Skip to main content
. 2021 Sep 20;12:686439. doi: 10.3389/fimmu.2021.686439

Figure 1.

Figure 1

Generation of a grafted A2-CAR. (A) Grafting strategy comparing the original VH and VL chain sequences of the SN607D8 hybridoma and of the Herceptin (trastuzumab) 4D5 scaffold. The grafted amino acid sequences are shown. The sequences of SN607D8 and 4D5 HER2 were aligned using the software Jalview and the level of conservation (C) and quality (Q) of each amino acid between SN607D8 and 4D5 sequences were compared. Conservation reflects similarity of the physicochemical properties of amino acid residues. Identical residues are shown as light-yellow columns and residues with more dissimilar physicochemical properties are marked with darker column colors. Quality measures the likelihood of observing a mutation in any particular amino acid residue position (38). CDRs were predicted using Paratome (29). (B) The conformation of the grafted antibody was predicted with ABodyBuilder (39) and displayed using PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA). (C) EGFRt and MYC-tag expression on Day 6 of culture of human Tregs transduced with the grafted A2-CAR-2A-EGFRt lentivirus. (D) On Day 9, A2-CAR Tregs were cultured for another 48 hours alone, with anti-CD3/CD28 beads, or with irradiated (4000 rad) parental A2– K562 or A2-expressing K562 cells. CD25, CD71, ICOS, CTLA-4, and FOXP3 expression were analyzed thereafter using flow cytometry. (E) OneLambda FlowPRA Single HLA Antigen bead panels FL1HD01 and FL1HD02. Percentage relative binding of A2-CAR Tregs to each HLA allele was calculated as described in the Materials and Methods section. Plotted averages of at least 5 independent experiments. Red coloring indicates HLA allele beads surpassing the 25% binding threshold to be considered binders. (F) On Day 9, A2-CAR or UT T cells were cultured for another 48 hours alone or with dissociated islet cells from 4 allogeneic donors. Expression of CD71 was analyzed thereafter using flow cytometry. HLA-A and -B alleles expressed by the 4 allogeneic donors are indicated above the histograms. For donors 1, 3, and 4, A2-CAR and UT Tregs were used in the assay and for donor 2, A2-CAR and UT CD4+ Tconv cells are used. Results are a summary of 4 independent experiments using T cells from unrelated healthy donors. scFv, single-chain variable fragment; CDR, complementarity-defining region; A2, HLA-A2; UT, untransduced; Tconv, conventional T cells.