Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy

Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vβ2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vβ chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vβ scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vβ2+ Jurkat cells and Vβ2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vβ2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.


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All manuscripts must include a data availability statement.This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Data supporting the findings of the study are available within the Article, Supplementary Information and Source data file.Human-related data that were collected but not shown in the paper might be subject to confidentiality (e.g., sex and age).All outstanding data are available upon request from the corresponding author, Michael Girardi (michael.girardi@yale.edu)due to the intellectual property protection applications that are currently under consideration for the disclosed nature portfolio | reporting summary April 2023 innovations.
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Reporting on sex and gender
Reporting on race, ethnicity, or other socially relevant groupings These findings are not sex or gender specific.Individuals of both sexes, regardless of gender, were invited to participate in the study.Sex assigned at birth from their medical record is used for reporting.Low sample size precludes sex-and genderbased analyses.
These findings are not race, ethnicity or other socially relevant groupings specific.

Ethics oversight
Patients previously diagnosed with CTCL or PTCL who have an identifiable malignant T cell population in their peripheral blood will be recruited.This includes both males and females of all races ranging in age from 18-90 years.Because disease incidence increases with increasing age, older subjects predominate.In this study, a limited number of anonymous adult volunteers aged from 35-55 were recruited as healthy donors without a history of cancer or immune disorders.
YNHH Oncology and Photopheresis Unit physicians or mid-level provider discuss the possibility of participating in this study with their patients during the normal course of their care for the treatment of CTCL or PTCL.We impartially chose patients with a significant presence of high blood cancer involvement.The healthy donors in this study were randomly selected from available volunteers.Informed consent was obtained.
The protocols involved in this study are all approved by Yale University Institutional Review Board and Human Investigation Committee Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Sample size
Sample sizes were primarily determined by availability of materials, e.g.patient-derived malignant cells.No power calculations were performed.The sample size for in vivo mouse studies utilizing the Jurkat-Vb2 tumor cell line were determined based on pilot experiments assessing the tumor cell line growth characteristics which we found to be sufficiently similar from mouse to mouse such that groups of N=5 mice could be used to reasonably assess the effects of different treatments.
Data exclusions No data were excluded from the analyses.

Replication
To ensure robustness of the data two different viral vectors (AAV and Lentivirus) were used and in this way CAR-T manufacturing was replicated.Both Vb2 expressing Jurkat cells and Vb2+ patient derived cells were used as a way to replicate both in vitro and in vivo experiments.Experiments were also repeated by using cells from multiple donors.All attempts at replication were successful.
Randomization Tumor-bearing mice were randomly allocated into experimental groups.Cells from limited donors were equally used into different experimental conditions for in vitro studies.

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Investigators were blinded to group identification for data collection and analysis.
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