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High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules

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Science Immunology  19 Jul 2019:
Vol. 4, Issue 37, eaav0860
DOI: 10.1126/sciimmunol.aav0860
  • Fig. 1 Overview of DS-A*02:01 production and usage for affinity measurements.

    (A) Expression plasmids of heavy chain and β2m are transfected into E. coli and proteins of interest expressed in inclusion bodies. HLA monomers are purified using size exclusion. (B) Functionally empty DS-A*02:01 molecules can be loaded with peptide ligands by incubation at room temperature. For affinity measurements, they can be immobilized on functionalized biosensors, e.g., by biotin streptavidin interaction, and used to record association and dissociation of TCRs or TCR-like molecules.

  • Fig. 2 Association and dissociation behavior of 1G4 TCR with different pMHCs.

    Raw data are displayed in black and curve fittings are in red. All measurements performed as 1:2 analyte dilution series starting at 24 μM. (A) Binding curve of the 1G4 TCR against immobilized DS-A*02:01 ESO 9V pMHC. (B) Binding curve of the 1G4 TCR against immobilized WT-A*02:01 ESO 9V pMHC. (C) Binding curve of the 1G4 TCR against immobilized empty DS-A*02:01. (D) Binding curve of the 1G4 TCR against immobilized DS-A*02:01 SL9 pMHC.

  • Fig. 3 Crystal structure of DS-A*02:01 ESO 9V and WT-A*02:01 ESO 9V in complex with 1G4.

    (A) Overlay of WT and DS-A*02:01 structure with focus on peptide and amino acid side-chain orientation. (B) Close-up of the F-pocket and the introduced disulfide bond between α1 and α2. (C) Overlay of the 1G4 CDR loops interacting with the peptide and the MHC backbone. (D) Overlay of both crystal structures from a lateral perspective.

  • Fig. 4 Affinities of the SV9 specific bs-868Z11-CD3 bsTCR with different MHC monomers and peptide ligands.

    (A) Binding curve of bs-868Z11-CD3 against immobilized DS-A*02:01 SL9 pMHC. Raw data are displayed in black and curve fittings are in red. Measured using 1:2 analyte dilution series starting at 500 nM. (B) Binding curve of bs-868Z11-CD3 against immobilized WT-A*02:01 SL9 pMHC. Raw data are displayed in black and curve fittings are in red. Measured using 1:2 analyte dilution series starting at 500 nM. (C) Correlation between affinities measured using DS-A*02:01 pMHCs or WT-A*02:01 pMHCs generated using UV exchange. Kd values were plotted for 140 different peptide ligands generated using both methods and measured during successive experiments. Kd values were fitted using 500 and 158 nM analyte concentrations. Peptides were included if peak signal levels reached at least 0.05 nM for both concentrations and individual fits had a R2 above 0.9. The in-picture R2 is the calculated correlation coefficient, and dashed line represents optimal 1:1 ratio.

  • Fig. 5 Binding motif of bs-868Z11-CD3 generated using DS-A*02:01– generated positional scanning library as soluble analyte and immobilized bsTCR.

    Measurements were performed using four soluble analyte concentrations. Interactions without fittable curves were assigned a Kd value of 5 μM. Soluble analyte concentration range produced by 1:3.16 analyte dilution series starting at 500 nM. (A) Heatmap depicting bs-868Z11-CD3 affinities for the respective SLYNTVATL mutants from the positional scanning library. (B) Visualization of the binding motif as Seq2Logo graph. Size of individual letters inversely represents measured affinity for the respective amino acid at this position, calculated using the inverse Kd value divided by 108 and the PSSM-Logo algorithm. (C) Binding curve of bs-868Z11-CD3 bsTCR against ALYNVLAKV DS-A*02:01 pMHC. Measured using 1:3.16 analyte dilution series starting at 500 nM. Raw data are displayed in black and curve fittings are in red.

  • Fig. 6 Result of coincubation assays with peptide-loaded target cells, Jurkat effector cells, and bs-868Z11-CD3 at six different concentrations.

    (A) Measured fold induction above background for Jurkat effector cells activated at different concentrations of bs-868Z11-CD3 against SL9 peptide–loaded T2 target cells. Error bars represent biological triplicates. (B) Measured binding affinities for peptide ligands from the positional scanning library and their respective bsTCR activation threshold and the lowest bsTCR concentration necessary to induce Jurkat effector cell activation threefold over background based on bioluminescence. Peptides are grouped on the basis of the location of the exchange in the wild-type sequence. (C) Measured binding affinities for peptide ligands from the positional scanning library and their respective NetMHC-predicted binding rank. Peptides are grouped on the basis of the bsTCR activation threshold. (D) Measured binding affinities for the cross-reactive peptide ligand candidates and their respective bsTCR activation threshold. (E) Measured fold induction above background for Jurkat effector cells stimulated at different concentrations of bs-868Z11-CD3 against ALYNVLAKV peptide–loaded T2 target cells. Error bars represent biological triplicates.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/37/eaav0860/DC1

    Methods

    Fig. S1. Binding of multiple different soluble TCRs and bsTCR bs-868Z11-CD3 to nonloaded DS-A*02:01 or DS-A*02:01 loaded with an irrelevant peptide.

    Fig. S2. Analysis of DS-A*02:01 peptide receptiveness after different storage durations at −80°C measured by fluorescence anisotropy.

    Fig. S3. Illustration of bsTCR bs-868Z11-CD3 construct.

    Fig. S4. Rmax values reported for immobilized DS-A*02:01 and UV exchange generated WT-A*02:01 pMHCs.

    Fig. S5. Analysis of UV exchange efficiency and Octet measurement results for 28 different peptides selected from SLYNTVATL-based positional scanning library.

    Fig. S6. Octet binding kinetics measurements for DS-A*02:01 SLYNTVATL pMHC with immobilized bs-868Z11-CD3 directly after exchange and after 2 weeks of storage at 4°C.

    Fig. S7. Flow cytometric peptide binding assay with an anti-human HLA-A2 antibody staining of T2 cells after exogenous peptide loading.

    Table S1. Data collection and refinement statistics 1G4/DS-A*02:01/ESO 9V.

    Table S2. bs-868Z11-CD3 binding affinity against SV9 peptide SLYNTVATL and peptides from positional scanning library.

    Table S3. Cross-reactive peptide ligand search motif for bs-868Z11-CD3 based on the binding affinities measured using the positional scanning library.

    Table S4. bs-868Z11-CD3 binding affinity for selected peptide ligands identified on the basis of the bs-868Z11-CD3 binding motif.

    Table S5. Raw data (Excel file).

  • Supplementary Materials

    The PDF file includes:

    • Methods
    • Fig. S1. Binding of multiple different soluble TCRs and bsTCR bs-868Z11-CD3 to nonloaded DS-A*02:01 or DS-A*02:01 loaded with an irrelevant peptide.
    • Fig. S2. Analysis of DS-A*02:01 peptide receptiveness after different storage durations at −80°C measured by fluorescence anisotropy.
    • Fig. S3. Illustration of bsTCR bs-868Z11-CD3 construct.
    • Fig. S4. Rmax values reported for immobilized DS-A*02:01 and UV exchange generated WT-A*02:01 pMHCs.
    • Fig. S5. Analysis of UV exchange efficiency and Octet measurement results for 28 different peptides selected from SLYNTVATL-based positional scanning library.
    • Fig. S6. Octet binding kinetics measurements for DS-A*02:01 SLYNTVATL pMHC with immobilized bs-868Z11-CD3 directly after exchange and after 2 weeks of storage at 4°C.
    • Fig. S7. Flow cytometric peptide binding assay with an anti-human HLA-A2 antibody staining of T2 cells after exogenous peptide loading.
    • Table S1. Data collection and refinement statistics 1G4/DS-A*02:01/ESO 9V.
    • Table S2. bs-868Z11-CD3 binding affinity against SV9 peptide SLYNTVATL and peptides from positional scanning library.
    • Table S3. Cross-reactive peptide ligand search motif for bs-868Z11-CD3 based on the binding affinities measured using the positional scanning library.
    • Table S4. bs-868Z11-CD3 binding affinity for selected peptide ligands identified on the basis of the bs-868Z11-CD3 binding motif.

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