Fine-tuning of T-cell receptor avidity to increase HIV epitope variant recognition by cytotoxic T lymphocytes

Abstract

Objective: T-cell receptor (TCR) gene therapy is an approach being considered for HIV-1, but epitope mutation is a significant barrier. We assessed whether HIV-specific TCR can be modified to have broader coverage of epitope variants by recombining polymorphisms between public clonotype TCR sequences.

Design: Public clonotype TCRs recognizing the same epitope often differ by polymorphisms in their third complementarity determining regions (CDR3). We assessed whether novel combinations of such polymorphisms could improve TCR recognition of epitope variation.

Methods: A TCR recognizing the HLA A*0201-restricted epitope SLYNTVATL (Gag 77-85, SL9) was engineered to have combinations of four polymorphisms in the CDR3 regions compared to another SL9-specific TCR. These novel TCRs were screened for functional avidities against SL9 epitope variants and abilities to mediate cytotoxic T-lymphocyte suppression of HIV-1 containing the same epitope variants.

Results: The TCRs varied modestly in functional avidities for SL9 variants, due to alterations in affinity. This translated to differences in antiviral activities against HIV-1 when functional avidity changes crossed the previously defined threshold required for efficient recognition of HIV-1-infected cells. Higher avidity TCR mutants had generally broader recognition of SL9 variants.

Conclusion: These results indicate that rationally targeted increases in functional avidities can be utilized to maximize the antiviral breadth of transgenic TCRs. In contrast to previously reported random mutagenesis to markedly increase functional avidities, tuning through recombining naturally occurring polymorphisms may offer a more physiologic approach that minimizes the risk of deleterious TCR reactivities.

Figure 1. Specificity and functionality of primary CD8⁺ T cells transduced with TCR 1.9

The lentiviral vector with TCR 1.9 (based on codon-optimized sequences of a TCR recognizing the SL9 epitope presented by A*02) was utilized to transduce CD8⁺ T cells from an HIV-1-uninfected healthy donor. A. The transduced cells were assessed by flow cytometry for expression of the HSA reporter and binding to SL9/A*02 tetramer. B. The transduced cells were seeded at varying concentrations in a 96-well filter plate for IFN-γ ELISpot analysis of reactivity to the SL9 peptide. C. The transduced cells were tested against A*02⁺ T1 target cells labeled with SL9 peptide in a chromium release cytotoxicity assay at an effector to target cell ratio of 10 to 1. D. The transduced cells were cultured for two weeks after stimulation with irradiated T1 cells pulsed with varying concentrations of SL9 peptide, in the presence of irradiated feeder PBMC and IL-2 at 50U/ml, and assessed for percentage of cells bound by SL9/A*02 tetramer.

Figure 2. Functional avidities of CD8⁺ T cells transduced with TCR 1.9 or TCR 1.9 mutants

CD8⁺ T cells from an HIV-1-uninfected control donor were transduced with TCR 1.9 or mutants of TCR 1.9 (listed in Table 1). A. The transduced cells were tested for cytotoxicity against A*02⁺ T1 target cells with varying concentrations of SL9 peptide or SL9 peptide variants. Three representative curves for different TCRs against the Consensus B SL9 peptide are shown. B.The SD₅₀ values for each TCR against each peptide are shown. C-F. From the data in B, the mean differences and standard deviations for the change in SD₅₀ (log₁₀ pg/ml) for all comparisons of TCRs differing by only the indicated mutation of interest are plotted. For example, the value plotted for “A1” is the mean for all comparisons of TCR that differ only by the absence/presence of A1 (i.e. A1 versus wild type TCR 1.9, A1A2 versus A2, A1A3 versus A3, A1A2A3 versus A2A3, A1B versus B, A1A2B versus A2B, A1A3B versus A3B, and A1A2A3B versus A2A3B). C. Mean differences for SL9 consensus B sequence. D. Mean differences for SL9 V82I/T84V. E. Mean differences for SL9 Y79F. F. Mean differences for SL9 V82I.

Figure 3. Varying structural avidity of TCR 1.9 mutants for the consensus B SL9 epitope

TCR-transduced Jurkat cells were evaluated for structural avidity through determination of SL9/A*02 tetramer binding. A. Representative equilibrium binding curves (tetramer concentration versus mean fluorescence intensity) are shown for TCR 1.9 and mutants A2A3B and A1. B. The calculated K_D values from tetramer binding curves are plotted for each of the TCR 1.9 mutants. C. The effects of individual TCR mutations on K_D are calculated by comparing all TCRs differing by a single mutation. D. For all TCR mutants, the functional avidities (SD₅₀) determined in Figure 2B are plotted against the structural avidities (K_D).

Figure 4. Varying antiviral activities of CTLs transduced with TCR 1.9 mutants against HIV-1 with SL9 variants

The TCR transduced CD8⁺ T cells were assessed for their antiviral activity in virus suppression assays against replicating HIV-1 containing SL9 variants (Consensus B,V82I/L84V, or Y79F). A. The efficiency of log₁₀ suppression of each TCR against each strain of HIV-1 is indicated. B. The antiviral efficiency of each TCR against each strain of HIV-1 is plotted against the corresponding functional avidity of the TCR for the SL9 variant.