Fig. 1: Strategic mutations on complementarity determining regions (CDR) of the JOVI.1 antibody result in switched specificity from T-cell receptor constant β chain (TRBC)1 to TRBC2, allowing for dual TRBC staining by flow cytometry.

A Simplified model of JOVI.1 antibody binding to TRBC1 (top), showing key amino acid residues on both molecules responsible for the discriminative binding to one isoform only. Rationally-designed antibody mutations on CDR1 (T28K and Y32F) and CDR3 (A96N and N99M) of JOVI.1’s variable heavy chain (VH) domain results in switched specificity to TRBC2 (bottom). B T-cell receptor αβ gene rearrangement showing the random selection of 1 of 2 mutually exclusive TRBC genes. Anti-TRBC1 (JOVI.1) and anti-TRBC2 antibodies can be utilized in conjunction to assess for TRBC-restriction by flow cytometry as a surrogate for T-cell clonality. C Half maximal effective concentration (EC50) measurements and dissociation constant (KD) estimations (non-linear regression one site binding analysis) of anti-TRBC2 (blue and light blue) or JOVI.1 (red and pink) binding to TRBC1-positive (triangles) or TRBC2-positive (circles) Jurkat cells; as assessed by geometric mean fluorescence intensity (gMFI) using an anti-mouse IgG fluorescent-labeled secondary antibody. D Flow cytometry histograms showing the specificity of the anti-TRBC2 antibody for TRBC2-positive (top) as compared to TRBC1-positive (bottom) Jurkat cells; and stability of an Alexa Fluor(AF)-647-conjugated anti-TRBC2 antibody. Secondary (2^ari) antibodies were AF-647 conjugates. A humanized JOVI.1 antibody (Hu-JOVI.1) is also shown as control.