Background/Purpose: Therapies that indiscriminately deplete all T cells carry a high risk of opportunistic infection, precluding their widespread use in T cell-mediated autoimmune diseases. Targeting autoreactive T cells with selectivity, conversely, is difficult given an incomplete understanding of their antigen specificities. Germline-encoded T cell receptor (TCR) regions provide an opportunity to target autoreactive T cells, even if the cognate autoantigen is unknown. TCR β-chain variable (TRBV) gene usage by autoreactive T cells can be highly skewed by HLA alleles. In ankylosing spondylitis (AS), CD8+ T cells against disease-relevant peptides presented on HLA-B27*05 are contained in the TRBV9+ T cell fraction. Analogously, HLA-DQ8:α-I-gliadin-reactive CD4+ T cells that drive celiac disease (CD) use TRBV9 (A). Deep depletion of the TRBV9+ T cell compartment has the potential to achieve long-standing disease remission in patients with AS and CD without increasing the risk of infection. We therefore developed a precision T cell-engaging BsAb therapy that selectively eliminates TRBV9+ T cells while sparing >95% of the T cell repertoire.

Methods: Anti-TRBV9 and anti-CD3 single-chain variable fragment (scFv) sequences were synthesized, cloned, and TRBV9xCD3 BsAbs expressed as single-chain diabodies (scDbs) and bispecific T-cell engagers (BiTEs). Human T cells were CRISPR-Cas12a-edited to replace their endogenous TCRs with autoreactive, TRBV9+ TCRs cloned from patients with AS or CD. Binding of anti-TRBV9 to edited T cells was determined by flow cytometry. The potency and specificity of TRBV9xCD3 BsAbs against primary human T cells was interrogated in culture, and cytotoxicity was quantified by flow cytometry and interferon (IFN)-γ ELISA. PBMCs from patients with HLA-B27+ AS and HLA-DQ8+ CD were treated with BsAbs, and selective depletion of TRBV9+ T cells quantified by flow cytometry and TCR repertoire sequencing.

Results: We developed BsAbs to redirect T cells to selectively eliminate TRBV9+ T cells that harbor the autoreactive T cell compartment in patients with AS and HLA-DQ8+ CD (B). Anti-TRBV9 bound engineered human T cells expressing autoreactive TRBV9+ TCRs cloned from patients with AS or CD, but not T cells expressing other TRBV alleles (C). In culture of primary human T cells, TRBV9xCD3 BiTEs resulted in the selective elimination of TRBV9+ T cells in a dose-dependent manner (IC₅₀: 7.2 ng/mL) (p< 0.0001), sparing T cells with other TRBV gene usage (e.g., TRBV4+ T cells) (p=ns) (D-E). Treatment of PBMCs from patients with AS and HLA-DQ8+ CD with TRBV9xCD3 BiTEs induced limited IFN-γ release (F) and depleted of TRBV9+ T cells (IC₅₀: 13-27 ng/mL) and disease-relevant autoreactive T cell clonotypes (p< 0.0001), but not non-TRBV9 T cells (p=ns) (G).

Conclusion: We describe an off-the-shelf, precision immunotherapy approach for the deep depletion of autoreactive TRBV9+ T cells in HLA-B27+ AS and HLA-DQ8+ CD. TRBV9xCD3 BsAbs are highly potent and specific at eliminating TRBV9+ T cells while sparing other T cell populations. Different to monoclonal antibodies targeting TRBV9, BsAbs may offer an opportunity to achieve sustained depletion and reset of the autoreactive T cell compartment in AS and CD.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/trbv9-targeted-bispecific-t-cell-engaging-antibodies-to-reset-the-autoreactive-t-cell-compartment-in-spondyloarthritis-and-hla-dq8-celiac-disease/