Engineered allogeneic T cells decoupling T-cell-receptor and CD3 signalling enhance the antitumour activity of bispecific antibodies.

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.

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing.

Chimeric antigen receptors (CARs) consist of an antigen-binding region fused to intracellular signaling domains, enabling customized T cell responses against targets. Despite their major role in T cell activation, effector function and persistence, only a small set of immune signaling domains have been explored. Here we present speedingCARs, an integrated method for engineering CAR T cells via signaling domain shuffling and pooled functional screening. Leveraging the inherent modularity of natural signaling domains, we generate a library of 180 unique CAR variants genomically integrated into primary human T cells by CRISPR-Cas9. In vitro tumor cell co-culture, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), enables high-throughput screening for identifying several variants with tumor killing properties and T cell phenotypes markedly different from standard CARs. Mapping of the CAR scRNA-seq data onto that of tumor infiltrating lymphocytes further helps guide the selection of variants. These results thus help expand the CAR signaling domain combination space, and supports speedingCARs as a tool for the engineering of CARs for potential therapeutic development.

Leveraging Single-Cell Sequencing for Chimeric Antigen Receptor T Cell Therapies.

Chimeric antigen receptor (CAR)-T cell therapies against cancer continue to make inroads in the clinic. However, progress is still hindered by subpar efficacy against many tumors. Gaining a better understanding of CAR-induced T cell activation would help identify and remediate the causes of treatment failure. Increasingly, technologies to analyze the transcriptome are used to molecularly profile the behavior of CAR-T cells, both before and after treatment. Here, we describe recent work on how gene expression signatures, especially those obtained from single-cell RNA sequencing (scRNA-seq), can be used to characterize CAR design, production conditions, therapy combinations, and finally disease outcome. In the future, scRNA-seq could become a standard tool for the development and clinical monitoring of CAR-T cell therapies.

A Functional Screening Strategy for Engineering Chimeric Antigen Receptors with Reduced On-Target, Off-Tumor Activation.

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.

NLRP7 in the spectrum of reproductive wastage: rare non-synonymous variants confer genetic susceptibility to recurrent reproductive wastage.

BACKGROUND: NLRP7 mutations are responsible for recurrent molar pregnancies and associated reproductive wastage. To investigate the role of NLRP7 in sporadic moles and other forms of reproductive wastage, the authors sequenced this gene in a cohort of 135 patients with at least one hydatidiform mole or three spontaneous abortions; 115 of these were new patients. METHODS/RESULTS: All mutations were reviewed and their number, nature and locations correlated with the reproductive outcomes of the patients and histopathology of their products of conception. The presence of NLRP7 mutations was demonstrated in two patients with recurrent spontaneous abortions, and some rare non-synonymous variants (NSVs), present in the general population, were found to be associated with recurrent reproductive wastage. These rare NSVs were shown to be associated with lower secretion of interleukin 1ß and tumour necrosis factor and therefore to have functional consequences similar to those seen in cells from patients with NLRP7 mutations. The authors also attempted to elucidate the cause of stillbirths observed in 13% of the patients with NLRP7 mutations by examining available placentas of the stillborn babies and live births from patients with mutations or rare NSVs. A number of severe to mild placental abnormalities were found, all of which are known risk factors for perinatal morbidity. CONCLUSIONS: The authors recommend close follow-up of patients with NLRP7 mutations and rare NSVs to prevent the death of the rare or reduced number of babies that reach term.