Engineering improved T cell receptors using an alanine-scan guided T cell display selection system

Abstract

T cell receptors (TCRs) on T cells recognize peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells and this interaction determines the T cell immune response. Due to negative selection, naturally occurring TCRs bind self (tumor) peptides with low affinity and have a much higher affinity for foreign antigens. This complicates isolation of naturally occurring, high affinity TCRs that mediate more effective tumor rejection for therapeutic purposes. An attractive approach to resolve this issue is to engineer high affinity TCRs in vitro using phage, yeast or mammalian TCR display systems. A caveat of these systems is that they rely on a large library by random mutagenesis due to the lack of knowledge regarding the specific interactions between the TCR and pMHC. We have focused on the mammalian retroviral display system because it uniquely allows for direct comparison of TCR-pMHC-binding properties with T-cell activation outcomes. Through an alanine-scanning approach, we are able to quickly map the key amino acid residues directly involved in TCR-pMHC interactions thereby significantly reducing the library size. Using this method, we demonstrate that for a self-antigen-specific human TCR (R6C12) the key residues for pMHC binding are located in the CDR3β region. This information was used as a basis for designing an efficacious TCR CDR3α library that allowed for selection of TCRs with higher avidity than the wild-type as evaluated through binding and activation experiments. This is a direct approach to target specific TCR residues in TCR library design to efficiently engineer high avidity TCRs that may potentially be used to enhance adoptive immunotherapy treatments.

Figure 1

Alanine scanning of R6C12 TCR indicates the CDR3β region to be more important for TCR-pMHC binding We used an alanine scanning screening method (CDR3α - light gray and CDR3β - dark gray) to map key amino acid residues in the TCR-pMHC binding interface. A. Mean fluorescence intensities (MFI) of PE HLA-A2-p209-2M tetramer binding for the indicated clones was quantified via flow cytometry. TCRs were expressed on hCD8 58−/− hybridoma cells. B. IL-2 cytokine production for the indicated clones after 20h incubation with T2 cells expressing A2Kb loaded with 1μM gp209-2M peptide. IL-2 cytokine release into the supernatant was quantified via ELISA. Experiments repeated three times, and a representative experiment is shown.

Figure 2

Kinetic features of wild-type and alanine mutated TCRs binding pMHC show similar trends as cell surface determined affinity binding Steady-state surface plasmon resonance experiments determines the affinities of alanine substituted TCRs and wild-type TCR for gp209-2M/HLA-A2*0201. 500 RU of the indicated soluble biotin-labeled TCRs was immobilized to a streptavidin sensor chip. Each data point represents the value of recorded experimental RU (response units) when injecting increasing amounts of gp209-2M/HLA-A2*0201 and the corresponding buffer baseline surface value of the reference flow cell was subtracted. n.d. = not determined.

Figure 3

Selection of T cell clones with enhanced T-cell activity from a R6C12 CDR3α library A. Analysis of T cell hybridoma transduced with the R6C12-CDR3α library. hCD8+, 58−/− TCR-negative T cell hybridoma were transduced with retrovirus generated from the R6C12-CDR3α library and populations were dual-stained with anti-cTCR APC mAb (Clone H57-597) and HLA-A2-gp209-2M tetramer using a MoFlo sorter. Cells were sorted once. B. Single cell sorted selected T cell hybridomas expressing mutated TCRs were incubated with 10uM gp209-2M -loaded T2 A2Kb cells for 24 hr and their level of IL-2 cytokine production was evaluated. A subset of clones were compared to the WT (green) and placed in color coded categories characterized by making less cytokine than the WT, 1-2, 2-4, or 4-10 times more cytokine than the WT.

Figure 4

Identification of specific and cross-reactive TCR clones T hybridoma cell populations of clonally expanded CDR3α mutants were incubated for 24h with T2 A2Kb cells and A. varying indicated concentrations of gp209-2M, B. no peptide (top); irrelevant Mart 1 (27L) (10 μM) peptide (middle) or no peptide and no T2 A2Kb cells (bottom). Secreted IL-2 was quantified using IL-2 ELISA. Specific clones (S) and cross-reactive clones (C) are defined as those that do not and do respectively, secrete IL-2 in the absence of relevant peptide, respectively. Error bars represent standard deviation of triplicate repeats. Experiments were repeated twice.

Figure 5

Cytokine production correlates with half-life and avidity for specific clones Half-life and binding avidity was determined via tetramer dissociation and binding assays to determine half life (t_1/2) and the avidity of binding, respectively, for the panel of representative specific (gray diamonds), cross-reactive (red diamonds), and WT TCR (black diamonds) expressed on hybridoma cells. IL-2 cytokine production was correlated to the experimentally determined dissociation half life (t_1/2) and tetramer avidity. Data was fitted using linear regression to derive R-squared values when including (A) or excluding (B) cross-reactive clones. The half-life value was calculated from the equation: t_1/2 = ln2/slope. Tetramer avidity is determined as the mean fluorescence intensity (MFI) of gp209-2M-HLA-A2 tetramer staining. (C) Correlation between TCR avidity and half-life for CDR3α engineered and wild-type TCRs.