GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling

Michele Procópio Machado; Matheus Henrique Dos Santos; Júlia Garcia Guimarães; Gabriela Yamazaki de Campos; Patrícia Kellen Martins Oliveira Brito; Camilly Melo Garcia Ferreira; Caroline Patini Rezende; Natália Fernandes Frota; Sandro Gomes Soares; Pappanaicken R Kumaresan; Marcos Roberto Lourenzoni; Thiago Aparecido da Silva

doi:10.1080/21655979.2023.2281059

GXMR-CAR containing distinct GXM-specific single-chain variable fragment (scFv) mediated the cell activation against Cryptococcus spp. And had difference in the strength of tonic signaling

Bioengineered. 2023 Dec;14(1):2281059. doi: 10.1080/21655979.2023.2281059. Epub 2023 Nov 18.

Authors

Affiliations

¹ Department of Cellular and Molecular Biology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao, Sao Paulo, Brazil.
² Department of Biochemistry and Immunology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao, Sao Paulo, Brazil.
³ Federal University of Ceara (UFC), Fortaleza, Ceara, Brazil.
⁴ Department of Biochemistry, University of Cambridge, Cambridge, UK.
⁵ Department of Hematopoietic Biology and Malignancy, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
⁶ Fundação Oswaldo Cruz Ceará (Fiocruz-CE), Research Group on Protein Engineering and Health Solutions (GEPeSS), Eusébio, Ceara, Brazil.
⁷ Department of Clinical Analysis, School of Pharmaceutical Sciences in Araraquara, Sao Paulo State University, Araraquara, Sao Paulo, Brazil.

Abstract

Cryptococcus spp. has a polysaccharide capsule composed of glucuronoxylomannan-GXM, a major virulence factor that can prevent the recognition of fungi by immune cells. Chimeric Antigen Receptor (CAR) redirects T cells to target Cryptococcus spp. as previously demonstrated by a CAR specific to GXM, GXMR-CAR. The current study evaluated the strength of the signal transduction triggered by GXMR-CAR, composed of a distinct antigen-binding domain sourced from a single-chain variable fragment (scFv). GXM-specific scFv derived from mAbs 2H1 and 18B7, 2H1-GXMR-CAR and 18B7-GXMR-CAR, respectively, were designed to express CD8 molecule as hinge/transmembrane, and the costimulatory molecule CD137 (4-1BB) coupled to CD3ζ. The 2H1-GXMR-CAR or 18B7-GXMR-CAR Jurkat cells recognized soluble GXM from C. gattii and C. neoformans, and the levels of IL-2 released by the modified cells did not differ between the GXMR-CAR constructs after exposure to Cryptococcus spp. 18B7-GXMR-CAR triggered tonic signaling was more pronounced in modified Jurkat cells, and a protein kinase inhibitor of the Src family (dasatinib) significantly reduced GXMR-CAR tonic signaling and inhibited cell activation against ligands. 18B7 scFv showed a structural modification of the variable heavy (VH) chain that clarified the difference in the strength of tonic signaling and the level of cell activation between 2H1-GXMR-CAR and 18B7-GXMR-CAR. GXMR-CAR constructs induced T-cell activation against clinical isolates of Cryptococcus spp. and serum from patients with cryptococcosis induced high levels of IL-2, mainly in cells modified with 18B7-GXMR-CAR. Thus, 18B7-GXMR-CAR and 2H1-GXMR-CAR mediated T cell activation against Cryptococcus spp. and 18B7 and 2H1 scFv influenced the strength of tonic signaling.

Keywords: Chimeric antigen receptor; GXM; T cells; cryptococcus spp; tonic signaling.

Plain language summary

2H1-GXMR-CAR and 18B7-GXMR-CAR are efficiently expressed on the cell surface;2H1-GXMR-CAR and 18B7-GXMR-CAR redirected T cells toward the ligands;18B7-GXMR-CAR provided highest levels of tonic signaling;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR;Binding pocket of 18B7 scFv favored the tonic signaling triggered by GXMR-CAR.

MeSH terms

Cryptococcus neoformans* / chemistry
Humans
Interleukin-2
Polysaccharides / chemistry
Receptors, Chimeric Antigen*
Signal Transduction
Single-Chain Antibodies*

Substances

Single-Chain Antibodies
Receptors, Chimeric Antigen
Interleukin-2
Polysaccharides

Grants and funding

This research was funded by São Paulo Research Foundation (FAPESP) (grant nos. 2018/18538-0; 2019/26074-7; 2021/07174-0; 2021/02758-4; 2020/16738-2; 2020/11307-3; 2020/09113-6).