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1.
Immunity ; 55(10): 1953-1966.e10, 2022 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-36174557

RESUMEN

A major challenge in adoptive T cell immunotherapy is the discovery of natural T cell receptors (TCRs) with high activity and specificity to tumor antigens. Engineering synthetic TCRs for increased tumor antigen recognition is complicated by the risk of introducing cross-reactivity and by the poor correlation that can exist between binding affinity and activity of TCRs in response to antigen (peptide-MHC). Here, we developed TCR-Engine, a method combining genome editing, computational design, and deep sequencing to engineer the functional activity and specificity of TCRs on the surface of a human T cell line at high throughput. We applied TCR-Engine to successfully engineer synthetic TCRs for increased potency and specificity to a clinically relevant tumor-associated antigen (MAGE-A3) and validated their translational potential through multiple in vitro and in vivo assessments of safety and efficacy. Thus, TCR-Engine represents a valuable technology for engineering of safe and potent synthetic TCRs for immunotherapy applications.


Asunto(s)
Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T , Antígenos de Neoplasias , Humanos , Inmunoterapia , Péptidos
2.
Mol Ther ; 28(12): 2564-2576, 2020 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-32827460

RESUMEN

In recent years, chimeric antigen receptor (CAR) T cell cancer immunotherapies have advanced substantially in the clinic. However, challenges related to safety persist; one major concern occurs when CARs trigger a response to antigen present on healthy cells (on-target, off-tumor response). A strategy to ameliorate this relies on the complex relationship between receptor affinity and signaling, such that one can engineer a CAR that is only activated by tumor cells expressing high antigen levels. Here, we developed a CAR T cell display platform with stable genomic expression and rapid functional screening based on interleukin-2 signaling. Starting with a CAR with high affinity toward its target antigen, we combined CRISPR-Cas9 genome editing and deep mutational scanning to generate a library of antigen-binding domain variants. This library was subjected to multiple rounds of selection based on either antigen binding or cell signaling. Deep sequencing of the resulting libraries and a comparative analysis revealed the enrichment and depletion of specific variants from which we selected CARs that were selectively activated by tumor cells based on antigen expression levels. Our platform demonstrates how directed evolution based on functional screening and deep sequencing-guided selection can be combined to enhance the selectivity and safety of CARs.


Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/inmunología , Ingeniería Celular/métodos , Inmunoterapia Adoptiva/métodos , Receptor ErbB-2/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Animales , Afinidad de Anticuerpos , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Técnicas de Cocultivo , Femenino , Edición Génica/métodos , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Células MCF-7 , Ratones , Receptor ErbB-2/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Anticuerpos de Cadena Única/inmunología
3.
Nat Biomed Eng ; 2024 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-39322719

RESUMEN

Bispecific antibodies (biAbs) used in cancer immunotherapies rely on functional autologous T cells, which are often damaged and depleted in patients with haematological malignancies and in other immunocompromised patients. The adoptive transfer of allogeneic T cells from healthy donors can enhance the efficacy of biAbs, but donor T cells binding to host-cell antigens cause an unwanted alloreactive response. Here we show that allogeneic T cells engineered with a T-cell receptor that does not convert antigen binding into cluster of differentiation 3 (CD3) signalling decouples antigen-mediated T-cell activation from T-cell cytotoxicity while preserving the surface expression of the T-cell-receptor-CD3 signalling complex as well as biAb-mediated CD3 signalling and T-cell activation. In mice with CD19+ tumour xenografts, treatment with the engineered human cells in combination with blinatumomab (a clinically approved biAb) led to the recognition and clearance of tumour cells in the absence of detectable alloreactivity. Our findings support the development of immunotherapies combining biAbs and 'off-the-shelf' allogeneic T cells.

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