiles.
• CAR-MSCs were also well-tolerated in healthy canine models, showing no hematologic or organ toxicity following administration.

Conclusion^1,2

The findings demonstrate EcCAR-MSCs as a novel therapeutic platform to enhance MSC antigen-specific immunosuppression in GvHD with no toxicity. EcCAR-MSCs will next be employed in a phase I clinical trial for patients with steroid-refractory acute GvHD, with alternative CAR-MSC designs also in progress for other autoimmune disease treatments.