Patent ID: 12188932

SEQ ID NOs 1 to 5 and 16 to 325 show peptide sequences as used in the examples, below.

EXAMPLES

1. Peptide Synthesis

All peptides were generated in house using standard Fmoc chemistry with a Syro II peptide synthesizer. Peptides were subsequently analyzed using HPLC and had an average purity of 74%. UV-light sensitive peptides contained a light-sensitive building block with a 2-nitrophenylamino acid residue. The dipeptide GM was procured from Bachem. Before use peptides were solved in DMSO (Sigma, Cat. Nr. 41640), 0.5% TFA (Sigma, Cat. Nr. T6508) at concentrations ranging from 2 mg/ml to 10 mg/ml depending on the desired use case.

2. Generation of MHC Complexes by Refolding and Purification

Recombinant HLA-A*02:01 wild type (WT-A*02:01, SEQ ID NO: 322) or disulfide modified HLA-A*02:01 heavy chains with C-terminal BirA signal sequences and human β2m light chain were produced inEscherichia colias inclusion bodies and purified as previously described (2). HLA-A*02:01 complex refolding reactions were performed as previously described with minor modifications (Saini et al 2013). In brief, WT-A*02:01 or disulfide-modified HLA-A*02:01 heavy chains, β2m light chain and peptide were diluted in refolding buffer (100 mM Tris-Cl pH 8, 0.5 M arginine, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione) and incubated for 2 to 8 days at 4° C. while stirring before concentration. The concentrated protein was purified by size exclusion chromatography (SEC) in 20 mM Tris-HCl, pH 8/150 mM NaCl on an ÄKTAprime system (GE Healthcare) using a HiLoad 26/600 200 pg column (GE Healthcare). Peak fraction was either concentrated directly to 2000 μg/ml, aliquoted and frozen at −80° C. or biotinylated by BirA biotin-protein ligase (Avidity) overnight at 4° C. according to the manufacturer's instructions and subjected to a second gel-filtration before final concentration to 2000 μg/ml, aliquotation and storage at −80° C.

To produce HLA-A*02:01 wild type peptide-MHC complexes 9mer (full length) peptides or UV-light sensitive 9mer peptides (full length) were added to the refolding buffer at a concentration of 30 μM. To produce empty Y84C/A139C HLA-A*02:01 (SEQ ID NO: 323) complexes the dipeptide GM was added to the refolding buffer at a concentration of 10 mM. To produce F22C/S71C HLA-A*02:01 (SEQ ID NO: 324) complexes no peptide was added to the refolding buffer. To produce F22C/S71C W51C/G175C HLA-A*02:01 (SEQ ID NO: 325) complexes no peptide was added to the refolding buffer.

TABLE 1Refolding MethodFull LengthPeptideDipeptideWithout PeptideHLA-A*02:01 wild+−−type (SEQ ID NO:322)HLA-A*02:01++−84/139 (SEQ IDNO: 323)HLA-A*02:01 22/71n.d.n.d.+(SEQ ID NO: 324)HLA-A*02:01 22/71n.d.n.d.+51/175 (SEQ IDNO: 325)+: Protein is refoldable;− protein is not refoldable.

Table 1 below shows the refolding methods of the different disulfide-modified HLA-A*02:01 molecules and the WT-A*02:01 molecule:

3. Generation of Peptide Exchanged HLA-A*02:01 pMHC Complexes Using UV Mediated Peptide Ligand Exchange or Empty Disulfide-Modified HLA-A*02:01 Molecules

Peptide exchange reactions with UV-light cleavable peptides were performed as previously described. In short desired nonamer peptides were mixed with biotinylated UV light-sensitive pMHC complexes at 100 to 1 molar ratio and subjected to at least 30 minutes of 366 nm UV light (Camag).

Peptide loading reactions with empty disulfide-modified HLA-A*02:01 MHC complexes were performed by addition and mixing of desired peptides of at least a 100 to 1 molar ratio to the monomer solution and 5-minute incubation at room temperature.

4. Soluble TCR Production

Soluble TCRs were produced as previously described (20). In short TCR alpha and TCR beta chain constructs were expressed separately inEscherichia colias inclusion bodies and purified. TCR alpha chains are mutated at position 48 by replacing a threonine with a cysteine and TCR beta chains at position 57 by replacing a serine with a cysteine to form an inter-chain disulfide bond.

5. bsTCR Design and Production

The bs-868Z11-CD3 molecule was generated by linking the scTv 868Z11 to the C-terminus of the F(ab′)-domain of a humanized antiCD3-antibody (22, 23). To this end the Vβ-domain of the scTv was directly fused to the upper CH2-region derived from human IgG2 (APPVAG, SEQ ID NO: 2). Cysteine-knock-outs C226S and C229S within the hinge prevent the formation of F(ab)2molecules. HCMV-driven expression vectors coding either for the construct described above or the light chain of the humanized antiCD3-antibody were transiently co-transfected in EXPICHO cells (Thermo). After 12 days supernatant was processed by tandem chromatography (protein L followed by preparative size exclusion, GE Biosciences) and highly pure monomeric bsTCR was formulated in PBS

6. OctetRED Based Bio-Layer Interferometry Kinetic Affinity Measurements

The affinity of sTCR or bsTCR molecules for different pMHC complexes was measured on an OctetRED 384 system (Pall Fortebio) using kinetic or steady state binding analysis. All analytes or ligands were diluted to their final concentration in kinetics buffer (PBS, 0.1% BSA, 0.05% TWEEN 20) if not specified otherwise. All biosensors were hydrated for at least 10 minutes in kinetics buffer before use. Loadings and measurements were performed in 384 tilted well plates (Pall Fortebio) with at least 40 μl at a 3 mm sensor offset. Plate temperature was set at 25° C. and shaker speed at 1000 rpm. To allow inter-step correction baselines before association phases and the following dissociation phase were performed in the same well. Kinetics buffer was used as dissociation buffer with DMSO at an appropriate concentration added if necessary to match the analyte composition.

In the case of pMHC immobilization dip and read streptavidin (SA; Pall Fortebio Cat. Nr. 18-5021) biosensors were used to immobilize biotinylated pMHC monomers at a presumed concentration of 25 μg/ml for 60 seconds followed by a 60 seconds baseline and association and dissociation phases of 60 seconds each if not specified otherwise.

In the case of bsTCR immobilization dip and read anti-human Fab-CH1 2ndgeneration (FAB2G; Pall Fortebio Cat. Nr. 18-5127) biosensors were used to immobilize bsTCR molecules at a concentration of 100 μg/ml for 60 seconds, followed by a 15 seconds baseline and association and dissociation phases of 60 seconds each if not specified otherwise. FAB2G biosensor were regenerated up to 4 times by incubating the loaded biosensor for 5 seconds each in 10 mM Glycine pH1.5 and kinetics buffer consecutively for three times. FAB2G were also pre-conditioned that way before their first ligand immobilization.

All sensorgrams were analyzed using the OctetRED software “Data Analysis HT” version 10.0.3.7 (Pall Fortebio). Raw sensor data was aligned at the Y axis by aligning the data to the end of the baseline step and inter-step correction was used to align the start of the dissociation to the end of the association phase. No Savitzky-Golay filtering was applied. Resulting sensorgrams were then fitted using a 1:1 Langmuir kinetics binding model.

7. Cell Lines

The TAP-deficient HLA-A*02:01 expressing cell line T2 was procured from ATCC (CRL-1992) and cultured in RPMI Medium 1640 GLUTAMAX (Thermo Fisher, Cat. Nr. 61870010) Supplemented with 10% heat inactivated FCS (Life Technologies, Cat. Nr. 10270106) and the antibiotics penicillin and streptomycin (Biozym, Cat. Nr. 882082, 100 μg ml−1each) up until passage number 16 if necessary. The GLORESPONSE NFAT-luc2 Jurkat cell line was procured from Promega (Cat. Nr. CS1764) at passage number 6 and cultured in RPMI Medium 1640 GLUTAMAX (Thermo Fisher, Cat. Nr. 61870010) supplemented with 10% heat inactivated FCS (Life Technologies, Cat. Nr. 10270106), 1% Sodium Pyruvate (C.C.Pro, Cat. Nr. Z-20M) and the antibiotics hygromycin B (Merck Millipore, Cat. Nr. 400052, 200 μg/ml), penicillin and streptomycin (Biozym, Cat. Nr. 882082, 100 μg/ml each) up until passage number 14, if necessary.

8. T Cell Activation Assay

T cell activation assays using GloResponse™ NFAT-luc2 Jurkat cells and peptide loaded T2 target cells were performed according to manufacturer instructions. In short, T2 cells were harvested from continuous cell culture, washed and resuspended in T2 culture medium at a concentration of 3.3×106cells/ml and transferred to 96 well round bottom plates (Corning Costar®, Cat. Nr. 3799). Peptide in DMSO, 0.5% TFA was added to a final concentration of 100 nM and the suspension incubated for 2 to 3 hours at 37° C., 5% CO2. bsTCR formulated in PBS was diluted in T2 culture medium to desired concentration and 25 μl of the respective dilution was distributed to white 96 well flat bottom plates (Brand, Cat. Nr. 781965). GloResponse™ NFAT-luc2 Jurkat cells were harvested from continuous cell culture, washed and resuspended in T2 culture medium at a concentration of 3.0×106cells ml−1and 25 μl of the cell suspension was distributed to the white 96 well flat bottom plates with bsTCR dilutions. After peptide loading T2 cells were resuspended and 25 μl distributed to the white 96 well flat bottom plates with bsTCR dilutions and GloResponse™ NFAT-luc2 Jurkat cells for a final effector to target ratio of 1:1 (75.000 cells each). Fully assembled plates were mixed for 5 minutes at 300 rpm on a plate shaker and the incubated for 18 to 20 h at 37° C., 5% CO2. After the incubation period 75 μl of Bio-Glo™ luciferase reagent was added to each well and the plates incubated for minutes at 300 rpm on a plate shaker in the dark before reading luminescence at a 0.5 second integration time with a Synergy2 plate reader (Biotek). Luminescence as measured in relative light units (RLU) was converted to fold induction for each well by dividing measured RLU through those of control wells.

9. Crystallization and Imaging

The Y84C/A139C HLA-A*02:01-SLLMWITQV (SEQ ID NO: 4) complex and the 1G4 TCR were concentrated and mixed in a 1:1 ratio to achieve a concentration of 7 mg/ml for crystallization. A sitting drop vapor diffusion experiment resulted in crystals in the presence of a mother liquor containing 0.1 M ammonium acetate, 0.1 M bis-tris (pH 5.5), and 17% polyethylene glycol (PEG) 10,000. A single crystal was transferred to a cryoprotectant solution containing 0.1 M ammonium acetate, 0.1 M bis-tris (pH 5.5), 20% (w/v) PEG 10,000, and 10% glycerol. The crystal was mounted and cryocooled at 100 K on the EMBL P14 beamline at Deutsche Elektronen-Synchrotron containing an EIGER 16M detector. An x-ray dataset was collected to a resolution of 2.5 Å (Table 2).

TABLE 2Data collection and refinement statistics 1G4/Y84C/A139CHLA-A*02:01/SLLMWITQV1G4/Y84C/A139C HLA-A*02:01/SLLMWITQVData collectionSpace groupP21Cell dimensionsa, b, c (Å)75.44, 53.67, 121.74α, β, γ (°)90.0 98.0 90.0Resolution (Å)2.50 (2.60-2.50)*Rpim0.037 (0.69)I/σI9.6 (1.1)CC (1/2)100.0 (0.68)Completeness (%)99.3 (99.1)Redundancy4.8 (5.0)RefinementResolution (Å)30-2.50No. reflections33552Rwork/Rfree0.229 (0.273)No. atomsProtein3180Ligand/ion19Water589B-factorsProtein98.1Ligand/ion97.8Water66.2R.m.s. deviationsBond lengths (Å)0.002Bond angles (°)0.47

The data were processed with XDS and scaled with AIMLESS (35, 36). Molecular replacement was performed using MOLREP with the coordinates of the TCR portion of the native complex first, followed by the pMHC [Protein Data Bank (PDB) 2BNR], and the structure was refined with REFMAC5 (37, 38). The engineered disulfide bond was manually built with Coot (39). The structure was refined to an R factor of 22.9% (Rfreeof 27.3%). MolProbity was used to validate the geometry and indicated that 93.9% of the residues were in the allowed regions of the Ramachandran plot [with one glycine residue (Gly143) in the disallowed regions] (40).

10. Motif-Based Identification of Potentially Cross-Reactive Peptide Ligands

Searches for nonamer peptide ligands matching one of the potential combinations allowed by the search motif were performed using the NCBI human protein database. This database covers all nonredundant GenBank CDS translations, as well as records from PDB, SwissProt, PIR, and PRF but excluding environmental samples from the whole-genome shotgun projects. The database was directly acquired from the NCBI servers.

11. Seq2Logo Generation

Seq2Logos visualizing the binding motif were created by taking the inverse value of measured Kdvalues for the respective interaction and dividing them by 108. These values were assembled in the form of a position-specific scoring matrix file and processed using the PSSM-Logo type at the Seq2Logo online resource of the Denmark Technical University Bioinformatics department (27).

12. Peptide Binding Measured by Fluorescence Anisotropy

Peptide binding was evaluated in fluorescence anisotropy assay with 300 nM of purified refolded Y84C/A139C HLA-A*02:01. 100 nM of the fluorescently labeled high-affinity peptide NLVPKFITCVATV (Genecast) was added to the folded Y84C/A139C HLA-A*02:01 and kinetic measurements were performed with Tecan Infinite M1000 PRO (Tecan, Crailsheim, Germany) multimode plate reader measuring anisotropy (FITC λex=494 nm, λem=517 nm). Y84C/A139C HLA-A*02:01 were either used directly after refolding or preserved at −80° C. in storage buffer (10% Glycerol, 50 mM Tris-HCL, pH 8.0) for the indicated amount of time before measurement. The kinetic measurements were performed at room temperature (22-24° C.) in 50 mM HEPES buffer, pH 7.5. Data was plotted using GraphPad Prism v7.

13. Anti-Beta-2 Microglobulin ELISA

Streptavidin (Molecular Probes, Cat. Nr. S888) at a final concentration of 2 μg/ml in PBS was added to Nunc MAXIsorp plates (Thermo Fisher, Cat. Nr. 439454) and sealed plates incubated over night at room temperature in a damp environment. The following day plates were washed 4 times with washing buffer (PBS, 0.05% TWEEN-20) using a ELx405 plate washer (Biotek). 300 μl blocking buffer (PBS with 2% BSA) was added to each well and sealed plates incubated at 37° C. for 1 hour. Blocking buffer was discarded before adding 100 μl of a 1:100 dilution in blocking buffer of the respective UV exchange pMHC preparation. A standard series ranging from 500 ng/ml to 15.6 ng/ml based on a conventionally refolded pMHC monomer was included on each plate. Edge wells were filled with 300 μl blocking buffer to reduce edge effects and sealed plates were incubated at 37° C. for 1 hour. Plates were again washed 4 times before adding 100 μl anti-beta 2 microglobulin HRP conjugated secondary antibody (Acris, Cat. Nr. R1065HRP) at a final concentration of 1 μg/ml to each well. Sealed plates were incubated at 37° C. for 1 hour. Plates were washed again 4 times with washing buffer before adding 100 μl of room temperature TMB substrate (Sigma, Cat. Nr. T0440) to each well. Plates were incubated for 5 minutes at room temperature before stopping by adding 50 μl 1N H2SO4to each well. Plates were immediately analyzed by reading absorbance at 450 nm for 5 seconds using a Synergy2 plate reader. pMHC concentration was calculated based on standard curve fitting (Log(Y)=A*Log(X)+B) using the Synergy2 software. Data was plotted using GraphPad Prism v7.

14. Flow Cytometric T2 Peptide Binding Assay

The TAP-deficient HLA-A*02:01-expressing cell line T2 was procured from ATCC (CRL-1992) and cultured in RPMI Medium 1640 GLUTAMAX (Thermo Fisher, Cat. Nr. 61870010) Supplemented with 10% heat inactivated FCS (Life Technologies, Cat. Nr. 10270106) and the antibiotics penicillin and streptomycin (Biozym, Cat. Nr. 882082, 100 μg/ml each) up until passage number 16 if necessary. T2 cells were harvested from continuous cell culture, washed and resuspended in T2 culture medium at a concentration of 3.3×106cells/ml and transferred to 96 well round bottom plates (Corning Costar®, Cat. Nr. 3799). Peptide in DMSO, 0.5% TFA was added to a final concentration of 10 μM and the suspension incubated for 2 hours 37° C., 5% CO2. Plates were washed twice with PFEA (PBS, 2% FCS, 2 mM EDTA, 0.01% sodium azide) before addition of 50 μl PE labelled anti-human HLA-A2 (Biolegend, Cat. Nr. 343305) Per well diluted 1:250 with PFEA to a final concentration of 0.8 μg/ml. Plates were incubated at 4° C. for 30 minutes before being washed twice with PFEA. Finally, cells were resuspended in fixation solution (PFEA, 1% formaldehyde) and kept at 4° C. before analysis using an iQue Screener (Intellicyt). T2 cells were gated based on the FSC-A/SSC-A signal and doublets removed using an FSC-H/FSC-A doublet exclusion. The PE channel positive gate coordinates were based on an unstained control. Data was plotted using GraphPad Prism v7.

15. Sequence Alignment

Multiple sequence alignments were performed by using Clustal Omega Multiple Sequence Alignment (ebi.ac.uk/Tools/msa/clustalo/) (Madeira et al. “The EMBL-EBI search and sequence analysis tools APIs in 2019”, Nucleic Acids Research, 47:W636-W641, 2019, doi: 10.1093/nar/gkz268).

16. Statistical Analysis

All data were plotted using the GraphPad Prism software version 7. Correlation between x and y datasets were calculated by computing the Pearson correlation coefficient and were reported as R2using the GraphPad Prism software version 7. R2and X2values for curve fittings of biolayer interferometry binding kinetics measurements were calculated using the Octet RED384 system software DataAnalysis HT version 10.0.3.7.

17. Design and Production of Disulfide-Stabilized Empty HLA-A*02:01 Molecules

Molecular dynamics simulations of empty and peptide loaded MHC class I molecules have indicated that the former has an increased mobility in the F-pocket that accommodates the C-terminus of the peptide ligand (16). In previous studies with the murine MHC class I molecule H-2Kbintroduction of a disulfide bond between opposing residues in the F-pocket by mutating a tyrosine at position 84 and an alanine at position 139 to cysteines was able to stabilize the complex. The mutant could be refolded without full length peptide and was capable of retroactive peptide binding (17, 18).

The inventors hypothesized that the same concept could be applied to the human MHC class I molecule HLA-A*02:01. Modifications resulting in mutations of the tyrosine at position 84 and alanine at position 139 into cysteines were introduced into an HLA-A*02:01 heavy chain expression plasmid. After production as inclusion bodies inE. coli, the heavy chain was incubated with similarly produced β2m but without peptide in refolding buffer. After size exclusion chromatography (SEC), no HLA-A*02:01 associated monomer fraction could be observed compared to a wild type control refolded with a 9mer peptide.

In a second approach, the dipeptide GM was added to the refolding: This dipeptide has a very low affinity for the MHC class I complex and assists the refolding (19). During SEC it dissociates quickly from the binding pocket by buffer exchange against the running buffer, yielding purified empty disulfide-stabilized Y84C/A139C HLA-A*02:01. Empty wild type A*02:01 complexes (WT-A*02:01) could not be produced in the same fashion. WT-A*02:01 complexes can be produced with the dipeptide but denature when attempting to remove the dipeptide by buffer exchange.

The inventors also introduced modifications resulting in mutations of phenylalanine at position 22 and serine at position 71 into cysteines into an HLA-A*02:01 heavy chain expression plasmid. After production as inclusion bodies inE. coli, the heavy chain was incubated with similarly produced β2m but without peptide in refolding buffer. SEC yielded purified empty disulfide-stabilized F22C/S71C HLA-A*02:01 complexes. The inventors also introduced modifications resulting in mutations of phenylalanine at position 22 and serine at position 71 as well as tryptophan at position 51 and glycine at position 175 into cysteines into an HLA-A*02:01 heavy chain expression plasmid. After production as inclusion bodies inE. coli, the heavy chain was incubated with similarly produced β2m but without peptide in refolding buffer. SEC yielded purified empty disulfide-stabilized F22C/S71C W51C/G175C HLA-A*02:01 complexes.

The absence of the dipeptide GM in the purified monomer could be shown by thermal stability analysis through buffer exchange: the empty Y84C/A139C HLA-A*02:01 molecule was less temperature stable (i.e., had a lower melting temperature) than the same molecule still complexed with dipeptide GM (41).

The resulting molecules were either biotinylated at 4° C. overnight and separated from excess biotin by a second SEC run or stored directly at −80° C. prior to use.

18. Peptide Loading and Affinity Measurements Using Soluble TCRs and Wild Type or Disulfide-Modified MHCs

Next, the inventors determined whether the disulfide-modified HLA-A*02:01 molecules were capable of peptide-MHC complex formation and TCR ligand binding. Affinity measurements were performed by bio-layer interferometry (BLI) on an OctetRED 384 using the refolded TCR 1G4 as soluble analyte. This TCR recognizes the HLA-A*02:01 specific peptide SLLMWITQC (ESO 9C, SEQ ID NO: 3) derived from the cancer testis antigen NY-ESO-1 or its synthetic variant SLLMWITQV (ESO 9V, SEQ ID NO: 4) (20,21). Biotinylated Y84C/A139C HLA-A*02:01 was either immobilized directly in its empty state or after a short incubation with the peptide ESO 9V on streptavidin-coated biosensors (FIG.1b). No differences could be detected between peptide incubations of 5 minutes, the minimal time needed to initiate the affinity measurements after assembly, or longer. Further analysis indicated that full exchange was indeed reached within one to two minutes when high peptide concentrations were used. Kinetics were measured across multiple 1G4 concentrations and wild type HLA-A*02:01 directly refolded with ESO 9V served as control.

1G4 TCR binding to either Y84C/A139C HLA-A*02:01 9V or WT-A*02:01 ESO 9V was very similar with respect to sensorgrams and Kds resulting from curve fittings (FIGS.2A and2B). A weak binding signal (but no dissociation) could be detected for the empty immobilized monomer at high concentrations of 1G4 (FIG.2C). This binding could be prevented by subsequently adding a peptide that is not recognized by 1G4 like SLYNTVATL (FIG.2D, SEQ ID NO: 5). The weak signal obtained with empty Y84C/A139C HLA-A*02:01 might be explained by unspecific interactions of the TCR with the empty binding pocket, a state that is typically not encountered by TCRs in vivo. Other A*02:01-restricted soluble TCRs with varying specificities behaved similarly, showing no binding to irrelevantly loaded Y84C/A139C HLA-A*02:01 pMHCs but association to functionally empty molecules, albeit but with a relatively lower response (FIG.11).

19. Correlation Between Disulfide-Modified HLA-A*02:01 and WT-A*02:01 Affinity Measurements for an Affinity Maturated TCR

Having established the usability of the Y84C/A139C HLA-A*02:01 molecule as ligand equivalent to WT-A*02:01 for unmodified TCRs the inventors wanted to expand this analysis towards mutated high affinity TCRs and a larger number of peptide ligands. The inventors employed the maturated single chain TCR (scTv) 868Z11, an affinity maturated variant of a TCR that recognizes the HIV p17 Gag-derived HLA-A*02:01 restricted peptide SLYNTVATL (SL9, SEQ ID NO: 5) (8, 22).

The inventors performed affinity measurements by immobilization of empty or SL9 peptide loaded disulfide-modified HLA-A*02:01 molecules on streptavidin biosensor and measurements against soluble bs-868Z11-CD3, a bsTCR variant of the 868Z11 scTv expressed in fusion with a humanised anti-CD3 antibody (FIG.9)(23). Binding affinity for SL9 disulfide-modified HLA-A*02:01 pMHC complexes using either Y84C/A139C HLA-A*02:01, F22C/S71C HLA-A*02:01 or F22C/S71C W51C/G175C HLA-A*02:01, was similar to the SL9 WT-A*02:01 pMHC produced by performing an UV-light mediated peptide ligand exchange (25) with 2.35 nM and 3.24 nM, respectively (FIG.3aand, alsoFIG.14). No binding was measurable with empty MHC molecules for this bsTCR (FIG.3c) and with irrelevantly loaded Y84C/A139C HLA-A*02:01 complexes at a high molar concentrations of 13.3 μM.

Next, the inventors analysed bs-868Z11-CD3 binding affinities towards a positional scanning library based on the SL9 peptide sequence. This library was created by exchanging an amino acid at one position of the wild type SL9 peptide against the 18 remaining proteinogenic amino acids while maintaining all other positions, resulting in 162 distinct peptides when performed at all positions of the nonamer (cysteine was excluded because of its propensity to dimerize) (24). pMHC complexes were generated by the inventors either by addition to Y84C/A139C HLA-A*02:01 molecules as before or by performing UV-light mediated peptide ligand exchange, a technology used for pMHC complex generation (25). Respective pMHC complexes were immobilized on streptavidin and kinetics measured at two different bs-868Z11-CD3 concentrations. As expected, using alternated peptide ligands resulted in a wide range of different Kds, ranging from undetectable within the sensitivity limits of the chosen setup to comparable or even stronger than the interaction with the unmodified SL9 peptide.

For direct comparison, all measured pMHC complexes were selected that had evaluable signals at both analyte concentrations and curve fittings with R2values of at least 0.9, representative of signals within the selected Kdsensitivity range. Kdvalues for the resulting 140 peptide ligands were very similar across the whole affinity range when plotted against each other, a finding supported by the high correlation coefficient value (FIG.3d). Discrepancies were within 2-fold range for over 90% of the pMHC pairs and 6.82-fold differences at most. Within the group with higher than 2-fold changes a trend towards a larger dissociation constant for measurements with the Y84C/A139C HLA-A*02:01 molecule was observed.

The amount of functional pMHC immobilized on each biosensor expressed by the reported Rmaxvalue for 140 different peptide ligands from the positional scanning library was comparable for both wild-type and disulfide-stabilized pMHCs (correlation coefficient R2=0.9459).

FIG.15shows Kdvalues of a high affinity TCR to different pMHC complexes. In each case the Kdof the WT-A*02:01 molecules or the Y84C/A139C HLA-A*02:01 molecule is shown on the X-axis and the Kdof the two different disulfide-modified HLA-A*02:01 MHC molecules is shown on the y-axis and each dot represents one of different peptides loaded in the MHC molecule. In each square inFIG.15the following peptides are represented:

A: HIV-005 WT(SLYNTVATL, SEQ ID NO: 5)B: HIV-005 6I(SLYNTIATL, SEQ ID NO: 110)C: HIV-005 8V(SLYNTVAVL, SEQ ID NO: 145)D: HIV-005 3F(SLFNTVATL, SEQ ID NO: 59)E: HIV-005 3F6I8V(SLFNTIAVL), SEQ ID NO: 318)F: HIV-005 3F8V(SLFNTVAVL, SEQ ID NO: 319)G: HIV-005 3F6I(SLFNTIATL, SEQ ID NO: 320)H: HIV-005 6I8V(SLYNTIAVL, SEQ ID NO: 321)

In the upper left panel the Kdfor each above-listed peptide for the WT-A*02:01 pMHC complex is plotted against the Kdof the disulfide-modified F22C/S71C HLA-A*02:01 pMHC complex. The disulfide-modified F22C/S71C HLA-A*02:01 pMHC complex shows almost identical KDvalues to the WT-A*02:01 pMHC complex for each of the investigated peptides. In the lower left panel the Kdfor each above-listed peptide for the WT-A*02:01 pMHC complex is plotted against the KDof the disulfide-modified F22C/S71C W51C/G175C HLA-A*02:01 pMHC complex and shows also almost identical Kdvalues to the WT-A*02:01 pMHC complex for each of the investigated peptides.

In the upper right panel the Kdfor each above-listed peptide for the Y84C/A139C HLA-A*02:01 pMHC complex is plotted against the Kdof the disulfide-modified F22C/S71C HLA-A*02:01 pMHC complex. In the lower right panel the Kdfor each above-listed peptide for the Y84C/A139C HLA-A*02:01 pMHC complex is plotted against the Kdof the disulfide-modified F22C/S71C W51C/G175C HLA-A*02:01 pMHC complex. The disulfide-modified pMHC complexes of the F22C/S71C and the F22C/S71C W51C/G175C mutant have almost identical Kdvalues compared to the Y84C/A139C HLA-A*02:01 pMHC complex for each of the investigated peptides. It can thus, be concluded that disulfide-modified HLA-A*02:01 molecules loaded with different peptides and forming pMHC complexes are comparably recognized by a respective affinity-maturated TCR to the WT HLA-A*02:01 pMHC complex. Therefore, the function of the disulfide-modified HLA-A*02:01 molecules loaded with peptides (pMHC complexes) is unaffected by the introduction of stabilizing amino acid mutations into the HLA-A*02:01 molecule.

The results shown inFIG.15make it credible for the skilled person that the disulfide-modified HLA-A*02:01 molecules according to the present invention loaded with peptide ligands and forming disulfide-modified pMHC complexes elicit a T-cell response upon binding to their respective TCR.

20. High-Throughput Kinetic Screenings for Binding Motif Generation

Quick and flexible generation of pMHCs facilitates the collection of large binding affinity datasets against many different pMHCs. One example of such a dataset is screening of a positional scanning library to generate a pMHC-bsTCR binding motif, which can serve as one component in a bsTCR safety screening approach. To perform such measurements, the pMHC should ideally be used as a soluble analyte because this offers multiple advantages. First, immobilizing the same ligand with known activity repeatedly, for example, a bsTCR, allows better interpretation of the fitting results, especially the reported Rmaxvalue. Second, using pMHC complexes as soluble analytes instead of immobilizing is preferable for quick and cost effective high throughput screenings, since a broad variety of regeneratable biosensors capable of reversibly immobilizing bispecific TCR constructs exists. These biosensors are typically coated with antibodies and can be used at least 20 times for kinetic measurements without loss of readout quality. Third, immobilizing the bsTCR is the only orientation available for measuring monovalent affinity when a bsTCR or antibody has multiple pMHC binding moieties, because, with immobilized pMHCs, only avidity can be measured.

While the UV mediated peptide ligand exchange can generate a high number of different pMHC complexes, the exchange efficiency varies depending on the peptide and its affinity for binding to the respective MHC class I allele, resulting in different pMHC concentrations in the samples (FIG.10). This uncertainty is a problem for affinity measurements with pMHCs used as soluble analytes, as precise knowledge of the concentration is desired to determine accurate affinities. Since the disulfide-stabilized Y84C/A139C HLA-A*02:01 mutant is stable without any peptide, this restriction does not apply. If the peptides are added at a concentration high enough to saturate the empty MHC complexes, the effective concentration of pMHC is known, significantly increasing the accuracy of the measurements and avoiding false negatives. Examples for this behavior could be detected in the positional scanning library, resulting in bad fitting data and miscalculation of the affinity when UV exchange preparations were used compared to Y84C/A139C HLA-A*02:01 peptide loadings (FIGS.5,6,10) (26). Accurately measuring bsTCR affinities for such peptides can be important in the context of binding motif generations, because these substitutions may result in relevant MHC binders when combined with substitutions at other positions. Tolerance of the amino acids by the bsTCR should thus, be reflected correctly in a comprehensive binding motif.

By immobilizing the bs-868Z11-CD3 bsTCR the inventors were able to analyze the positional scanning library at four different soluble pMHC concentrations for each peptide ligand, ranging from 500 to 15.8 nM, within 4 hours of unattended measurement time at a 20-fold reduced price tag. All curves reaching at least a signal level of 0.05 nm were included in the fittings, resulting in a comprehensive TCR binding motif (FIGS.4a,8, Table 3).

Table 3: bs-868Z11-CD3 binding affinity for SV9 peptide SLYNTVATL (SEQ ID NO: 5) and peptides from positional scanning library (SEQ ID NOS: 16-177). Table includes KD, konand koffvalues determined by curve fittings following a 1:1 Langmuir binding model using the Fortebio Data Analysis HT 10.0.3.7 software. Respective errors are reported as well as accuracy of the fit according to the model. Peptides reported as “No fit” had no evaluable curves reaching at least a peak signal of 0.05 nm at any concentration.

TABLE 3bs-868Z11-CD3 binding affinity for SV9 peptide SLYNTVATL (SEQ ID NO: 5)and peptides from positional scanning library (SEQ ID NOS: 16-177). Tableincludes KD, Konand koffvalues determined by curve fittings following a 1:1Langmuir binding model using the Fortébio Data Analysis HT 10.0.3.7 software.Respective errors are reported as well as accuracy of the fit according to themodel. Peptides reported as ″No fit″ had no evaluable curves reaching at leasta peak signal of 0.05 nm at any concentration.konkonkoffkoffPeptideKD (M)KD Error(M-1s-1)Error(s-1)ErrorFull X2Full R2SLYNTVATL3.81E-091.49E-101.03E+051.45E+023.91E-041.53E-050.2620.9993(5)GLYNTVATL3.05E-083.55E-101.04E+053.42E+023.19E-033.56E-051.09150.9966(16)PLYNTVATL8.54E-093.46E-109.65E+043.03E+028.24E-043.33E-051.23630.9969(17)ALYNTVATL5.82E-093.18E-101.04E+053.14E+026.04E-043.29E-051.27910.9969(18)VLYNTVATL5.74E-092.24E-101.05E+052.27E+026.04E-042.35E-050.67190.9984(19)LLYNTVATL4.99E-082.99E-101.04E+052.67E+025.17E-032.80E-050.56230.9981(20)ILYNTVATL1.35E-082.35E-101.06E+052.40E+021.43E-032.47E-050.67480.9982(21)MLYNTVATL4.19E-082.95E-101.09E+052.93E+024.56E-032.96E-050.69220.9978(22)FLYNTVATL5.22E-083.07E-101.15E+053.20E+026.02E-033.13E-050.64520.9976(23)YLYNTVATL1.24E-075.65E-101.15E+054.01E+021.43E-024.21E-050.49310.9972(24)WLYNTVATL4.62E-074.57E-091.66E+051.48E+037.66E-023.27E-040.12160.9955(25)HLYNTVATL3.43E-072.50E-091.34E+058.87E+024.60E-021.43E-040.19080.9961(26)KLYNTVATL1.91E-082.14E-109.03E+041.67E+021.73E-031.91E-050.32390.999(27)RLYNTVATL4.42E-095.15E-108.86E+043.93E+023.92E-044.56E-052.06990.9944(28)QLYNTVATL1.38E-075.55E-109.85E+043.05E+021.36E-023.50E-050.35340.9981(29)NLYNTVATL3.13E-083.42E-109.84E+042.98E+023.08E-033.23E-050.94660.9973(30)ELYNTVATL4.85E-074.84E-099.29E+048.66E+024.50E-021.63E-040.45250.9948(31)DLYNTVATL4.49E-083.46E-109.55E+042.77E+024.28E-033.06E-050.78160.9977(32)TLYNTVATL6.94E-092.07E-101.02E+051.98E+027.07E-042.10E-050.5440.9988(33)SGYNTVATL1.86E-084.56E-108.40E+043.18E+021.56E-033.79E-051.38760.9964(34)SPYNTVATL1.65E-072.27E-096.80E+047.29E+021.12E-029.66E-052.20250.9852(35)SAYNTVATL1.00E-081.28E-101.02E+051.23E+021.02E-031.30E-050.20520.9995(36)SVYNTVATL8.47E-091.64E-101.01E+051.55E+028.57E-041.65E-050.33270.9992(37)SIYNTVATL8.68E-099.77E-111.02E+059.42E+018.89E-049.97E-060.11920.9997(38)SMYNTVATL6.55E-092.07E-101.01E+051.95E+026.61E-042.08E-050.48080.9987(39)SFYNTVATL8.52E-093.97E-109.54E+043.41E+028.13E-043.77E-051.52510.996(40)SYYNTVATL3.26E-083.90E-105.83E+041.62E+021.90E-032.21E-050.230.9989(41)SWYNTVATL8.16E-081.74E-094.46E+044.66E+023.64E-036.77E-050.98270.991(42)SHYNTVATL2.73E-088.86E-106.92E+044.66E+021.89E-035.99E-052.19470.9915(43)SKYNTVATL7.43E-081.57E-095.08E+045.00E+023.77E-037.06E-052.01620.9899(44)SRYNTVATL1.02E-072.33E-094.84E+046.42E+024.95E-039.17E-050.69460.9837(45)SQYNTVATL9.41E-092.19E-101.09E+052.35E+021.03E-032.37E-050.69760.9984(46)SNYNTVATL2.45E-086.68E-106.85E+043.45E+021.68E-034.50E-051.73670.9953(47)SEYNTVATL4.09E-081.77E-095.16E+046.23E+022.11E-038.78E-054.56910.9843(48)SDYNTVATL1.01E-071.68E-096.51E+046.61E+026.56E-038.69E-053.25070.9854(49)SSYNTVATL8.17E-091.97E-109.64E+041.72E+027.88E-041.89E-050.40630.999(50)STYNTVATL5.41E-091.49E-109.87E+041.35E+025.34E-041.47E-050.24270.9994(51)SLGNTVATLNo fit(52)SLPNTVATLNo fit(53)SLANTVATLNo fit(54)SLVNTVATL5.11E-075.80E-091.95E+052.01E+039.96E-024.80E-040.07690.9966(55)SLLNTVATL1.32E-078.45E-101.21E+056.09E+021.60E-026.33E-051.0380.9944(56)SLINTVATL4.77E-075.50E-091.40E+051.48E+036.69E-023.15E-040.3250.9939(57)SLMNTVATL1.07E-065.52E-082.35E+051.12E+042.50E-015.13E-030.12440.979(58)SLFNTVATL3.47E-081.92E-109.54E+041.59E+023.31E-031.75E-050.24450.9992(59)SLWNTVATL3.36E-081.91E-109.34E+041.53E+023.14E-031.71E-050.24790.9992(60)SLHNTVATL9.09E-083.31E-101.16E+052.84E+021.06E-022.85E-050.36760.9984(61)SLKNTVATLNo fit(62)SLRNTVATL5.55E-075.54E-099.64E+049.00E+025.35E-021.88E-040.16750.9957(63)SLQNTVATL6.29E-079.45E-092.62E+053.51E+031.65E-011.11E-030.03840.9961(64)SLNNTVATL4.74E-075.90E-091.81E+052.05E+038.59E-024.48E-040.10490.9953(65)SLENTVATLNo fit(66)SLDNTVATLNo fit(67)SLSNTVATLNo fit(68)SLTNTVATL3.01E-063.45E-061.88E+052.15E+055.66E-016.50E-020.11230.9199(69)SLYGTVATL5.33E-071.20E-082.07E+054.15E+031.11E-011.12E-030.56780.9842(70)SLYPTVATL5.54E-071.44E-083.62E+058.18E+032.00E-012.58E-030.08840.9892(71)SLYATVATL1.15E-076.46E-101.40E+056.09E+021.60E-025.72E-050.93540.9952(72)SLYVTVATL1.80E-079.65E-101.31E+055.98E+022.35E-026.64E-050.46690.9962(73)SLYLTVATL6.70E-083.12E-101.11E+052.85E+027.44E-032.89E-050.51520.9981(74)SLYITVATL5.25E-075.68E-091.18E+051.18E+036.19E-022.55E-040.22080.9949(75)SLYMTVATL1.88E-062.10E-063.58E+053.96E+056.72E-011.11E-010.090.876(76)SLYFTVATLNo fit(77)SLYYTVATLNo fit(78)SLYWTVATLNo fit(79)SLYHTVATL8.11E-086.16E-101.38E+057.03E+021.12E-026.26E-051.84220.9923(80)SLYKTVATLNo fit(81)SLYRTVATLNo fit(82)SLYQTVATL2.84E-073.23E-091.71E+051.73E+034.86E-022.53E-040.67210.9898(83)SLYETVATLNo fit(84)SLYDTVATLNo fit(85)SLYSTVATL5.95E-082.69E-101.27E+053.13E+027.57E-032.87E-050.54040.9981(86)SLYTTVATL1.61E-077.59E-101.35E+055.34E+022.18E-025.63E-050.39650.9968(87)SLYNGVATL6.03E-071.50E-075.42E+051.29E+053.27E-012.34E-020.04520.9399(88)SLYNPVATLNo fit(89)SLYNAVATL9.66E-085.82E-101.27E+055.41E+021.22E-025.20E-050.99440.995(90)SLYNVVATL3.07E-084.63E-109.09E+043.58E+022.79E-034.06E-051.33330.9958(91)SLYNLVATL1.46E-083.05E-109.80E+042.73E+021.43E-032.97E-050.9170.9976(92)SLYNIVATL4.85E-083.37E-108.41E+042.21E+024.08E-032.62E-050.43140.9984(93)SLYNMVATL4.26E-083.52E-101.20E+054.09E+025.12E-033.86E-051.24150.9962(94)SLYNFVATL7.26E-073.75E-082.97E+051.36E+042.16E-015.09E-030.33460.9697(95)SLYNYVATL4.60E-069.88E-061.37E+052.94E+056.31E-019.34E-020.11130.8904(96)SLYNWVATLNo fit(97)SLYNHVATL6.23E-074.07E-084.00E+052.26E+042.49E-018.17E-030.21060.9526(98)SLYNKVATL2.24E-071.05E-091.35E+055.55E+023.02E-026.82E-050.25720.9973(99)SLYNRVATL7.78E-077.24E-083.54E+053.19E+042.76E-016.54E-030.020.9899(100)SLYNQVATL4.72E-077.25E-092.10E+052.90E+039.91E-026.64E-040.1310.9936(101)SLYNNVATL1.19E-075.68E-101.32E+054.88E+021.58E-024.76E-050.59560.9966(102)SLYNEVATLNo fit(103)SLYNDVATL3.91E-055.33E-041.23E+041.67E+054.79E-015.67E-020.16850.904(104)SLYNSVATL6.91E-083.75E-101.21E+053.86E+028.39E-033.68E-050.71810.997(105)SLYNTGATL1.34E-076.48E-101.37E+055.33E+021.84E-025.26E-050.52670.9965(106)SLYNTPATL1.54E-081.56E-101.19E+051.93E+021.83E-031.82E-050.35610.999(107)SLYNTAATL5.48E-083.74E-101.10E+053.59E+026.05E-033.62E-050.88430.9967(108)SLYNTLATL9.08E-091.01E-101.12E+051.15E+021.02E-031.13E-050.150.9996(109)SLYNTIATL8.74E-091.86E-109.97E+041.72E+028.71E-041.85E-050.37880.999(110)SLYNTMATL2.72E-083.66E-109.75E+043.17E+022.65E-033.46E-050.95510.9966(111)SLYNTFATL5.79E-076.47E-097.96E+048.39E+024.61E-021.69E-040.14160.9946(112)SLYNTYATL4.43E-078.76E-094.61E+048.61E+022.04E-021.33E-040.12860.9831(113)SLYNTWATL1.74E-051.28E-051.91E+031.41E+033.33E-022.40E-040.00630.9878(114)SLYNTHATL1.75E-071.46E-097.39E+044.94E+021.30E-026.42E-050.36530.9929(115)SLYNTKATLNo fit(116)SLYNTRATLNo fit(117)SLYNTQATL2.71E-071.50E-091.19E+055.86E+023.22E-027.94E-050.13920.9969(118)SLYNTNATL1.79E-077.80E-101.20E+054.42E+022.15E-025.03E-050.29830.9974(119)SLYNTEATL1.43E-065.11E-085.54E+041.93E+037.94E-025.83E-040.07720.99(120)SLYNTDATL6.04E-077.08E-091.12E+051.22E+036.77E-022.89E-040.11390.995(121)SLYNTSATL1.66E-076.43E-101.43E+054.69E+022.38E-024.93E-050.26730.9979(122)SLYNTTATL3.37E-084.58E-101.07E+054.51E+023.59E-034.64E-051.76730.9938(123)SLYNTVGTL8.29E-094.59E-108.24E+043.12E+026.83E-043.77E-051.22830.996(124)SLYNTVPTL3.71E-094.42E-101.22E+055.76E+024.51E-045.36E-054.0520.9904(125)SLYNTVVTL5.99E-071.07E-081.44E+052.37E+038.64E-026.15E-040.18830.9892(126)SLYNTVLTLNo fit(127)SLYNTVITLNo fit(128)SLYNTVMTL1.02E-074.28E-106.90E+041.80E+027.04E-032.31E-050.13330.9988(129)SLYNTVFTL5.14E-071.01E-081.72E+053.07E+038.85E-027.06E-040.12740.9897(130)SLYNTVYTLNo fit(131)SLYNTVWTLNo fit(132)SLYNTVHTL1.14E-072.51E-108.42E+041.27E+029.63E-031.53E-050.07630.9995(133)SLYNTVKTL1.20E-065.58E-085.35E+042.43E+036.42E-026.41E-040.07920.9775(134)SLYNTVRTL1.28E-062.41E-082.49E+044.61E+023.20E-029.10E-050.05470.9967(135)SLYNTVQTL5.38E-087.00E-106.84E+043.40E+023.68E-034.43E-050.92960.9952(136)SLYNTVNTL4.11E-088.02E-107.22E+044.32E+022.97E-035.51E-051.58780.9921(137)SLYNTVETL1.61E-062.46E-075.74E+038.80E+029.22E-031.00E-040.0070.989(138)SLYNTVDTLNo fit(139)SLYNTVSTL1.04E-084.47E-109.80E+044.00E+021.02E-034.36E-051.96340.9944(140)SLYNTVTTL6.90E-082.99E-109.23E+042.09E+026.37E-032.36E-050.28930.9987(141)SLYNTVAGL1.14E-081.42E-101.14E+051.65E+021.30E-031.61E-050.3020.9992(142)SLYNTVAPL2.34E-071.20E-091.35E+056.11E+023.16E-027.71E-050.31550.9969(143)SLYNTVAAL8.50E-091.51E-101.14E+051.75E+029.69E-041.72E-050.35040.9991(144)SLYNTVAVL6.98E-091.19E-101.05E+051.20E+027.31E-041.25E-050.18810.9995(145)SLYNTVALL1.58E-081.20E-109.58E+041.03E+021.51E-031.14E-050.12590.9996(146)SLYNTVAIL4.16E-097.48E-109.74E+046.62E+024.05E-047.28E-055.86070.9834(147)SLYNTVAML7.69E-095.22E-109.75E+044.63E+027.50E-045.08E-052.71810.9922(148)SLYNTVAFL1.93E-071.68E-099.29E+046.77E+021.80E-028.45E-050.94560.9906(149)SLYNTVAYL4.00E-073.75E-099.61E+048.32E+023.85E-021.39E-040.24510.994(150)SLYNTVAWL2.09E-071.94E-099.65E+047.69E+022.01E-029.70E-051.03580.9893(151)SLYNTVAHL1.09E-085.55E-109.19E+044.47E+021.00E-035.07E-052.63880.9925(152)SLYNTVAKL1.73E-082.87E-101.02E+052.76E+021.77E-032.90E-050.90540.9975(153)SLYNTVARL7.93E-093.98E-101.06E+054.06E+028.37E-044.19E-052.12010.9946(154)SLYNTVAQL1.59E-086.14E-101.01E+055.74E+021.61E-036.13E-054.00590.9888(155)SLYNTVANL1.08E-086.43E-101.01E+056.03E+021.09E-036.46E-054.59430.9874(156)SLYNTVAEL4.73E-082.37E-109.22E+041.79E+024.36E-032.02E-050.2910.999(157)SLYNTVADL2.12E-083.17E-108.90E+042.40E+021.88E-032.77E-050.68890.9979(158)SLYNTVASL4.68E-092.55E-101.09E+052.71E+025.08E-042.76E-050.9180.9977(159)SLYNTVATG7.71E-094.30E-101.01E+054.05E+027.79E-044.34E-052.11990.9943(160)SLYNTVATP5.03E-081.57E-093.41E+043.34E+021.72E-035.08E-050.69610.9945(161)SLYNTVATA6.74E-094.88E-101.13E+055.56E+027.61E-045.49E-053.69050.9904(162)SLYNTVATV8.41E-096.00E-101.04E+055.95E+028.76E-046.23E-054.7520.988(163)SLYNTVATI6.70E-092.87E-101.13E+053.25E+027.53E-043.22E-051.27120.9968(164)SLYNTVATM7.45E-092.48E-109.88E+042.26E+027.36E-042.44E-050.69220.9982(165)SLYNTVATF1.19E-082.46E-107.18E+041.37E+028.51E-041.76E-050.22280.9992(166)SLYNTVATY1.02E-083.37E-107.11E+041.85E+027.24E-042.39E-050.46250.9985(167)SLYNTVATW3.32E-085.59E-103.70E+041.34E+021.23E-032.02E-050.08240.9991(168)SLYNTVATH1.37E-083.64E-104.75E+041.19E+026.51E-041.72E-050.0890.9993(169)SLYNTVATK4.57E-081.20E-092.70E+042.00E+021.23E-033.11E-050.09290.9982(170)SLYNTVATR5.71E-092.30E-109.59E+041.99E+025.48E-042.20E-050.55320.9986(171)SLYNTVATQ5.88E-093.12E-108.96E+042.41E+025.27E-042.79E-050.73970.9978(172)SLYNTVATN9.10E-093.77E-109.76E+043.36E+028.88E-043.67E-051.59710.9961(173)SLYNTVATE6.96E-069.43E-063.79E+025.13E+022.64E-038.02E-050.19970.9908(174)SLYNTVATD7.18E-068.48E-063.95E+024.67E+022.83E-037.30E-050.11370.9924(175)SLYNTVATS7.19E-092.13E-101.16E+052.54E+028.33E-042.46E-050.76370.9981(176)SLYNTVATT5.66E-091.27E-101.12E+051.42E+026.32E-041.41E-050.26270.9994(177)

TABLE 4Cross-reactive peptide ligand search motif forbs-868Z11-CD3 based on the affinities measuredusing the positional scanning library. All aminoacids of the 19 proteinogenic amino acidsinvestigated at each position that increased therespective affinity of the bsTCR above 50 nMwere removed to reach the search motif.PeptidePermittedPositionAmino Acids1GPAVLIMKRNDST2GAVLIMFYHQNEST3FYW4N5VLIMT6PVLIMT7GPANS8GAVLIMHKRQNEDST9GAVLIMFYWHKRQNST

Soluble Y84C/A139C HLA-A*02:01 pMHC preparations can be stored for at least 2 weeks at 4° C. without loss of quality and used for multiple analyses (FIG.12; Day 1: KD=1.35E−09M, R2=0.9992; Day 14: KD=1.08E−09M, R2=0.9991).

The 868Z11 TCR displayed an expected pattern of recognition: changes of amino acids between positions 3 to 7 had the biggest influence on the bsTCR binding affinity. Interestingly, only one amino acid change resulted in an increased binding affinity by bs-868Z11-CD3 compared to the interaction with the wild type peptide, showcasing the remarkable affinity the TCR has for the target in its affinity maturated state. This behavior can also be graphically illustrated when visualizing the binding motif as Seq2Logo graph (FIG.4B) (27).

21. Identification of Peptide Ligands Cross-Reactive with Bs-868Z11-CD3

The inventors further wanted to explore whether they could use the generated binding motif to identify cross-reactive peptide ligands from the human genome. The inventors created a peptide ligand search motif from the affinity dataset by introducing an exemplary Kdthreshold of 50 nM: all single amino acid substitutions increasing the bs-868Z11-CD3 Kdabove that threshold were excluded from the motif (Table 4). Based on this motif the inventors performed a search in the NCBI human non-redundant protein sequence database for nonamer sequences matching combinations allowed by the motif. The search identified over 400 hits within the human genome, with sequence identity to the wild type sequence SLYNTVATL (SEQ ID NO: 5) ranging from 1 to 6 identical positions. 140 peptides were selected, sampled to be representative of the sequence identity distribution in the larger group, synthesized and used for affinity measurements (Table 5; SEQ ID NOS: 178-317). The inventors were able to detect binding affinities of single digit μM Kds or higher for 91 of those peptides.

TABLE 5the bs-868Z11-CD3 binding motif. Peptide sequences and associated genes accordingto the NCbi data base are reported and peptides are sorted by decreasing Kds. Tableincludes KD, Kon, and koffvalues determined by curve fittings following a 1:1 Langmuirbinding model using the Fortébio Data Analysis HT 10.0.3.7 software. Respective errorsare reported as well as accuracy of the fit according to the model. Peptides reported as″No fit″ had no evaluable curves reaching at least a peak signal of 0.05 nm at anyconcentration.AssociatedkonkonkoffkoffFullFullPeptideGeneKD (M)KD Error(M-1s-1)Error(s-1)ErrorX2R2RVYNTVPLVHIPK31.32E-081.76E-109.50E+041.69E+021.26E-031.66E-050.29080.9993(178)RMYNLVSRICUL11.91E-082.52E-109.25E+042.28E+021.76E-032.29E-050.54290.9986(179)SLYNMVPSIOVOS1.97E-081.71E-101.31E+052.89E+022.57E-032.16E-050.5860.9987(180)TVYNMVPSIOVOS2.07E-081.54E-101.28E+052.50E+022.66E-031.91E-050.42280.999(181)ALYNVIAMASECISBP2L2.11E-081.51E-109.28E+042.25E+021.96E-031.32E-050.1140.9997(182)AIYNLLPDINCAPD22.33E-081.82E-101.01E+051.90E+022.36E-031.79E-050.32990.9992(183)STYNLVSTSKIAA20182.47E-082.16E-107.07E+041.28E+021.75E-031.49E-050.17720.9995(184)SVYNMVPSIOVOS22.68E-081.96E-101.32E+053.26E+023.53E-032.43E-050.64710.9984(185)RTYNVLAILATP8B13.11E-081.55E-107.54E+049.93E+012.34E-031.12E-050.09370.9997(186)SVYNLVSIAKPTN3.65E-082.01E-107.97E+042.13E+022.91E-031.40E-050.09260.9997(187)RAYNLIGTVLOC1001285013.72E-081.71E-108.94E+041.40E+023.33E-031.43E-050.17210.9995(188)ALFNLIPVGFGF123.83E-083.22E-106.64E+041.71E+022.54E-032.04E-050.2660.999(189)RIYNVIGTLFOLH1, FOLH1B4.53E-083.00E-105.75E+041.28E+022.61E-031.62E-050.10950.9994(190)RIYNVVGTINAALAD25.15E-084.13E-105.94E+041.81E+023.06E-032.27E-050.27230.9989(191)TLFNLVPNSCLASP25.40E-083.31E-109.76E+042.90E+025.28E-032.82E-050.48220.9981(192)SLFNVISILKCNK12, KCNK135.83E-083.11E-106.94E+041.64E+024.05E-031.94E-050.20680.9992(193)STFNLVAISCCKAR6.06E-082.91E-104.96E+049.98E+013.01E-031.31E-050.04360.9996(194)TLFNLIPVGFGF12, FGF13,6.32E-083.97E-106.72E+041.98E+024.25E-032.36E-050.30440.9988(195)FGF14TIFNLIPNSCLASP16.41E-082.67E-108.97E+041.99E+025.75E-032.03E-050.26550.999(196)ALYNVLAKVIFFO1, IFFO26.59E-082.96E-101.02E+052.63E+026.75E-032.49E-050.36020.9986(197)AVFNLLPHTSMYD47.11E-082.72E-108.53E+041.82E+026.07E-031.93E-050.23440.9991(198)RMYNLLGHMZNF7108.71E-087.55E-105.30E+042.55E+024.62E-033.33E-050.22780.9977(199)STWNTPPNMKIAA09228.98E-083.45E-109.05E+042.28E+028.13E-032.36E-050.25910.9989(200)NIYNLIAIIBICD29.32E-083.91E-101.06E+053.10E+029.84E-032.95E-050.38940.9983(201)RIYNLPPELWRAP539.95E-083.78E-109.47E+042.51E+029.42E-032.56E-050.30260.9988(202)TTFNLPSAAWDR171.02E-076.71E-107.87E+043.47E+028.04E-033.91E-050.5790.997(203)MFFNVIAIVUGGT21.06E-071.29E-095.21E+043.95E+025.52E-035.23E-050.07720.9934(204)SLWNTVSGIHHLA11.08E-075.04E-108.88E+042.94E+029.60E-033.14E-050.38730.9982(205)MLWNLLALRCOX7A21.17E-071.03E-081.26E+061.05E+051.47E-014.25E-030.04480.9675(206)VFWNLLPTVC12orf741.20E-077.20E-101.24E+055.97E+021.49E-025.29E-050.91160.9955(207)STFNTTSNGQSER11.52E-076.60E-091.76E+057.05E+032.67E-024.39E-040.03110.9225(208)GFFNLLSHVPCP21.59E-079.88E-096.24E+053.64E+049.94E-022.08E-030.03210.9717(209)LLYNVPAVAAPP1.67E-071.27E-087.41E+055.29E+041.24E-013.21E-030.01640.9712(210)ALFNTISQGVTA11.83E-078.26E-107.15E+042.68E+021.31E-023.28E-050.22140.9984(211)TTFNTLAGSMUC161.97E-078.70E-109.03E+043.44E+021.78E-023.98E-050.15560.9981(212)SLWNLLGNALMAN2L2.14E-073.02E-081.02E+061.35E+052.19E-011.07E-020.03240.952(213)SLYNLLNLTSLC4A52.19E-078.40E-106.75E+042.24E+021.48E-022.85E-050.0940.9988(214)GVWNLLSIVZSWIM82.52E-074.59E-081.09E+061.86E+052.74E-011.73E-020.05450.9403(215)ALFNVVNSISLC38A112.55E-072.10E-096.83E+044.99E+021.74E-026.59E-050.28970.9956(216)VIYNLLGLASH3TC22.64E-071.88E-091.07E+057.25E+022.83E-025.92E-050.21150.9983(217)SIFNIVAIAGPR502.84E-071.60E-094.44E+042.37E+021.26E-022.25E-050.03690.9995(218)TVYNTVSEGSLC39A63.04E-072.50E-094.71E+043.50E+021.43E-024.99E-050.25380.997(219)DLWNTLSSLEFCAB13, ITGB33.39E-072.60E-084.15E+053.02E+041.41E-013.55E-030.02380.9752(220)IFFNLLAVLPOMT13.50E-074.75E-088.47E+051.08E+052.97E-011.35E-020.0230.9688(221)DLFNLLPDVPSMD73.60E-071.08E-087.69E+042.15E+032.77E-023.14E-040.08810.9367(222)LSWNVVPNASPCS33.67E-072.91E-084.13E+053.09E+041.52E-013.89E-030.02340.9734(223)MLWNLLALHCOX7A2P23.67E-072.04E-081.07E+064.75E+043.94E-011.33E-020.11590.9595(224)TIFNTVNTSTIMMDC13.87E-072.77E-094.20E+042.80E+021.63E-024.19E-050.03040.9978(225)KTFNLIPAVMRPL44.13E-072.59E-091.12E+056.52E+024.62E-021.07E-040.11850.9979(226)NLFNVTPLIZNF66P4.28E-071.38E-071.05E+063.19E+054.49E-014.68E-020.04470.9139(227)SYWNIISTVOR2D34.39E-074.84E-094.56E+044.74E+022.00E-027.23E-050.13730.9952(228)GVENLIAVLAC002365.5,4.59E-074.94E-097.59E+047.70E+023.48E-021.26E-040.32680.9946(229)LOC100288814RLFNITSSAIFITM104.74E-074.12E-082.51E+052.08E+041.19E-013.08E-030.01670.9706(230)NLWNLVAVIWDR174.97E-071.16E-082.07E+054.34E+031.03E-011.04E-030.2560.9836(231)RIFNLIGMMHCN1, HCN34.98E-071.26E-082.29E+045.51E+021.14E-028.55E-050.07120.9889(232)RLFNVVSRGTRPV25.02E-076.55E-096.50E+048.05E+023.26E-021.34E-040.18750.9931(233)LVFNVIPTLABCB65.35E-073.99E-091.33E+059.21E+027.13E-022.00E-040.04450.9982(234)TTWNILSSACOX15.36E-074.08E-082.26E+051.65E+041.21E-012.63E-030.02140.9794(235)KLFNVLSTLNUP210P25.76E-073.35E-082.97E+051.65E+041.71E-012.83E-030.01310.9912(236)RVYNLTAKSVWA3B5.95E-074.57E-094.53E+043.35E+022.69E-025.65E-050.02190.9981(237)LTFNTISLSENTHD17.09E-072.12E-074.78E+051.37E+053.39E-012.72E-020.03870.929(238)AQFNLLSSTTP737.13E-079.97E-098.59E+041.15E+036.12E-022.58E-040.16580.9947(239)VVYNVLSELSP100, SP140L7.35E-076.29E-081.84E+051.52E+041.35E-012.89E-030.02550.9785(240)KVYNTPSTSAEBP27.51E-071.39E-081.71E+052.91E+031.28E-019.13E-040.07180.9945(241)GIFNIIPSTCAPN77.90E-078.67E-091.32E+051.36E+031.04E-014.00E-040.03640.9979(242)NIYNTLSGLUBR48.73E-071.71E-081.59E+052.89E+031.38E-019.89E-040.05640.9947(243)RLFNLTSTFFLJ44715, FUT119.32E-072.82E-081.72E+054.83E+031.60E-011.79E-030.05250.9894(244)TVWNTLSSLDNAH99.39E-071.22E-084.52E+045.73E+024.25E-021.20E-040.03440.9971(245)RLFNMLSAVCFAP221, PCDP19.71E-073.06E-081.73E+055.09E+031.68E-011.94E-030.07140.9892(246)SIWNVTAIAHTR5A1.10E-065.12E-073.21E+051.45E+053.54E-013.47E-020.05760.9051(247)ALFNLMSGIEGR41.19E-063.21E-089.57E+042.48E+031.14E-018.53E-040.06450.9931(248)IVYNLLSAMSLC39A101.30E-061.62E-071.61E+051.98E+042.10E-014.82E-030.02450.987(249)ISFNMLPSIGPR981.37E-064.65E-081.24E+054.04E+031.70E-011.70E-030.05810.991(250)NTYNILPGSC9orf1731.38E-061.17E-071.14E+059.57E+031.57E-012.30E-030.0250.9925(251)RLWNMVNVTIL12RB21.39E-062.57E-071.52E+052.77E+042.11E-016.87E-030.0490.9763(252)SAFNITSLIWAC1.41E-063.21E-071.65E+053.70E+042.32E-019.29E-030.03140.9682(253)NIFNLPNIVOMD1.48E-066.62E-074.19E+051.85E+056.20E-014.90E-020.09050.9596(254)GVYNLPGASGPX21.58E-063.07E-071.17E+052.23E+041.84E-015.65E-030.04880.9756(255)GTYNVISLVTRPC4, TRPC51.64E-064.18E-071.23E+053.10E+042.02E-018.00E-030.06660.965(256)SIFNTLSDISGSM31.97E-065.86E-084.07E+041.20E+038.01E-023.78E-040.08560.9957(257)TIFNILSGIABCA32.66E-062.37E-074.68E+044.13E+031.24E-011.66E-030.17280.9807(258)LLFNLISSSMON1A2.79E-062.57E-061.51E+051.38E+054.20E-014.10E-020.05990.9183(259)RTFNLTAGSPDXDC12.85E-065.89E-074.63E+049.54E+031.32E-012.56E-030.03560.9845(260)TVFNILPGGPAFAH23.23E-061.06E-063.69E+041.21E+041.19E-013.22E-030.0250.968(261)GLFNIPPASCYP2S13.91E-064.13E-068.52E+048.97E+043.33E-012.74E-020.03950.9216(262)RMFNIISDSRASA13.99E-064.08E-071.51E+041.54E+036.02E-024.39E-040.05090.9862(263)TTFNIVGTTGABRA36.79E-063.10E-068.68E+033.96E+035.89E-028.72E-040.03510.9743(264)ALFNLMSGVEGR47.87E-061.14E-053.17E+044.60E+042.50E-011.45E-020.11150.9454(265)SVFNITAIAMTNR1B1.96E-052.39E-042.58E+043.15E+055.06E-011.06E-010.28050.7869(266)KIYNTPSASNCAM12.56E-052.88E-058.86E+039.95E+032.27E-015.62E-030.24740.9662(267)LLYNLLGSSABCC91.41E-046.82E-033.21E+031.55E+054.54E-015.27E-020.11530.9007(268)SLYNMMGEATMTC2No fit(269)SLWNLMGNALMAN2LNo fit(270)GLYNIVGNASUMF1No fit(271)LTWNLTPKADLEC1No fit(272)LIFNVTGLAZDHHC23No fit(273)SIFNITGIAMTNR1ANo fit(274)LTFNLVSDACASP8AP2No fit(275)MQWNILAQACCRN4LNo fit(276)LSWNLVPEACOL7A1No fit(277)DLWNTLSEATRHDENo fit(278)GLFNIPPAFCYP2S1No fit(279)LIWNILASFTTC29No fit(280)LLFNMLPGGEXT2No fit(281)LVYNIMSSGFAM120BNo fit(282)IIYNVPGTGRNF133No fit(283)VIYNVTSDGTTNNo fit(284)GTFNLPSDGBAG6No fit(285)KLWNTLNLIENPP5No fit(286)LMWNIISIIVTCN1No fit(287)GLFNTTSNISEMA3ENo fit(288)LIFNTLSLIPDCD6IPNo fit(289)SVFNLMNAISLC38A6No fit(290)LTFNILGQIDOCK11No fit(291)GLFNMVSSLRRN3No fit(292)KIFNIINSLFER1L5No fit(293)AVWNVLGNLBAGSNo fit(294)KVFNIVSDLFSIP2No fit(295)DLWNVVSHLDDX60LNo fit(296)LQFNTVSKLJAM2No fit(297)MSFNTVSELZNF33A, ZNF33BNo fit(298)ASWNIVNLLTRPA1No fit(299)ISFNIISALMS4A18No fit(300)AFFNILNELFNBP1LNo fit(301)LVFNLLPIMABCB7No fit(302)KIFNTVPDMARHGAP26No fit(303)MLFNLIGLSOR10J1No fit(304)LLFNLPPGSVGLL1No fit(305)MTFNLIGESCR1, CR1LNo fit(306)KVYNIPGISKLI1L10No fit(307)GIYNIPGDSTNS1No fit(308)GLYNLMNITINSRNo fit(309)LTWNMINTTLRIT3No fit(310)IVFNVLSDTHCN3No fit(311)IVFNVVSDTHCN2, HCN4No fit(312)LIFNITASVSVEP1No fit(313)IVFNLTNNVMNAT1No fit(314)KSFNVLSSVZNF557No fit(315)LAFNILGMVSLC46A1No fit(316)VSWNITGTVSEH1LNo fit(317)

One of them, ALYNVLAKV (SEQ ID NO: 1), was worth of special notice. It was selected as a theoretical peptide but found in addition on tissue samples and cell lines according to the XPRESIDENT® immunopeptidomics database. This database combines quantitative HLA peptidomics based on LC-MS analysis and quantitative transcriptomics provided by RNAseq from healthy tissues and tumor tissues to identify peptides presented exclusively or predominately on tumor tissue (28, 29).

ALYNVLAKV (SEQ ID NO: 1), an antigen from intermediate filament family orphan 1 or 2 (IFFO1/2), was detected on multiple healthy tissue and tumor tissue samples, ranging from head and neck, spleen, or kidney to non-small cell lung carcinoma or renal cell carcinoma. The pMHC-bsTCR binding affinity was measured with a KDof 65.9 nM (FIG.4c). The inventors were able to identify a second LC-MS detected peptide, KTFNLIPAV (SEQ ID NO: 226), with a lower Kdof 413 nM detected on three tumor tissue samples.

22. Correlation of bsTCR Affinity with T Cell Activation

The pMHC-bsTCR binding affinity can be measured using this high-throughput screening platform, but should be consistent with the in vitro activity as functional T cell engaging bsTCR to be even more useful. Commonly, in vitro co-incubations of target and effector cells coupled with an appropriate readout are used to characterize these constructs. GloResponse™ NFAT-luc2 Jurkat effector cells, a cell line that expresses a luciferase reporter gene driven by a NFAT-response element, and peptide-loaded T2 target cells, a TAP-deficient A*02:01 cell line with restorable pMHC presentation through exogenous peptide loading, were incubated in the presence of bs-868Z11-CD3 to corroborate the significance of the kinetic screening in this context. T2 cells were loaded separately with respective peptides from the positional scanning library at a concentration of 100 nM and subsequently co-incubated with Jurkats and different bsTCR concentrations for 18 hours before readout. As expected the inventors encountered a broad spectrum of results, ranging from no detectable T cell activation at any bsTCR concentration to strong responses starting at low concentrations, e.g. for the wild type peptide (FIG.5A). Since EC50 values could not be determined for many of the interactions in the selected bsTCR concentration range the inventors categorized the individual peptides by onset of T cell activation, defined as the lowest bsTCR concentration that was able to induce a 3-fold increased signal above. Onset values were plotted against the respectively measured Kos (FIG.5B).

Overall, the inventors detected a good correlation between the determined Kdvalues and T cell activation with one notable group of outliers with strong pMHC-bsTCR binding affinities but late T cell activation onset or no activation at all. The inventors were able to identify a direct connection between these peptides and their NetMHC predicted binding strength to the MHC (FIG.5C) (26). This offered a potential explanation because different peptide binding affinities could result in different presentation levels of the respective pMHCs on the target cells after exogenous loading. These levels might, in turn, influence pMHC-bsTCR complex numbers and ultimately Jurkat effector (T cell) activation. To corroborate the hypothesis, the inventors performed a flow cytometric T2 peptide binding assay using an anti-HLA-A2 antibody and could detect less elevated HLA-A2 surface levels after peptide loading for peptides with lower binding affinities, especially NetMHC ranks of 2 and above, supporting the initial hypothesis. pMHC-bsTCR binding affinity correlated well with T cell activation onset for peptide ligands between NetMHC rank 0.05 and 2, whereas above that threshold T cell activation decreased with further increasing NetMHC ranks largely irrespective of pMHC-bsTCR binding affinity.

The inventors also performed T cell activation assays for the 140 peptide ligands selected by binding motif search, 24 were capable of inducing a 3-fold T cell activation over background with at least one of the supplied bsTCR concentrations (FIG.5D). Measured Kds correlated with the onset of T cell activation similarly to the results obtained by the positional scanning library. The previously highlighted IFFO1 antigen ALYNVLAKV (SEQ ID NO: 1) was also reactive in the reporter assay (FIG.5E).

The inventors showed that pMHC-bsTCR binding affinity is a good indicator for the in vitro function of the scTv 868Z11 coupled with an anti-CD3 T cell engager. This highlights the value of the pMHC-bsTCR binding kinetics screening platform because it allows quick but adequate characterization of bsTCRs early in the development of such molecules.

23. Crystal Structure of the 1G4 Y84C/A139C HLA-A*02:01:01 ESO 9V TCR-pMHC

To further confirm that the 1G4 TCR recognizes ESO 9V Y84C/A139C HLA-A*02:01 indistinguishably from ESO 9V WT-A*02:01. TCR and disulfide-stabilized MHC refolded with ESO 9V were cocrystallized, as reported previously for the wild-type ESO 9V HLA-A*02:01 molecule and analyzed by x-ray crystallography (Table 2) (21). Comparison of the crystal structures revealed a high degree of structural overlap between both complexes. The backbone of both HLA-A*02:01 molecules aligned almost perfectly with a root mean square deviation (RMSD) value of 1.14 Å calculated over Ca (constant portion of the α chain of a T cell receptor;FIG.7A). The same was true for both bound peptides including their side chains with an RMSD value of 1.27 Å calculated over all atoms, even when in close vicinity to the disulfide bond (FIG.7B).

Similar conclusions could be made for the interaction with the 1G4 TCR. The complementarity-determining region (CDR) loop regions interacting with the peptide and the MHC backbone did show slight deviations of the interface and a small change in the docking angle of 4.13°, when comparing WT-A*02:01 1G4 with the Y84C/A139C HLA-A*02:01 1G4 crystal structure. This shift was still within the range of expected deviations for the same complex when crystallized repeatedly (FIGS.7, C and D). Together, determined binding affinities and crystal structure showcase peptide receptiveness and similar properties of the Y84C/A139C HLA-A*02:01 pMHC complexes compared with wild-type complexes with respect to TCR binding. The crystal structure of the 1G4 Y84C/A139C HLA-A*02:01 ESO 9V complex has been deposited in the PDB under the accession number 6Q3S.

24. Discussion

Here, the inventors have presented disulfide-stabilized and functionally empty HLA-A*02:01 molecules, which can be refolded and purified without the use of typically required high-affinity peptides e.g. the dipeptide GM. The resulting monomers can form pMHCs after addition of peptides in a one-step loading procedure. Although the disulfide bridge enhances the stability of the MHC molecule, introduction does not inhibit or significantly alter binding of TCRs to disulfide-modified HLA*02:01 pMHC complexes compared with the wild type. This technique represents a great tool to quickly produce large pMHC libraries that are suitable for affinity measurements. Combining disulfide modified HLA*02:01-produced pMHC complex libraries with biolayer interferometry-based analysis results in a platform capable of high-throughput pMHC-bsTCR binding kinetics screenings. This setup could also be useful for the analysis of other biologics targeting pMHC complexes, like monoclonal antibodies or bispecifics, such as bispecific T cell engagers. In one application of this platform, the inventors were able to quickly collect a pMHC-bsTCR binding affinity dataset for the HIV-specific bsTCR bs-868Z11-CD3. bsTCR binding affinities for respective pMHCs were indicative of in vitro activity when coupled with the presented T cell engager and tested in a cellular reporter assay, making these datasets valuable for bsTCR characterization. Analysis of the relationship between binding affinity and bsTCR-mediated cellular activation over a wide range of pMHC-bsTCR affinities has been difficult, thus far as a result of the limited tools available to feasibly collect such datasets.

The collected binding motif revealed similarities to the binding motif of the wild-type TCR 868. Analysis of an 868-SV9 crystal structure, as well as an accompanying alanine scan by Cole et al. (34), revealed prominent interactions between the CDR3a region and the amino acids 4N and 5T of SLYNTVATL (SEQ ID NO: 5). This behavior seems to be conserved although a significant part of the CDR3a is mutated in the 868Z11 construct. Using the binding motif and a model search strategy, the inventors were able to identify multiple peptides from the human proteome, which demonstrated high-affinity interactions with the bsTCR and the potential to induce bsTCR-mediated Jurkat effector activation when presented on target cells.

Note that TCR binding motifs derived from single amino acid substitution libraries may still not reflect all possible peptides a specific TCR (sTCR) can recognize, because the exchange of multiple amino acids, at the same time, might have different effects than the isolated exchanges. Alternative approaches include screening of more complex libraries, for example, through target cell loading with high diversity peptide pools, each randomized at all but one position of the peptide, or screenings against randomized peptide libraries presented as pMHC complexes on yeast surfaces (10, 32, 33). Further research directly comparing these approaches will be necessary to gain a deeper understanding of the respective strengths and weaknesses. Ultimately, safety screenings of clinical candidates should always be composed of multiple approaches, for example, by combining binding motif guided analysis together with cellular screenings of large panels of healthy tissue-derived cell lines, to minimizing risks. The results presented herein highlight the capability of this approach to identifying potentially relevant off-target interactions in combination with the pMHC-bsTCR binding kinetics screening platform. Because it offers quick analysis of complex pMHC libraries, it can be used early in the development process to select promising candidates and thus, complements established methods. This platform can also facilitate larger and more comprehensive screenings of late-stage candidates, potentially against mass spectrometry data-driven tissue-specific pMHC libraries covering the known immunopeptidome. Because of its stability and low-effort peptide loading procedure, the disulfide-modified HLA*02:01 molecules could potentially enable even higher-throughput platforms. Thanks to these properties, it could be perfectly suited for the creation of high complexity pMHC microarrays with thousands of different pMHC complexes, for example, by combining large-scale coating of disulfide-modified HLA*02:01 molecules and modern high-throughput peptide microarray inkjet printers.

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