Kaufmann, M. M. | bioRxiv Science

bioRxiv2025

Clinical development of gene edited tacrolimus-resistant Treg (FKBP12KO-Treg) to enable simultaneous immunosuppression and support of immune regulation

Background: Unwanted immune responses play a central role in the pathogenesis of solid organ allograft rejection. These are managed by life-long immunosuppression with considerable burden for the patient and society. Adoptive therapy with regulatory T-cells (Treg) is a promising approach to restore sustainable immune balance and avoid long-term adverse effects of immunosuppression. While Treg effectively inhibit activation of unwanted immune responses, they are less effective in controlling pre-existing/activated memory effector T-cells (Teff). Thus, co-administration of Treg with immunosuppressants is required to achieve a sustainable organ acceptance. Calcineurin inhibitors (CNI) are powerful in controlling de novo generated and preformed Teff. However, CNI also dampen Treg immunoregulatory function. Thus, we hypothesize improved results of adoptive Treg therapy in immunosuppressed patients applying tacrolimus-resistant Treg. Methods: While retaining CNI modulation of Teff with tacrolimus, we knocked-out FKBP12 in Treg (FKBP12KO-Treg) by gene-editing using ribonucleoprotein-based CRISPR/Cas9 technology to generate tacrolimus-resistant Treg and characterised them using flow cytometry, functional assays and in-depth phenotyping. Results: This detailed in vitro analysis showed FKBP12KO-Treg were comparable to non-gene edited Treg and impervious to tacrolimus while maintaining immunoregulatory function and sensitivity to alternative CNIs raising no safety concerns. Furthermore, we aligned our methodology to achieve GMP compliance laying the basis for a manufacturing license in preparation of a clinical trial. Conclusion: Based on the presented preclinical dataset implying safety and efficacy of FKBP12KO-Treg, we are now seeking to undertake a proof-of-concept clinical trial to evaluate the co-administrationof FKBP12KO-Treg and tacrolimus to enhance the management of living donor kidney transplant recipients.

immunology↗

bioRxiv2024

Rare twin cysteine residues in the HIV-1 envelope variable region 1 link to neutralization escape and breadth development

The identification of HIV-1 Envelope glycoprotein (Env) traits associated with development of neutralization cross-reactivity in natural infection is critical for vaccine design. Here we describe the presence of additional Cysteine (Cys) residues in V1 that are enriched among people with elite neutralization breadth. Using >65,000 V1 sequences from the CATNAP database, the AMP trials and three large longitudinal HIV infection cohorts, the SHCS, ZPHI and CAPRISA studies, we show that Env variants with extra V1 Cys are present at low levels throughout infection and fluctuate in frequency over time within participants. We demonstrate an independent association of extra V1 Cys with elite plasma neutralization, and a strong preference for two versus one extra Cys, suggesting certain Envs introduce an additional disulfide bond for stabilization. We observed high levels of neutralization resistance among Envs from 34 bNAb donors, of which 17.6% had elongated V1 regions with extra Cys. We show that extra V1 Cys moderately increase neutralization resistance in an Env from a V2- Apex bNAb-inducer. Modulation of the accessibility of bNAb epitopes on this Env by extra V1 Cys enhanced epitope shielding of several regions, but increased V2 exposure. This suggests that escape from autologous neutralizing activity drove insertion of the extra V1 Cys, creating a modified antigen that may have favored V2 bNAb induction in this donor. Overall, we identify a rare motif of twin Cys in V1 that confers increased neutralization resistance and Env stabilization, is associated with bNAb induction, and may hold potential for incorporation into future HIV bNAb immunogens.

immunology↗