Patent Description:
Presentation of peptides on cell surface MHC molecules plays a fundamental role for the immune response against viral infection or cancer (<NUM>). MHC class I molecules are trimeric complexes that consist of a polymorphic heavy chain, beta-<NUM> microglobulin (β<NUM>m) and a peptide ligand, typically between <NUM> and <NUM> amino acids long and derived from degradation of cytosolic proteins. T cells can recognize specific peptide-MHC complexes (pMHC) with their clone-specific T cell receptor (TCR) and initiate an immune response.

Production of soluble pMHC complexes is important for many different applications in scientific and clinical fields that are centered around the interaction between pMHCs and TCRs. They were first generated using protein expression and refolding techniques in <NUM> and have since then been used for many applications, e.g. identification of antigen-specific T cells through flow cytometry or affinity measurements of the TCR-pMHC interaction (<NUM> to <NUM>).

The affinity of the TCR towards its cognate pMHC has a substantial impact on the functionality of the expressing T cell (<NUM>). Thus, efforts have been made to improve the affinity of low-affinity TCRs to reach optimal levels for clinical applications (<NUM>). Extensive maturation experiments have produced TCRs with picomolar affinities, a range normally reserved to antibodies. They bind targeted pMHCs with long interaction half-lives even in monomeric form and have thus attracted attention as tumor cell engaging component in bi-specific T cell engager formats (<NUM>, <NUM>).

<CIT> discloses recombinant MHC class I molecules which are produced in bacteria and are present as an insoluble attachment body for a detection of epitope-specific CTL. These are first denatured in a solution of a chaotropic agent. The chaotrope is then removed in the presence of the desired peptide (renaturing, refolding) and the peptide class I complex is separated by gel filtration chromatography from the unfolded protein. <CIT> presents a gene for encoding an MHC class I molecule, the MHC class I molecule having an alpha <NUM> helix and an alpha <NUM> helix and the gene being encoded such that a bond is formed between the alpha <NUM> helix and the alpha <NUM> helix in the MHC class I molecule. Thus, a kit for analysis of T cell frequencies can be provided. Amino acid <NUM> is substituted by a cysteine so as to provide Cys-<NUM>, the amino acid <NUM> is substituted by the cysteine so as to provide Cys-<NUM> or the amino acid <NUM> is substituted by the cysteine so as to provide Cys-<NUM>, the disulfide bridge is formed between the alpha-<NUM> helix and the alpha-<NUM> helix in the MHC class I heavy chain between Cys-<NUM> and Cys-<NUM> or between Cys-<NUM> and Cys-<NUM>.

<CIT> discloses a so-called disulfide trap, comprising an antigen peptide covalently attached to an MHC class I heavy chain molecule by a disulfide bond extending between two cysteines. In some configurations, a disulfide trap, such as a disulfide trap single chain trimer (dtSCT), can comprise a single contiguous polypeptide chain. Upon synthesis in a cell, a disulfide trap oxidizes properly in the ER, and can be recognized by T cells. In some configurations, a peptide moiety of a disulfide trap is not displaced by high-affinity competitor peptides, even if the peptide binds the heavy chain relatively weakly. In various configurations, a disulfide trap can be used for vaccination, to elicit CD8 T cells, and in multivalent MHC/peptide reagents for the enumeration and tracking of T cells. Also disclosed are nucleic acids comprising a sequence encoding a disulfide trap. Such nucleic acids, which can be DNA vectors, can be used as vaccines.

<NPL>) describe a variant of the murine MHC-I allotype H-2Kb, in which the α1 and α2 helices are connected by a disulfide bond close to the F-pocket, restricting their mobility. The C84-C139 disulfide bond allows normal PLC interaction and antigen presentation but renders MHC-I surface expression TAP- and tapasin-independent, accelerates anterograde transport, and greatly decreases the rate of MHC-I endocytosis.

<CIT> discloses disulfide bond stabilized recombinant MHC class II molecules that are linked by a disulfide bond between cysteine residues located in the α2 domain of said α chain and the β2 domain of said β chain, wherein said cysteine residues are not present in native MHC class II α2 and β2 domains.

<CIT> discloses a Kon-rate assay and an improved TCR ligand koff-rate assay, which enables a broader application through a novel combination with UV peptide exchange technology. The disclosure enables Koff-rate MHC monomer preparation in a high throughput manner, which can then be used to screen TCR candidates for extended peptide libraries in assays such as the TCR ligand Koff-rate assay that was previously not feasible. Further, the UV peptide exchange with the Koff-rate MHC monomers allows the analysis of TCR candidates recognizing specific peptides carrying the amino acid cysteine, which previously could interfere with or even abolish the koff-rate measurement.

Newell et al. (in: <NPL>) disclose high content combinatorial peptide-MHC tetramer staining using mass cytometry.

Bakker et al. (in: <NPL>) disclose conditional ligands that disintegrate upon exposure to long-wavelength UV light that can be designed for the human MHC molecule HLA-A2. This peptide-exchange technology allegedly can be developed into a generally applicable approach for high throughput MHC based applications for an analysis of cytotoxic T cell immunity.

<NPL>) disclose tools to study the molecular mechanisms responsible for initiation of activation processes in T-cells. A topologically diverse set of oligomers of the human MHC protein HLA-DR1, varying in size from dimers to tetramers, was produced by varying the location of an introduced cysteine residue and the number and spacing of sulfhydryl-reactive groups carried on novel and commercially available cross-linking reagents. Fluorescent probes incorporated into the cross-linking reagents facilitated measurement of oligomer binding to the T-cell surface. Oligomeric MHC-peptide complexes, including a variety of MHC dimers, trimers and tetramers, bound to T-cells and initiated T-cell activation processes in an antigen-specific manner.

<NPL>) disclose a high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides from up to <NUM> samples in a plate format, suitable for both cell lines and tissues. The method is directed at improving the allegedly most critical step in the immunopeptidomics pipeline, the sample preparation, as it determines the overall peptide yield and reproducibility.

Luimstra et al. (in: <NPL>) disclose an allegedly simple, fast, flexible, and efficient method to generate many different MHC class I (MHC I) multimers in parallel using temperature-mediated peptide exchange. They designed conditional peptides for HLA-A*<NUM>:<NUM> and H-<NUM>b that form stable peptide-MHC I complexes at low temperatures, but dissociate when exposed to a defined elevated temperature. The resulting conditional MHC I complexes, either alone or prepared as ready-to-use multimers, can swiftly be loaded with peptides of choice without additional handling and within a short time frame.

<NPL>disclose a HLA-B27:<NUM> disulfide mutant.

<CIT> discloses a gene coded for a MHC class I molecule so that a bond is formed between the ALPHA-<NUM> helix and the ALPHA-<NUM> helix in the MHC class I molecule.

<NPL>, disclose a molecular dynamics simulation study of the conformational flexibility of the MHC Class I α1- α2 domain in peptide bound and free states.

<NPL> disclose the peptide-receptive transition state of MHC Class I molecules.

A potential downside of TCR affinity enhancement is the introduction of off-target toxicities. Due to the inherent cross-reactivity of TCRs these can arise by unknowingly increasing the affinity towards other pMHCs as well (<NUM>). Multiple cases like these have already been reported in clinical studies (<NUM> to <NUM>).

Comprehensive screening is therefore necessary not only to ensure efficacy but also specificity and safety of therapeutic candidates (<NUM>). This is a task of high complexity given the currently established size of the immunopeptidome, with at least <NUM>,<NUM> MHC class I ligand peptides identified by mass spectrometry, and the available methods for pMHC generation (<NUM>).

The large-scale generation of pMHC libraries and subsequent high throughput binding screenings of TCRs, e.g. for binding motif generation or the direct identification and characterization of potentially cross-reactive peptides are still difficult to achieve using common technologies in the art, like the ones above. This difficulty extends to the preparation of high quality pMHC complexes even in lower numbers for individuals or institutions without the necessary technically challenging facilities to produce pMHC, e.g. for time sensitive on demand production in clinical settings. It is therefore an object of the present invention, to provide improved strategies in this field. Other objects and aspects of the present invention will become apparent to the person of skill upon reading the following description of the invention.

The present invention relates to the subject matter of claims <NUM> to <NUM>.

According to a first aspect thereof, the above object of the invention is solved by a method for screening for a TCR-binding peptide ligand/MHC molecule complex (pMHC), comprising the steps of:.

Preferred is a method according to the invention, wherein said stabilized MHC molecule encompasses at least one artificially introduced covalent disulfide bridge between two amino acids, more preferable at least one artificially introduced covalent bridge between amino acids between α-helices, by (i) mutating an amino acid at position <NUM>, a phenylalanine in the majority of HLAs (see <FIG>) and an amino acid at position <NUM>, a serine in the majority of HLAs (see <FIG>), or (ii) mutating an amino acid at position <NUM>, a phenylalanine in the majority of HLAs (see <FIG>) and an amino acid at position <NUM>, a serine in the majority of HLAs (see <FIG>) and mutating an amino acid at position <NUM>, a tryptophan in the majority of HLAs (see <FIG>), and an amino acid at position <NUM>, a glycine in the majority of HLAs (see <FIG>) of MHC I (based on IGMT numbering excluding the first <NUM> amino acids). Such a stabilized MHC molecule may be referred to as disulfide-modified MHC molecule or disulfide-modified MHC mutant. Either the TCR or the MHC molecule can be suitably immobilized on a solid surface, such as a chip, glass slide, biosensor or bead, in particular as a high-throughput screening format.

In a second aspect the present invention provides a polypeptide comprising or consisting of a stabilized MHC molecule or a peptide binding fragment thereof, which is a HLA-A protein and comprises at least one artificially introduced covalent bridge between two amino acids in the alpha1 domain of the MHC I by mutating an amino acid in position <NUM> and an amino acid in position <NUM>.

Two amino acid positions that are modified, e.g. by artificially introducing a cysteine residue instead of the naturally occurring amino acid, to form a covalent bridge are selected based on their relative distance. If two amino acids in an MHC I that are not linked to each other by peptide bonds naturally have a distance to each other that is similar to the distance of a covalent bond, it is preferred that they are substituted by an amino acid that can form a covalent bond, e.g. a cysteine. Thus, preferably two amino acids are modified that have a distance of between <NUM> to <NUM>Å in the folded protein (determined between the alpha carbons of the respective amino acids). The 3D structures of a large number of MHC I molecules are known and the skilled person can use standard software to determine the distance between two given amino acids within the folded molecules.

According to the invention, two amino acids are modified in the alpha1 domain of the MHC I, namely one amino acid is modified in the β1 unit and one in the α1 unit of MHC I. Within the β1 unit the amino acid position is <NUM>. Within the α1 unit the amino acid position is <NUM>.

Further one amino acid can be modified in the alpha1 domain of an MHC I and one amino acid in the alpha2 domain of an MCH I within amino acid positions <NUM> to <NUM>, it is preferred that the one amino acid in the alpha1 domain is modified in the α1 unit, preferably within amino acid positions <NUM> to <NUM>, more preferably within amino acid positions <NUM> to <NUM>, more preferably <NUM> to <NUM> and most preferably amino acid position <NUM>. It is preferred that the other amino acid in the alpha2 domain is modified in the α2 unit, suitable regions are within amino acid positions <NUM> to <NUM>, preferably within amino acid positions <NUM> to <NUM>, more preferably within amino acid positions <NUM> to <NUM> and most preferably amino acid position <NUM>. In each case the two amino acids are preferably selected within the respectively indicated amino acid stretches to have a distance of between <NUM> to <NUM>Å in the folded MHC I protein. Thus, in a particularly preferred embodiment the stabilized MHC I comprises further a modified amino acid at position <NUM> and at position <NUM>.

It is further preferred that within one MHCl two pairs of modified amino acids are comprised.

Particularly, it is preferred that the second pair of modified amino acids comprise one amino acid that is modified in the alpha1 domain of an MHC I and one amino acid in the alpha2 domain of an MCH I within amino acid positions <NUM> to <NUM>. It is preferred that the one amino acid in the alpha1 domain is modified in the α1 unit, preferably within amino acid positions <NUM> to <NUM>, more preferably within amino acid positions <NUM> to <NUM>, more preferably <NUM> to <NUM> and most preferably amino acid position <NUM>. Particularly, suitable regions for modifying the other amino acid within the alpha2 domain are within amino acid positions <NUM> to <NUM>, preferably within amino acid positions <NUM> to <NUM>, more preferably within amino acid positions <NUM> to <NUM> and most preferably amino acid position <NUM>. Thus, in a particularly preferred embodiment the stabilized MHC I comprises a first pair of modified amino acids at position <NUM> and <NUM> and a second pair of modified amino acid at position <NUM> and at position <NUM>.

Any of above modifications of MHC I may further be combined with a pair of modifications wherein the first modified amino acid is within amino acid positions <NUM> to <NUM>, preferably within amino acid positions <NUM> to <NUM>, and more preferably amino acid position <NUM> and the second amino acid is within amino acid positions <NUM> to <NUM>, preferably within amino acid positions <NUM> to <NUM>, and more preferably amino acid position <NUM>.

It was surprising that the modification of amino acids in the above-described amino acid regions of MHC I and at the respectively indicated positions and, thus the introduction of covalent bonds between amino acids at position which are not naturally connected by covalent bonds allows the generation of modified MHC I molecules that: (i) are properly folded, (ii) bind peptides with high affinity and (iii) are recognized by TCR molecules with high specificity and selectivity.

The preferred modified MHC I molecules of the second aspect can also be used in all other aspects of the present invention.

The present invention also comprises peptide binding fragments of the modified MHC I molecules. As known in the art, MHC I bind to peptides and are in turn bound by TCRs that interact both with the MHC I and the peptide. However, only parts of the MHC I molecule are required for binding to the peptide that is "presented" to the TCR. In MHC I the alpha1 and alpha2 domain fold to form a binding groove that binds the peptide. Thus, peptide binding fragments of MHC I comprise at least the alpha1 and alpha2 domain. Accordingly, the binding fragment may lack the transmembrane domain or additionally the alpha3 domain in MHC I. Fragments lacking at least the transmembrane domain are soluble and are particularly suitable to be used in a pharmaceutical composition, in particular in a vaccine.

In a third aspect thereof, the present invention provides a method for detecting or generating a specific amino acid binding motif for a TCR, comprising performing the method according to the first aspect thereof comprising a preselected TCR, and the additional step of determining and comparing the amino acid sequences of those peptide ligands in said peptide ligand/MHC molecule complexes for which a TCR binding was detected, thereby identifying the specific amino acid binding motif for said preselected TCR.

In a fourth aspect thereof, the present invention provides a method for detecting or determining cross-reactivity of a TCR, comprising performing the method according to the second aspect of the invention, and the additional step of determining and comparing the amino acid sequences of those peptide ligands in said peptide ligand/MHC molecule complexes for which a TCR binding was detected, thereby identifying cross-reactivity of said TCR.

In a fifth aspect thereof, the present invention provides a method for detecting or determining cross-reactivity of a TCR, comprising performing the method according to the first aspect of the invention comprising a preselected TCR, and the additional step of determining and comparing the amino acid sequences of those peptide ligands in said peptide ligand/MHC molecule complexes for which a TCR binding was detected, thereby identifying cross-reactivity of said TCR.

The methods according to the present invention can be used for screening or in vitro priming of cellular drug products. The stabilized HLA complexes bound to beads, filaments, nanoparticles or other carriers can be readily loaded with a peptide of interest mimicking antigen presenting cells, and afterwards conveniently used in combination with costimulatory molecules (e.g. anti CD28, anti <NUM>1BB) as "ready to use" artificial antigen presenting cells for in vitro priming and expansion.

Current methods for the large-scale generation of pMHC libraries, a high throughput binding motif determination of a high affinity TCR, and the identification and characterization of potentially cross-reactive peptides suffer from stability problems, requiring multimers to be swiftly loaded with peptides of choice without additional handling and within a short time frame (as in Luimstra et al. , above), which also makes technologies like UV exchange unsuitable.

With the present technology, the inventors gain multiple advantages over the wild type molecule or other existing exchange technologies: the empty monomer can be produced in bulk way ahead of the desired experiment and pMHC generation is not restricted by any other method aside from procuring desired peptides and quick peptide loading reactions. The inventors have successfully stored the empty monomer for at least a year at -<NUM> and used them with no degradation or impaired peptide receptiveness detected. The inventors have also successfully stored the resulting pMHC complexes for at least two weeks at <NUM> and reused them for affinity measurements without loss of signal. In addition to all these advantages achieved by introducing the modification, pMHC complexes generated displayed by the mutant are substantially representative of wild type complexes with respect to TCR ligand binding.

In one aspect, the invention provides a method for screening for a TCR-binding peptide ligand/MHC molecule complex for TCR-binding.

The method comprises the use of a suitably stabilized MHC molecule that comprises at least one artificially introduced covalent bridge between two amino acids of the alpha1 domain of said stabilized MHC I molecule by mutating an amino acid in position <NUM> and an amino acid in position <NUM>. Major histocompatibility complex class I and class II share an overall similar fold. The binding platform is composed of two domains, originating from a single heavy α-chain (HC) in the case of MHC class I. The two domains evolved to form a slightly curved β-sheet as a base and two α-helices on top, which are far enough apart to accommodate a peptide chain in-between. Hence, suitable stabilization for the method according to the present invention can be achieved.

In one embodiment, the present invention involves the use of disulfide-stabilized, initially empty, MHC molecules that can be loaded by simply adding suitable peptide before the use thereof. pMHCs generated using this disulfide-modified MHC molecule are representative of the non-modified wild type variant, and are suitable for screening, e.g. high throughput binding motif determination of a high affinity TCR as well as identification and characterization of potentially cross-reactive peptides.

The empty MHCs do not substantially degrade on commonly used surfaces, like glass plates, are representative for the non-modified wild type variant when loaded with peptide, and are suitable for screening, and allow to generate pMHCs quickly, even when immobilized on a surface. In the context of the present invention, this is achieved by and understood as a "suitably stabilized" or "stabilized" pMHC.

In previous studies with the murine MHC class I molecule H-<NUM>b introduction of a disulfide bond between opposing residues in the F-pocket by mutating a tyrosine at position <NUM> and an alanine at position <NUM> to cysteines was able to stabilize the complex. Thus, an artificially introduced covalent bridge between amino acids was introduced between α-helices, for example by mutating a tyrosine at position <NUM> and an alanine at position <NUM> into cysteines of MHC I. While in some cases, it may be difficult to isolate monomers without any peptide ligand, this could be efficiently overcome by adding a low affinity peptide.

The term "MHC" is an abbreviation for the phrase "major histocompatibility complex". MHC's are a set of cell surface receptors that have an essential role in establishing acquired immunity against altered natural or foreign proteins in vertebrates, which in turn determines histocompatibility within a tissue. The main function of MHC molecules is to bind to antigens derived from altered proteins or pathogens and display them on the cell surface for recognition by appropriate T-cells. The human MHC is also called HLA (human leukocyte antigen) complex or HLA. The MHC gene family is divided into three subgroups: class I, class II, and class III. Complexes of peptide and MHC class I are recognized by CD8-positive T-cells bearing the appropriate TCR, whereas complexes of peptide and MHC class II molecules are recognized by CD4- positive-helper-T-cells bearing the appropriate TCR. Since both types of response, CD8 and CD4 dependent, contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens and corresponding TCRs is important in the development of cancer immunotherapies such as vaccines and cell therapies. The MHC I molecule consists of an alpha chain, also referred to as MHC I heavy chain and a beta chain, which constitutes a beta <NUM> microglobulin molecule. The alpha chain, interchangeably used with heavy chain in the context of the present invention, comprises three alpha domains, i.e. alpha1 domain, alpha2 domain and alpha3 domain. Alpha1 and alpha2 domain mainly contribute to forming the peptide pocket to produce a peptide ligand MHC (pMHC) complex. The alpha1 domain of a MHC I spans amino acid positions <NUM> to <NUM> and comprises as secondary structure a β-sheet spanning amino acid positions <NUM>-<NUM> (termed herein "β1 unit") followed by an α-helix structure spanning amino acid positions <NUM>-<NUM> (termed herein "α1 unit"). The alpha2 domain of a MHC I spans amino acid positions <NUM> to <NUM> and comprises as secondary structure a β-sheet spanning amino acid positions <NUM>-<NUM> (termed herein "β2 unit") followed by an α-helix structure spanning amino acid positions <NUM>-<NUM> (termed herein "α2 unit"). The beta1 domain of a MHC II is on a separate polypeptide and fulfills within MHC II the structural role of the alpha2 domain of MHC I. It spans amino acid positions <NUM> to <NUM> and comprises as secondary structure a β-sheet spanning amino acid positions <NUM> to <NUM> (termed herein "β3 unit") followed by an α-helix structure spanning amino acid positions 50to <NUM> (termed "α3 unit"). Here and in each other case in which reference is made to an amino acid position in an MHC I or MHC II molecule the positions are indicated based on IGMT numbering excluding the N-terminal first signal peptide, which typically varies in length between <NUM> to <NUM> amino acids.

HLAs are molecules which differ between different human beings in amino acid sequence. However, HLAs can be identified by an internationally agreed nomenclature, the IMGT nomenclature, of HLA. The categorization to, e.g. HLA-A, is based on the identity of a given HLA to official reference sequences of each HLA, that were produced by sequence alignments. Thus, a given HLA sequence with the highest sequence identity to the HLA-A sequence according to SEQ ID NO: <NUM> will be categorized as HLA-A. The official HLA reference sequences as well as information to the categorization system are available: www. uk/ipd/imgt/hla/nomenclature/alignments. The website provides the following information regarding how to categorize any given HLA sequence:
"<NPL>.

For MHC class I proteins the following HLA reference protein sequences are indicated on July <NUM>, <NUM> on the web site in each case indicating the accession number that will not change for each HLA over time:.

The HLA-A gene is located on the short arm of chromosome <NUM> and encodes the larger, α-chain, constituent of HLA-A. Variation of HLA-A α-chain is key to HLA function. This variation promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of certain structures, greater variety of HLAs means greater variety of antigens to be 'presented' on the cell surface. Each individual can express up to two types of HLA-A, one from each of their parents. Some individuals will inherit the same HLA-A from both parents, decreasing their individual HLA diversity. However, the majority of individuals receive two different copies of HLA-A. The same pattern follows for all HLA groups. In other words, every single person can only express either one or two of the <NUM> known HLA-A alleles coding for currently <NUM> active proteins. HLA-A*<NUM> signifies a specific HLA allele, wherein the letter A signifies to which HLA gene the allele belongs to and the prefix "*<NUM> prefix" indicates the A2 serotype. In MHC class I dependent immune reactions, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized by T-cells bearing specific TCRs.

In the second step of the preferred method according to the invention, the suitably stabilized MHC molecule is contacted with a multitude of peptide ligands, in order to form peptide ligand/MHC (pMHC) molecule complexes. 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.

"Contacting" in the context of the present invention shall mean that peptide(s) is (are) brought in contact with the empty and/or low affinity peptide-loaded MHC molecules in such a way that a substantial portion of the peptides form complexes (are "loaded") with said empty and/or low affinity peptide-loaded MHC molecules. As one preferred example, loading MHC complexes was performed by addition and mixing of desired peptides of at least a <NUM> to <NUM> molar ratio to the monomer solution in a suitable buffer, and a minimum of <NUM> minutes incubation at room temperature.

The groove in-between the two helices accommodates peptides based on (i) the formation of a set of conserved hydrogen bonds between the side-chains of the MHC molecule and the backbone of the peptide and (ii) the occupation of defined pockets by peptide side chains (anchor residues P2 or P5/<NUM> and PΩ in MHC class I). The type of interactions of individual peptide side-chains with the MHC depend on the geometry, charge distribution, and hydrophobicity of the binding groove. In MHC class I, the binding groove is closed at both ends by conserved tyrosine residues leading to a size restriction of the bound peptides to usually <NUM>-<NUM> residues with its C-terminal end docking into the F-pocket. In contrast, MHC class II proteins usually accommodate peptides of <NUM>-<NUM> residues in length in their open binding groove, with the peptide N-terminus usually extruding from the P1 pocket. It has been reported that the interactions at the F pocket region in MHC class I and the P1 region (including the P2 site) in MHC class II appear to have a dominant effect on the presentation of stable pMHC complexes and on the immunodominance of certain peptidic epitopes. Interestingly, these pockets are located at opposite ends of the binding groove of the respective MHC class I and MHC class II structures.

The multitude of peptide ligands can comprise at least about <NUM>,<NUM> different MHC binding peptides, preferably at least about <NUM>,<NUM> different MHC binding peptides, more preferred at least about <NUM>,<NUM> different MHC binding peptides, and most preferred an immunopeptidome preparation with at least about <NUM>,<NUM> MHC binding peptides. Said peptides comprise a binding motif of <NUM>-<NUM> residues in length for MHC class I proteins and <NUM>-<NUM> residues in length for MHC class II proteins, and can be of a length of between <NUM> and <NUM>, preferably of between <NUM> and <NUM>, more preferred between <NUM> and <NUM> residues. Most preferred are peptides that consist of the actual binding motif.

Ligand peptides as used in the context of the present invention can be derived from polypeptides that are cancer-related, infection-related (bacterial or viral), and even immune- (e.g. autoimmune-) disease related. The term also includes suitably mutated or naturally occurring mutated ligand peptides, i.e. different from their underlying sequence as occurring in the respective polypeptide.

Preferred is the method according to the present invention, wherein said contacting comprises loading said MHC binding peptides onto the MHC at between about <NUM> to <NUM>, preferably at about room temperature (<NUM>° to <NUM>, preferably <NUM>).

It was surprisingly found that the loaded HLA/peptide molecules (pMHC or pMHC complex) are very stable for more than about <NUM> day, and preferably for more than <NUM> week at (e.g. more than <NUM> weeks) at about <NUM>. This allows an effective and convenient use in many more applications than in known methods as described above.

It was also found in the context of the present invention, and somewhat in contrast to the literature as above, that the present method was clearly superior to the popular method of UV exchange using a WT pMHC molecule, allowing to perform it (in particular in a high-throughput format) on a surface, like a chip or glass slide. 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. This uncertainty is a problem for affinity measurements with pMHCs used as soluble analytes, as precise knowledge of the concentration is required to determine accurate affinities. Since the disulfide-stabilized MHC mutant is stable without 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.

In the next step of the method of the present invention, said pMHC molecule complexes are screened for a TCR-binding. The binding and kinetic attributes of this interaction are parameters for protective T cell-mediated immunity, with stronger TCR-pMHC interactions showing increased interaction half-life and thus conferring superior T cell activation and responsiveness than weaker ones. The interaction strength between the TCR and pMHC ligand is typically described and measured as the dissociation constant Kd, an equilibrium constant that is a ratio between the on-rate constant kon and off-rate constant koff of a specific interaction. The dissociation constant Kd inversely correlates with the binding strength of a specific interaction, as smaller Kd values represent stronger binding.

The screening can comprise any suitable and known method for measuring and/or detecting pMHC/TCR-binding, e.g. structural TCR-pMHC affinity/avidity measurements. One example is screening of a peptide-MHC library for TCR binding by bio-layer interferometry (BLI), a special form of reflective interferometry (RI), as disclosed herein, where binding interactions for said TCR were detected stronger than a sensitivity threshold suitable for the method of Kd <NUM> x <NUM>-<NUM>, with measured Kd values ranging from <NUM> x <NUM>-<NUM> to <NUM> x <NUM>-<NUM>, or no binding interactions for said TCR were detected when weaker than the sensitivity threshold.

Other methods involve other forms of RI, like surface plasmon resonance (SPR), or reflective interferometric spectroscopy (RIfS), or single-color reflectometry (SCORE, Biametrics, Tübingen, Germany), or marker-based assays, e.g. flow cytometric analysis with NTAmers (TCMetrix, Epalinges, Switzerland), or pMHC or TCR tetramers, or other forms of fluorescent readouts, like protein microarrays. Of course, ideally these methods can be performed in/can be readily adjusted to high-throughput formats.

In the context of the present invention, the term "about" shall mean to include +/- <NUM>% of a given value, unless otherwise noted.

The present invention as an example presents the use of disulfide-stabilized empty HLA-A*<NUM>:<NUM> molecules which can be loaded by simply adding peptide before use. pMHCs generated using this modified MHC molecule are representative of the non-modified wild type variant and thus, demonstrate suitability for high throughput binding motif determination of a high affinity TCR as well as identification and characterization of potentially cross-reactive peptides.

Preferred is a method according to the present invention, wherein said MHC molecule is a multimer of HLA-A selected from the group consisting of a dimer, a trimer and a tetramer. Methods using more than one MHC molecule at once in screenings are known in the art, e.g. from <NPL>. Similarly, dimers or trimers can be used.

The MHC molecules as used include at least one artificially introduced covalent bridge between amino acids. This bridge is selected from a recombinantly introduced disulfide bridge, the introduction of non-natural amino acids to be crosslinked, the introduction of photo-crosslinking amino acids, and chemically introduced crosslinks. The introduction of crosslinks using cysteines is described herein; examples for dimeric cross-linking reagents are DPDPB and HBVS, and the trimeric cross-linker TMEA.

Preferred is a method according to the present invention, wherein said at least one artificially introduced covalent bridge between amino acids is introduced between α-helices, for example by (i) mutating an amino acid at position <NUM> of MHC I, a phenylalanine in the majority of HLAs (see <FIG>) and an amino acid at position <NUM> of MHC I, a serine in the majority of HLAs (see <FIG>), or (ii) mutating an amino acid at position <NUM> of MHC I, a phenylalanine in the majority of HLAs (see <FIG>) and an amino acid at position <NUM> of MHC I, a serine in the majority of HLAs (see <FIG>) and mutating an amino acid at position <NUM> of MHC I, a tryptophan in the majority of HLAs (see <FIG>), and an amino acid at position <NUM> of MHC I, a glycine in the majority of HLAs (see <FIG>) of MHC I (based on IGMT numbering excluding the first <NUM> amino acids). Molecular dynamics simulations of the α<NUM> and α<NUM> domain or of entire MHC-I have suggested one eminent difference between empty and peptide-bound MHC-I: in the absence of a peptide, the helical sections that flank the F-pocket region (residues <NUM>-<NUM> and <NUM>-<NUM> in the α<NUM> and α<NUM> helices, respectively) are significantly more mobile. It seems that bound peptides restrict the mobility of this region, and that a similar advantageous and stabilizing conformational restriction might be achieved by linking different structural features of the peptide binding pocket with a covalent bond, preferably a disulfide bond.

To determine amino acids at positions corresponding to above mentioned residues <NUM>, <NUM>, <NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM> in each given HLA allele the respective sequence is aligned with the above indicated reference antibodies. An example of the alignment of multiple sequences of official HLA (MHC class I) reference protein sequences and murine MHC I H2Kb protein; highlighting amino acid positions <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> (bold) and further regions suitable for introducing stabilizing mutations (grey) is shown in <FIG> will enable the skilled person to identify the amino acids at positions corresponding to above mentioned residues <NUM>, <NUM>, <NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM> in each given HLA allele.

According to the present invention, the MHC I molecule is a MHC class I HLA-A protein. These preferred HLA proteins can be mutated in their α<NUM> domain and α<NUM> domain, respectively, according to the reference sequences of the IMGT nomenclature. These HLA proteins are mutated, one amino acid is mutated at position <NUM> and one amino acid is mutated at position <NUM>. Preferably, one amino acid is mutated at position <NUM> and one amino acid is mutated at position <NUM> and one amino acid is mutated at position <NUM> and one amino acid is mutated at position <NUM>. Preferred amino acid mutations are substitutions of one amino acid at positions <NUM> to cysteine. Even more preferred amino acid mutations are substitutions of one amino acid at positions <NUM> to cysteine. Even more preferred amino acid mutations are substitutions of one amino acid at positions <NUM> to cysteine. Even more preferred amino acid mutations are substitutions of one amino acid at positions <NUM> to cysteine.

In another preferred embodiment the HLA-A protein is selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, and HLA-A11. These preferred HLA-A proteins can be mutated in their α<NUM> domain and α<NUM> domain, respectively, according to the reference sequences of the IMGT nomenclature. It is even more preferably that the HLA-A protein is a HLA -A*<NUM> protein. Preferred HLA-A alleles are HLA-A*<NUM>:<NUM> ; HLA-A*<NUM>:<NUM> or HLA-A*<NUM>:<NUM>.

In the context of the present invention, the term "TCR" shall include any proteinaceous molecule/construct that comprises a TCR-derived or TCR-like binding domain, wherein the molecule/construct is suitable for the analysis/detection of pMHC/TCR binding according to the invention as described herein. In the case of the α- and/or β-chain of a TCR, this may include a molecule where both chains remain able to form a T-cell receptor (either with a non- modified α- and/or β-chain or with a fusion protein or modified α- and/or β-chain) which exerts its biological function, in particular binding to a (specific) pMHC, and/or functional signal transduction upon peptide activation. Preferred is a method according to the present invention, wherein said TCR is selected from a native TCR, a soluble TCR molecule, a single-chain TCR, and a TCR-like molecules comprising a TCR-derived or TCR-like binding domain (e.g. derived from an antibody), such as a bispecific (bs) TCR, for example like the ones as described herein.

The methods according to the present invention in preferred embodiments allow for a parallel detection, analysis and/or screening of a much larger number of peptide ligands and/or pMHC, when compared to common technologies, including UV exchange-related methods. The collection of peptides presented to the cell surface by class I and class II human leukocyte antigen (HLA) molecules are referred to as the immunopeptidome. In May <NUM>, already <NUM>,<NUM> high-confidence HLA class I peptides and <NUM>,<NUM> high-confidence HLA class II peptides were reported (<NPL>), and therefore it can be expected that the human immunopeptidome exceeds <NUM>,<NUM> MHC binding peptides for each of class I and II. Current methods can analyze about <NUM> peptides a day, so that there is a demand for "true" high throughput methods, i.e. a multitude of peptide ligands as analyzed that comprises at least about <NUM>,<NUM> different MHC binding peptides, preferably at least about <NUM>,<NUM> different MHC binding peptides, more preferred at least about <NUM>,<NUM> different MHC binding peptides, and most preferred a substantially complete immunopeptidome preparation with at least about <NUM>,<NUM> MHC binding peptides.

The inventive methods allow for immunopeptidome-wide screening for as short of a period as within a day.

In view of the number of pMHC/TCR bindings to be detected/analyzed, preferred is a method according to the present invention, wherein said method is performed as a high-throughput screening (HTS) format. In HTS, up to hundreds of thousands of experimental samples can be subjected to simultaneous testing for pMHC/TCR binding under given conditions. The samples are usually and preferably handled by laboratory robotics that automate sample preparation, handling and data analysis. HTS thus easily and reliably generates and uses large datasets to answer complex biological questions, e.g. pMHC/TCR binding kinetics and biological function as described herein.

HTS classically requires samples to be prepared in an arrayed format. If necessary, the arrayed samples can be grown either on microtiter plates in liquid, or on solid agar. The density of plates can range from <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM>, or <NUM>,<NUM>. All these densities are multiples of <NUM>, reflecting the original <NUM>-well microtiter plate arranged in <NUM> x <NUM> with <NUM> spacing (see also, for example, <NPL>).

For uses relating to pMHC/TCR binding kinetics as detected/analyzed and as described herein, a solid surface, such as a chip, biosensor, glass slide or bead can be used, onto which some of the analysis reagents (e.g. either the TCR or the MHC molecule) can be suitably immobilized, e.g. spotted. For immobilization, any suitable technique can be used, e.g. by biotin streptavidin interaction. Examples of the embodiments as described here are binding assays involving binding of at least one soluble TCR(s) against at least one immobilized pMHC(s), or binding of at least one immobilized TCR(s) against at least one soluble pMHC(s).

Preferred is the method according to the present invention, wherein said TCR and/or the MHC molecule is/are not labelled or suitably labelled with a detectable marker. Respective markers are known in the art and include direct or indirect labelling with radioactive, fluorescent or chemical groups (e.g. dyes). Also, enzymatic markers or antigenic markers (for a detection with antibodies) as well as mass markers can be used. Another option is coding markers (e.g. specific nucleic acids). In case of no labelling, a detection of the binding based on changes in the physical state upon complex formation/binding can be used in order to identify binding, such as a change in mass, charge, or changes in optical properties, for example of the optical thickness of the biolayer by analyte binding, and thus of the interference pattern or reflection coefficient.

Methods to detect a binding, in particular a "specific" binding of a pMHC with a TCR are known in the art. In the present invention, preferred is a method according to the present invention, wherein Kd values as well as kon and koff values can be measured for said TCR, preferably with sensitivity between Kds of <NUM> x <NUM>-<NUM> M and <NUM> x <NUM>-<NUM> M, where sensitivity can be directed by analyte concentration.

As one preferred example, the affinity is measured using <NUM>:<NUM> analyte dilution series starting at <NUM>, or using <MAT> analyte dilution series starting at <NUM>. As one preferred example, the peptide ligand/MHC molecule complexes are used in parallel assay reactions having different concentrations.

In yet another important aspect of the method according to the present invention, said method further comprises the step of measuring T cell activation comprising a TCR and a TCR-binding peptide ligand/MHC molecule complex that binds said TCR. Methods to detect such T cell activation through a binding, in particular a "specific" binding of a pMHC to a TCR are known in the art. In the present invention, as an example, co-incubation assays with peptide loaded target cells, Jurkat effector cells and bs-868Z11-CD3 at six different concentrations were performed, and a correlation of measured affinity for the peptide ligands from the positional scanning library with the lowest bsTCR concentration necessary to induce <NUM>-fold luminescence increase over background was taken as a cut-off.

Yet another important aspect of the invention is a method for detecting or generating a specific amino acid binding motif for a TCR, comprising performing the method according to the present invention as described herein, wherein a preselected TCR is chosen, for which a specific amino acid binding motif is to be detected or generated. The method comprises a) providing a suitably stabilized MHC I molecule, wherein said MHC molecule is a HLA-A protein and comprises at least one artificially introduced covalent bridge between two amino acids of the alpha1 domain of said stabilized MHC molecule by mutating an amino acid in position <NUM> and an amino acid in position <NUM>, b) contacting said suitably stabilized MHC molecule with a multitude of peptide ligands thereof, to form peptide ligand/MHC (pMHC) molecule complexes, and c) screening said pMHC molecule complexes for TCR-binding using said pre-selected TCR. In an additional step, the amino acid sequences of those peptide ligands in said peptide ligand/MHC molecule complexes for which a TCR binding was detected are determined and optionally and preferably compared, resulting in identifying the specific amino acid binding motif for said preselected TCR.

One additional embodiment comprises a mutagenesis of a particular amino acid sequence after the identification thereof, and contacting said mutated peptides with a suitably stabilized MHC molecule, and screening said pMHC molecule complexes for TCR-binding with a preselected TCR to obtain an amino acid binding motif for said preselected TCR. The mutagenesis of peptides can easily be performed, for example by synthesizing mutated peptides, or chemically modifying existing amino acids in respective peptide binders. The mutagenesis can also involve adding markers or other groups to the peptide(s) in order to identify diagnostically effective binders. This aspect relates to the method according to the present invention as described herein, wherein said method steps are repeated comprising a pool of peptides consisting of modified amino acid sequences for said preselected TCR as identified. The modification can furthermore be guided by one of the known computer algorithms and/or programs used to calculate improved binding parameters based on modifications of the amino acid sequence(s).

One example thereof is the screening of a pMHC complex library, comprised of peptides created in said fashion, against a preselected TCR for TCR binding by bio-layer interferometry (BLI) as disclosed herein, where binding interactions for said TCR were detected stronger than a sensitivity threshold suitable for the method of Kd <NUM> x <NUM>-<NUM> M, with measured Kd values ranging from <NUM> x <NUM>-<NUM> M to <NUM> x <NUM>-<NUM> M, or no binding interactions for said TCR were detected when weaker than the sensitivity threshold. In said embodiment the present invention shows particular improvement over existing methods, as generation of pMHC complexes with a suitably stabilized MHC molecule generates predictable amounts of pMHC, thus increasing Kd measurement accuracy compared to existing methods (<FIG>).

In one additional embodiment, the multitude of peptide ligands is mostly composed of known peptide ligands from the immunopeptidome, as identified e.g. by mass spectrometry, wherein a preselected TCR is screened for TCR-binding to directly identify existing cross-reactive peptide ligands for said TCR. Preferred is the method according to this embodiment where the number of different peptides comprises at least about <NUM>,<NUM> different MHC binding peptides, preferably at least about <NUM>,<NUM> different MHC binding peptides that are measured in parallel.

Yet another important aspect of the invention is a method for detecting or determining cross-reactivity of a TCR, comprising performing the method for detecting or generating a specific amino acid binding motif for a TCR as described herein, and the additional step of determining and comparing the amino acid sequences of those peptide ligands in said peptide ligand/MHC molecule complexes for which a TCR binding was detected, thereby identifying cross-reactivity of said TCR. This aspect detects variants of a peptide that are recognized by a single TCR.

Yet another important aspect of the invention is a method for detecting or determining cross-reactivity of a TCR, comprising performing the method for screening for a TCR-binding peptide ligand/MHC molecule complex for TCR-binding according to the present invention as described herein comprising a preselected TCR, and the additional step of determining and comparing the amino acid sequences of those peptide ligands in said peptide ligand/MHC molecule complexes for which a TCR binding was detected, thereby identifying cross-reactivity of said TCR. This aspect also detects variants of a peptide that are recognized by a single TCR.

One example thereof is identification of a cross-reactive peptide ligand based on the amino acid binding motif, previously determined by screening a preselected TCR for TCR-binding with a mutagenesis derived pMHC complex library according to the present invention, and searching for a matching peptide ligand in a database of known or assumed peptide ligands.

Yet another important aspect of the invention is a method for detecting or determining cross-reactivity of a peptide ligand/MHC molecule complex, comprising performing the method for screening for a TCR-binding peptide ligand/MHC molecule complex for TCR-binding according to the present invention as described herein comprising a preselected pMHC, and the additional step of identifying of those TCRs for which a pMHC binding was detected, thereby identifying cross-reactivity of said TCR. This aspect detects variants of TCRs that recognize a single peptide.

In these aspects, the same methods to detect a binding of a pMHC with a preselected TCR can be used as above. Nevertheless, as the TCR binding is not necessarily required to be specific, the cut-off value and sensitivity for measuring and evaluating binding does not need to be optimal, and should be chosen as best suited under the respective circumstances, which will be comprehensible to a person of skill.

In another important aspect of the methods according to the present invention, said methods can further comprise the step of measuring T cell activation comprising a TCR and a TCR-binding peptide ligand/MHC molecule complex that binds said TCR. Methods to detect such T cell activation through a binding, in particular a "specific" binding of a pMHC to a TCR are known in the art. In the present invention, as an example, co-incubation assays with peptide loaded target cells, Jurkat effector cells and bs-868Z11-CD3 at six different concentrations were performed, and a correlation of measured affinity for the peptide ligands from the positional scanning library with the lowest bsTCR concentration necessary to induce <NUM>-fold luminescence increase over background was taken as a cut-off.

Also disclosed herein is a method for the improved personalized identification of T cell receptors, or activation of T-cells, and/or T-cell therapeutics against proliferative diseases, such as cancer, by stimulation with pMHC complexes to generate cellular drug products for a specific patient. Such stimulation can be based on pMHC complexes loaded with peptides identified by obtaining/providing a sample of cancer tissue and/or cancer cells from said patient, providing obtaining/providing a sample of normal tissue and/or cells from said patient, detecting peptides as presented in the context of MHC in said sample(s) using the XPRESIDENT® or comparable method, and determining the sequence(s) of at least one of said peptides, optionally, detecting the expression of the underlying genes of said peptides as determined, detecting the MHC presentation level/number of the peptides as detected in said sample(s), optionally comparing said MHC presentation level/number of the peptides as detected in said tumor and normal tissue and/or cell samples, screening for an optimized TCR-binding peptide ligand/MHC molecule complex, comprising a method according to the present invention. Said T-cells include those recovered directly from said patient which can be re-administered after said stimulation as cellular drug product. Said stimulation can include the use of preproduced stimulation frameworks, produced by immobilization of a suitably stabilized MHC molecule, preferably produced under clinical grade conditions (e.g. GMP), onto a carrier, for example filaments or beads, that are then loaded with peptide on demand, for example directly at the clinical site. These stimulation frameworks can also include other costimulatory molecules (e.g. anti CD28 antibodies, anti 41BB antibodies) immobilized together with the suitably stabilized MHC molecule.

Said peptide, e.g. a peptide specific for a certain type of cancer or other kind of proliferative disease, may already be known to the entity performing the procedure through previous identification in another patient or patients. Said peptide can thus be selected and produced quickly for a different patient bearing the same type of cancer, loaded on said stimulation framework and used to produce a cellular drug product.

Said process of activation of T-cells, and/or T-cell therapeutics recovered directly from said patient may also comprise transducing the T-cells to express a tumor-specific exogenous T-cell receptor (TCR), and, optionally, suitably formulating said resulting T-cell therapeutic.

The term "T cell" refers to T lymphocytes as defined in the art and is intended to include recombinant T cells. As used herein, the terms "T-cell receptor" and "TCR" refer to a molecule found on the surface of the T cell responsible for recognizing the antigens that bind to MHC molecules, and customarily refer to a molecule capable of recognizing a peptide when presented by a MHC molecule. The molecule is a heterodimer including α and β chains (or selectively, γ and δ chains) or a TCR construct that generates signals. The TCR of the present invention is a hybrid TCR including the sequences derived from other species. For example, as mouse TCRs are more effectively expressed than human TCRs in human T cells, the TCR includes a human variable region and a murine constant region. The term also includes soluble TCR molecules, and derivatives thereof, as long as they include the complementarity determining regions (CDRs) as necessary for binding.

The XPRESIDENT® technology is described, amongst others, in <CIT>, <CIT>, and <CIT>.

Said developing improved personalized T-cell receptors, T-cells, and/or T-cell therapeutics against proliferative diseases may further comprise transducing the patient's autologous (own) T-cells to express a tumor-specific exogenous T-cell receptor (TCR), and, optionally, suitably formulating said resulting T-cell therapeutics.

The present inventors demonstrate that the disulfide-modified HLA-A*<NUM>:<NUM> molecule as an example can be readily generated as a stable and empty MHC monomer, loaded with ligand peptides after refolding, and used to generate affinity data in good agreement with data collected using wild type pMHC complexes.

Both disulfide-modified HLA-A*<NUM>:<NUM> molecules and bispecific TCRs can be used jointly with BLI-based screenings to measure pMHC-bsTCR binding affinities, a platform with much higher throughput than surface plasmon resonance measurements presently used for these interactions in the literature. Disulfide-modified HLA-A*<NUM>:<NUM> molecules are a piece of this platform, providing reliable yet high-throughput pMHC generation. This platform could also be useful for the analysis of other biologics if targeting pMHCs, like monoclonal antibodies or bispecifics (e.g. BITEs). The pMHC-bsTCR binding affinities correlated well with cellular assays when both were performed by the inventors with a functional bispecific T cell engager. To the inventors' knowledge, this is the first in depth analysis of the connection between pMHC-bsTCR binding affinity and the in vitro activity over a wide range of affinities. Compared to the cellular screenings, the affinity screening platform was easier to use and performed significantly quicker, therefore qualifying as an early screening tool. Due to the capability of the disulfide-modified HLA-A*<NUM>:<NUM> molecules to predictably present even low affinity peptide ligands as pMHC complexes, the inventors can precisely measure pMHC-bsTCR binding affinities without having to account for variations encountered in exogenous peptide loading approaches, resulting in no loss of potentially valuable information. The inventors believe that the ease of use of the presented affinity analysis platform will aid the development of safe and effective T cell receptor based bispecific molecules from the early stages on.

As an example, the inventors show that it is possible to quickly generate pMHC-bsTCR binding affinity datasets and extrapolate cross-reactivity search motifs from them. Guided by the inventor's HLA peptidomics-based XPRESIDENT® platform, the search motifs can be used to identify potentially cross-reactive peptide ligands. In the presented execution of this strategy, the inventors were able to identify a large number of peptides strongly recognized by the bsTCR and capable of inducing T cell activation, with sequence consensus compared to the original target as low as one out of nine positions.

This exciting innovative technology could even lead to screenings of the entire discovered immunopeptidome: pMHC libraries of such dimensions are currently only available by yeast display using randomly mutated single-chain peptide MHC libraries (<NUM>, <NUM>). While useful for broad TCR analysis, they are far more complicated in use and of less predictable peptide ligand composition compared to the peptide microarrays typically used in antibody development. Due to its stability and low-effort peptide loading process, the disulfide-modified HLA-A*<NUM>:<NUM> molecules of the present invention may be the ideal fit for the creation of pMHC microarrays with high complexity in the future, for example by combining large scale coating of empty MHCs and the high-throughput of modern peptide microarray inkjet printers.

Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8+ T cells. MHC class I complexes presenting tumor-associated peptides (TUMAPs) are key targets of cancer immunotherapy approaches currently in development, making them important for efficacy as well as safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive. The inventors have developed a disulfide bond stabilized HLA-A*<NUM>:<NUM> molecules that are stable without peptide but can form peptide-MHC complexes with ligands of choice within minutes. The inventors illustrate the concurrence between the engineered mutants and the wild type variant with respect to the binding affinity of wild type or maturated high affinity TCRs. The inventors demonstrate their potential as analytes in high throughput affinity screenings of bispecific TCR molecules and generate a comprehensive TCR binding motif to identify off-target interactions.

The present invention will now be further described in the examples with reference to the accompanying figures, nevertheless, without wanting to be limited thereto.

SEQ ID NOs <NUM> to <NUM> and <NUM> to <NUM> show peptide sequences as used in the examples, below.

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 <NUM>%. UV-light sensitive peptides contained a light-sensitive building block with a <NUM>-nitrophenylamino acid residue. The dipeptide GM was procured from Bachem. Before use peptides were solved in DMSO (Sigma, Cat. <NUM>), <NUM>% TFA (Sigma, Cat. T6508) at concentrations ranging from <NUM>/ml to <NUM>/ml depending on the desired use case.

Recombinant HLA-A*<NUM>:<NUM> wild type (WT-A*<NUM>:<NUM>, SEQ ID NO: <NUM>) or disulfide modified HLA-A*<NUM>:<NUM> heavy chains with C-terminal BirA signal sequences and human β<NUM>m light chain were produced in Escherichia coli as inclusion bodies and purified as previously described (<NUM>). HLA-A*<NUM>:<NUM> complex refolding reactions were performed as previously described with minor modifications (Saini et al <NUM>). In brief, WT-A*<NUM>:<NUM> or disulfide-modified HLA-A*<NUM>:<NUM> heavy chains, β<NUM>m light chain and peptide were diluted in refolding buffer (<NUM> Tris·Cl pH <NUM>, <NUM> arginine, <NUM> EDTA, <NUM> oxidized glutathione, <NUM> reduced glutathione) and incubated for <NUM> to <NUM> days at <NUM> while stirring before concentration. The concentrated protein was purified by size exclusion chromatography (SEC) in <NUM> Tris-HCl, pH <NUM>/<NUM> NaCl on an ÄKTAprime system (GE Healthcare) using a HiLoad <NUM>/<NUM><NUM> pg column (GE Healthcare). Peak fraction was either concentrated directly to <NUM>µg/ml, aliquoted and frozen at -<NUM> or biotinylated by BirA biotin-protein ligase (Avidity) overnight at <NUM> according to the manufacturer's instructions and subjected to a second gel-filtration before final concentration to <NUM>µg/ml, aliquotation and storage at -<NUM>.

To produce HLA-A*<NUM>:<NUM> 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 <NUM>. To produce empty Y84C/A139C HLA-A*<NUM>:<NUM> (SEQ ID NO: <NUM>) complexes the dipeptide GM was added to the refolding buffer at a concentration of <NUM>. To produce F22C/S71C HLA-A*<NUM>:<NUM> (SEQ ID NO: <NUM>) complexes no peptide was added to the refolding buffer. To produce F22C/S71C W51C/G175C HLA-A*<NUM>:<NUM> (SEQ ID NO: <NUM>) complexes no peptide was added to the refolding buffer.

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 <NUM> to <NUM> molar ratio and subjected to at least <NUM> minutes of <NUM> UV light (Camag).

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

Soluble TCRs were produced as previously described (<NUM>). In short TCR alpha and TCR beta chain constructs were expressed separately in Escherichia coli as inclusion bodies and purified. TCR alpha chains are mutated at position <NUM> by replacing a threonine with a cysteine and TCR beta chains at position <NUM> by replacing a serine with a cysteine to form an inter-chain disulfide bond.

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 (<NUM>, <NUM>). 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: <NUM>). Cysteine-knock-outs C<NUM>S and C<NUM>S within the hinge prevent the formation of F(ab)<NUM> molecules. 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 <NUM> 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.

The affinity of sTCR or bsTCR molecules for different pMHC complexes was measured on an OctetRED <NUM> system (Pall Fortébio) using kinetic or steady state binding analysis. All analytes or ligands were diluted to their final concentration in kinetics buffer (PBS, <NUM>% BSA, <NUM>% TWEEN <NUM>) if not specified otherwise. All biosensors were hydrated for at least <NUM> minutes in kinetics buffer before use. Loadings and measurements were performed in <NUM> tilted well plates (Pall Fortébio) with at least <NUM>µl at a <NUM> sensor offset. Plate temperature was set at <NUM> and shaker speed at <NUM> 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 Fortébio Cat. <NUM>-<NUM>) biosensors were used to immobilize biotinylated pMHC monomers at a presumed concentration of <NUM>µg/ml for <NUM> seconds followed by a <NUM> seconds baseline and association and dissociation phases of <NUM> seconds each if not specified otherwise.

In the case of bsTCR immobilization dip and read anti-human Fab-CH1 <NUM>nd generation (FAB2G; Pall Fortébio Cat. <NUM>-<NUM>) biosensors were used to immobilize bsTCR molecules at a concentration of <NUM>µg/ml for <NUM> seconds, followed by a <NUM> seconds baseline and association and dissociation phases of <NUM> seconds each if not specified otherwise. FAB2G biosensor were regenerated up to <NUM> times by incubating the loaded biosensor for <NUM> seconds each in <NUM> Glycine pH1. <NUM> 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 <NUM>. <NUM> (Pall Fortébio). 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 <NUM>:<NUM> Langmuir kinetics binding model.

The TAP-deficient HLA-A*<NUM>:<NUM> expressing cell line T2 was procured from ATCC (CRL-<NUM>) and cultured in RPMI Medium <NUM> GlutaMAX™ (Thermo Fisher, Cat. <NUM>) supplemented with <NUM>% heat inactivated FCS (Life Technologies, Cat. <NUM>) and the antibiotics penicillin and streptomycin (Biozym, Cat. <NUM>, <NUM>µg ml-<NUM> each) up until passage number <NUM> if necessary. The GloResponse™ NFAT-luc2 Jurkat cell line was procured from Promega (Cat. CS1764) at passage number <NUM> and cultured in RPMI Medium <NUM> GlutaMAX™ (Thermo Fisher, Cat. <NUM>) supplemented with <NUM>% heat inactivated FCS (Life Technologies, Cat. <NUM>), <NUM>% Sodium Pyruvate (C. Z-<NUM>) and the antibiotics hygromycin B (Merck Millipore, Cat. <NUM>, <NUM>µg/ml), penicillin and streptomycin (Biozym, Cat. <NUM>, <NUM>µg/ml each) up until passage number <NUM>, if necessary.

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 <NUM> x <NUM><NUM> cells/ml and transferred to <NUM> well round bottom plates (Corning costar®, Cat. Peptide in DMSO, <NUM>% TFA was added to a final concentration of <NUM> and the suspension incubated for <NUM> to <NUM> hours at <NUM>, <NUM>% CO<NUM>. bsTCR formulated in PBS was diluted in T2 culture medium to desired concentration and <NUM>µl of the respective dilution was distributed to white <NUM> well flat bottom plates (Brand, Cat. GloResponse™ NFAT-luc2 Jurkat cells were harvested from continuous cell culture, washed and resuspended in T2 culture medium at a concentration of <NUM> x <NUM><NUM> cells ml-<NUM> and <NUM>µl of the cell suspension was distributed to the white <NUM> well flat bottom plates with bsTCR dilutions. After peptide loading T2 cells were resuspended and <NUM>µl distributed to the white <NUM> well flat bottom plates with bsTCR dilutions and GloResponse™ NFAT-luc2 Jurkat cells for a final effector to target ratio of <NUM>:<NUM> (<NUM> cells each). Fully assembled plates were mixed for <NUM> minutes at <NUM> rpm on a plate shaker and the incubated for <NUM> to <NUM> at <NUM>, <NUM>% CO<NUM>. After the incubation period <NUM>µl of Bio-Glo™ luciferase reagent was added to each well and the plates incubated for minutes at <NUM> rpm on a plate shaker in the dark before reading luminescence at a <NUM> 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.

The Y84C/A139C HLA-A*<NUM>:<NUM>-SLLMWITQV complex and the 1G4 TCR were concentrated and mixed in a <NUM>:<NUM> ratio to achieve a concentration of <NUM>/ml for crystallization. A sitting drop vapor diffusion experiment resulted in crystals in the presence of a mother liquor containing <NUM> ammonium acetate, <NUM> bis-tris (pH <NUM>), and <NUM>% polyethylene glycol (PEG) <NUM>,<NUM>. A single crystal was transferred to a cryoprotectant solution containing <NUM> ammonium acetate, <NUM> bis-tris (pH <NUM>), <NUM>% (w/v) PEG <NUM>,<NUM>, and <NUM>% glycerol. The crystal was mounted and cryocooled at <NUM> on the EMBL P14 beamline at Deutsche Elektronen-Synchrotron containing an EIGER <NUM> detector. An x-ray dataset was collected to a resolution of <NUM>Å (Table <NUM>).

The data were processed with XDS and scaled with AIMLESS (<NUM>, <NUM>). 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 (<NUM>, <NUM>). The engineered disulfide bond was manually built with Coot (<NUM>). The structure was refined to an R factor of <NUM>% (Rfree of <NUM>%). MolProbity was used to validate the geometry and indicated that <NUM>% of the residues were in the allowed regions of the Ramachandran plot [with one glycine residue (Gly143) in the disallowed regions] (<NUM>).

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.

Seq2Logos visualizing the binding motif were created by taking the inverse value of measured Kd values for the respective interaction and dividing them by <NUM><NUM>. 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 (<NUM>).

Peptide binding was evaluated in fluorescence anisotropy assay with <NUM> of purified refolded Y84C/A139C HLA-A*<NUM>:<NUM>. <NUM> of the fluorescently labeled high-affinity peptide NLVPKFITCVATV (Genecast) was added to the folded Y84C/A139C HLA-A*<NUM>:<NUM> and kinetic measurements were performed with Tecan Infinite M1000 PRO (Tecan, Crailsheim, Germany) multimode plate reader measuring anisotropy (FITC λex = <NUM>, λem = <NUM>). Y84C/A139C HLA-A*<NUM>:<NUM> were either used directly after refolding or preserved at -<NUM> in storage buffer (<NUM>% Glycerol, <NUM> Tris-HCL, pH <NUM>) for the indicated amount of time before measurement. The kinetic measurements were performed at room temperature (<NUM>-<NUM>) in <NUM> HEPES buffer, pH <NUM>. Data was plotted using GraphPad Prism v7.

Streptavidin (Molecular Probes, Cat. S888) at a final concentration of <NUM>µg/ml in PBS was added to Nunc MAXIsorp plates (Thermo Fisher, Cat. <NUM>) and sealed plates incubated over night at room temperature in a damp environment. The following day plates were washed <NUM> times with washing buffer (PBS, <NUM>% TWEEN-<NUM>) using a ELx405 plate washer (Biotek). <NUM>µl blocking buffer (PBS with <NUM>% BSA) was added to each well and sealed plates incubated at <NUM> for <NUM> hour. Blocking buffer was discarded before adding <NUM>µl of a <NUM>:<NUM> dilution in blocking buffer of the respective UV exchange pMHC preparation. A standard series ranging from <NUM> ng/ml to <NUM> ng/ml based on a conventionally refolded pMHC monomer was included on each plate. Edge wells were filled with <NUM>µl blocking buffer to reduce edge effects and sealed plates were incubated at <NUM> for <NUM> hour. Plates were again washed <NUM> times before adding <NUM>µl anti-beta <NUM> microglobulin HRP conjugated secondary antibody (Acris, Cat. R1065HRP) at a final concentration of <NUM>µg/ml to each well. Sealed plates were incubated at <NUM> for <NUM> hour. Plates were washed again <NUM> times with washing buffer before adding <NUM>µl of room temperature TMB substrate (Sigma, Cat. T0440) to each well. Plates were incubated for <NUM> minutes at room temperature before stopping by adding <NUM>µl 1N H<NUM>SO<NUM> to each well. Plates were immediately analyzed by reading absorbance at <NUM> for <NUM> 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.

The TAP-deficient HLA-A*<NUM>:<NUM>-expressing cell line T2 was procured from ATCC (CRL-<NUM>) and cultured in RPMI Medium <NUM> GlutaMAX™ (Thermo Fisher, Cat. <NUM>) supplemented with <NUM>% heat inactivated FCS (Life Technologies, Cat. <NUM>) and the antibiotics penicillin and streptomycin (Biozym, Cat. <NUM>, <NUM>µg/ml each) up until passage number <NUM> if necessary. T2 cells were harvested from continuous cell culture, washed and resuspended in T2 culture medium at a concentration of <NUM> x <NUM><NUM> cells/ml and transferred to <NUM> well round bottom plates (Corning costar®, Cat. Peptide in DMSO, <NUM>% TFA was added to a final concentration of <NUM> and the suspension incubated for <NUM> hours <NUM>, <NUM>% CO<NUM>. Plates were washed twice with PFEA (PBS, <NUM>% FCS, <NUM> EDTA, <NUM>% sodium azide) before addition of <NUM>µl PE labelled anti-human HLA-A2 (Biolegend, Cat. <NUM>) per well diluted <NUM>:<NUM> with PFEA to a final concentration of <NUM>µg/ml. Plates were incubated at <NUM> for <NUM> minutes before being washed twice with PFEA. Finally, cells were resuspended in fixation solution (PFEA, <NUM>% formaldehyde) and kept at <NUM> 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.

Multiple sequence alignments were performed by using Clustal Omega Multiple Sequence Alignment (www. uk/Tools/msa/clustalo/) (<NPL>).

All data were plotted using the GraphPad Prism software version <NUM>. Correlation between x and y datasets were calculated by computing the Pearson correlation coefficient and were reported as R<NUM> using the GraphPad Prism software version <NUM>. R<NUM> and X<NUM> values for curve fittings of biolayer interferometry binding kinetics measurements were calculated using the Octet RED384 system software DataAnalysis HT version <NUM>.

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 (<NUM>). In previous studies with the murine MHC class I molecule H-<NUM>b introduction of a disulfide bond between opposing residues in the F-pocket by mutating a tyrosine at position <NUM> and an alanine at position <NUM> to cysteines was able to stabilize the complex. The mutant could be refolded without full length peptide and was capable of retroactive peptide binding (<NUM>, <NUM>).

The inventors hypothesized that the same concept could be applied to the human MHC class I molecule HLA-A*<NUM>:<NUM>. Modifications resulting in mutations of the tyrosine at position <NUM> and alanine at position <NUM> into cysteines were introduced into an HLA-A*<NUM>:<NUM> heavy chain expression plasmid. After production as inclusion bodies in E. coli, the heavy chain was incubated with similarly produced β<NUM>m but without peptide in refolding buffer. After size exclusion chromatography (SEC), no HLA-A*<NUM>:<NUM> 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 (<NUM>). 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*<NUM>:<NUM>. Empty wild type A*<NUM>:<NUM> complexes (WT-A*<NUM>:<NUM>) could not be produced in the same fashion. WT-A*<NUM>:<NUM> 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 <NUM> and serine at position <NUM> into cysteines into an HLA-A*<NUM>:<NUM> heavy chain expression plasmid. After production as inclusion bodies in E. coli, the heavy chain was incubated with similarly produced β<NUM>m but without peptide in refolding buffer. SEC yielded purified empty disulfide-stabilized F22C/S71C HLA-A*<NUM>:<NUM> complexes. The inventors also introduced modifications resulting in mutations of phenylalanine at position <NUM> and serine at position <NUM> as well as tryptophan at position <NUM> and glycine at position <NUM> into cysteines into an HLA-A*<NUM>:<NUM> heavy chain expression plasmid. After production as inclusion bodies in E. coli, the heavy chain was incubated with similarly produced β<NUM>m but without peptide in refolding buffer. SEC yielded purified empty disulfide-stabilized F22C/S71C W51C/G175C HLA-A*<NUM>:<NUM> 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*<NUM>:<NUM> molecule was less temperature stable (i.e., had a lower melting temperature) than the same molecule still complexed with dipeptide GM (<NUM>).

The resulting molecules were either biotinylated at <NUM> overnight and separated from excess biotin by a second SEC run or stored directly at -<NUM> prior to use.

Next, the inventors determined whether the disulfide-modified HLA-A*<NUM>:<NUM> molecules were capable of peptide-MHC complex formation and TCR ligand binding. Affinity measurements were performed by bio-layer interferometry (BLI) on an OctetRED <NUM> using the refolded TCR 1G4 as soluble analyte. This TCR recognizes the HLA-A*<NUM>:<NUM> specific peptide SLLMWITQC (ESO 9C, SEQ ID NO: <NUM>) derived from the cancer testis antigen NY-ESO-<NUM> or its synthetic variant SLLMWITQV (ESO 9V, SEQ ID NO: <NUM>) (<NUM>,<NUM>). Biotinylated Y84C/A139C HLA-A*<NUM>:01was either immobilized directly in its empty state or after a short incubation with the peptide ESO 9V on streptavidin-coated biosensors (<FIG>). No differences could be detected between peptide incubations of <NUM> 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*<NUM>:<NUM> directly refolded with ESO 9V served as control.

1G4 TCR binding to either Y84C/A139C HLA-A*<NUM>:<NUM>9V or WT-A*<NUM>:<NUM> ESO 9V was very similar with respect to sensorgrams and Kds resulting from curve fittings (<FIG>). A weak binding signal (but no dissociation) could be detected for the empty immobilized monomer at high concentrations of 1G4 (<FIG>). This binding could be prevented by subsequently adding a peptide that is not recognized by 1G4 like SLYNTVATL (<FIG>, SEQ ID NO: <NUM>). The weak signal obtained with empty Y84C/A139C HLA-A*<NUM>:<NUM> 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*<NUM>:<NUM>-restricted soluble TCRs with varying specificities behaved similarly, showing no binding to irrelevantly loaded Y84C/A139C HLA-A*<NUM>:<NUM> pMHCs but association to functionally empty molecules, albeit but with a relatively lower response (<FIG>).

Having established the usability of the Y84C/A139C HLA-A*<NUM>:<NUM> molecule as ligand equivalent to WT-A*<NUM>:<NUM> 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*<NUM>:<NUM> restricted peptide SLYNTVATL (SL9, SEQ ID NO: <NUM>) (<NUM>, <NUM>).

The inventors performed affinity measurements by immobilization of empty or SL9 peptide loaded disulfide-modified HLA-A*<NUM>:<NUM> 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>)(<NUM>). Binding affinity for SL9 disulfide-modified HLA-A*<NUM>:<NUM> pMHC complexes using either Y84C/A139C HLA-A*<NUM>:<NUM>, F22C/S71C HLA-A*<NUM>:<NUM> or F22C/S71C W51C/G175C HLA-A*<NUM>:<NUM>, was similar to the SL9 WT-A*<NUM>:<NUM> pMHC produced by performing an UV-light mediated peptide ligand exchange (<NUM>) with <NUM> and <NUM>, respectively (<FIG> and, also <FIG>). No binding was measurable with empty MHC molecules for this bsTCR (<FIG>) and with irrelevantly loaded Y84C/A139C HLA-A*<NUM>:<NUM> complexes at a high molar concentrations of <NUM>.

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 <NUM> remaining proteinogenic amino acids while maintaining all other positions, resulting in <NUM> distinct peptides when performed at all positions of the nonamer (cysteine was excluded because of its propensity to dimerize) (<NUM>). pMHC complexes were generated by the inventors either by addition to Y84C/A139C HLA-A*<NUM>:<NUM> molecules as before or by performing UV-light mediated peptide ligand exchange, a technology used for pMHC complex generation (<NUM>). 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 R<NUM> values of at least <NUM>, representative of signals within the selected Kd sensitivity range. Kd values for the resulting <NUM> 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>). Discrepancies were within <NUM>-fold range for over <NUM>% of the pMHC pairs and <NUM>-fold differences at most. Within the group with higher than <NUM>-fold changes a trend towards a larger dissociation constant for measurements with the Y84C/A139C HLA-A*<NUM>:<NUM> molecule was observed.

The amount of functional pMHC immobilized on each biosensor expressed by the reported Rmax value for <NUM> different peptide ligands from the positional scanning library was comparable for both wild-type and disulfide-stabilized pMHCs (correlation coefficient R<NUM> = <NUM>).

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

In the upper left panel the Kd for each above-listed peptide for the WT-A*<NUM>:<NUM> pMHC complex is plotted against the Kd of the disulfide-modified F22C/S71C HLA-A*<NUM>:<NUM> pMHC complex. The disulfide-modified F22C/S71C HLA-A*<NUM>:<NUM> pMHC complex shows almost identical KD values to the WT-A*<NUM>:<NUM> pMHC complex for each of the investigated peptides. In the lower left panel the Kd for each above-listed peptide for the WT-A*<NUM>:<NUM> pMHC complex is plotted against the KD of the disulfide-modified F22C/S71C W51C/G175C HLA-A*<NUM>:<NUM> pMHC complex and shows also almost identical Kd values to the WT-A*<NUM>:<NUM> pMHC complex for each of the investigated peptides.

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

The results shown in <FIG> make it credible for the skilled person that the disulfide-modified HLA-A*<NUM>:<NUM> 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.

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 Rmax value. 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 <NUM> 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>). 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*<NUM>:<NUM> 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*<NUM>:<NUM> peptide loadings (<FIG>, <FIG>, <FIG>) (<NUM>). 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 <NUM> to <NUM>, within <NUM> hours of unattended measurement time at a <NUM>-fold reduced price tag. All curves reaching at least a signal level of <NUM> were included in the fittings, resulting in a comprehensive TCR binding motif (<FIG>, <FIG>, Table <NUM>).

Soluble Y84C/A139C HLA-A*<NUM>:<NUM> pMHC preparations can be stored for at least <NUM> weeks at <NUM> without loss of quality and used for multiple analyses (<FIG>; Day <NUM>: KD = <NUM>. 35E-<NUM> M, R<NUM> = <NUM>; Day <NUM>: KD = <NUM>. 08E-<NUM> M, R<NUM> = <NUM>).

The 868Z11 TCR displayed an expected pattern of recognition: changes of amino acids between positions <NUM> to <NUM> 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>) (<NUM>).

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 Kd threshold of <NUM>: all single amino acid substitutions increasing the bs-868Z11-CD3 Kd above that threshold were excluded from the motif (Table <NUM>). 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 <NUM> hits within the human genome, with sequence identity to the wild type sequence SLYNTVATL ranging from <NUM> to <NUM> identical positions. <NUM> peptides were selected, sampled to be representative of the sequence identity distribution in the larger group, synthesized and used for affinity measurements (Table <NUM>; SEQ ID NOS: <NUM>-<NUM>). The inventors were able to detect binding affinities of single digit µM Kds or higher for <NUM> of those peptides.

One of them, ALYNVLAKV (SEQ ID NO: <NUM>), 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 (<NUM>, <NUM>). ALYNVLAKV, an antigen from intermediate filament family orphan <NUM> or <NUM> (IFFO1/<NUM>), 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 KD of <NUM> (<FIG>). The inventors were able to identify a second LC-MS detected peptide, KTFNLIPAV (SEQ ID NO: <NUM>), with a lower Kd of <NUM> detected on three tumor tissue samples.

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*<NUM>:<NUM> 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 <NUM> and subsequently co-incubated with Jurkats and different bsTCR concentrations for <NUM> 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>). 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 <NUM>-fold increased signal above. Onset values were plotted against the respectively measured KDs (<FIG>).

Overall, the inventors detected a good correlation between the determined Kd values 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>) (<NUM>). 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 <NUM> and above, supporting the initial hypothesis. pMHC-bsTCR binding affinity correlated well with T cell activation onset for peptide ligands between NetMHC rank <NUM> and <NUM>, 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 <NUM> peptide ligands selected by binding motif search, <NUM> were capable of inducing a <NUM>-fold T cell activation over background with at least one of the supplied bsTCR concentrations (<FIG>). 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: <NUM>) was also reactive in the reporter assay (<FIG>).

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.

To further confirm that the 1G4 TCR recognizes ESO 9V Y84C/A139C HLA-A*<NUM>:<NUM> indistinguishably from ESO 9V WT-A*<NUM>:<NUM>. TCR and disulfide-stabilized MHC refolded with ESO 9V were cocrystallized, as reported previously for the wild-type ESO 9V HLA-A*<NUM>:<NUM> molecule and analyzed by x-ray crystallography (Table <NUM>) (<NUM>). Comparison of the crystal structures revealed a high degree of structural overlap between both complexes. The backbone of both HLA-A*<NUM>:<NUM> molecules aligned almost perfectly with a root mean square deviation (RMSD) value of <NUM>Å calculated over Cα (constant portion of the α chain of a T cell receptor; <FIG>). The same was true for both bound peptides including their side chains with an RMSD value of <NUM>Å calculated over all atoms, even when in close vicinity to the disulfide bond (<FIG>). 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 <NUM>°, when comparing WT-A*<NUM>:<NUM>1G4 with the Y84C/A139C HLA-A*<NUM>:<NUM>1G4 crystal structure. This shift was still within the range of expected deviations for the same complex when crystallized repeatedly (<FIG>, C and D). Together, determined binding affinities and crystal structure showcase peptide receptiveness and similar properties of the Y84C/A139C HLA-A*<NUM>:<NUM> pMHC complexes compared with wild-type complexes with respect to TCR binding. The crystal structure of the 1G4 Y84C/A139C HLA-A*<NUM>:<NUM> ESO 9V complex has been deposited in the PDB under the accession number 6Q3S.

Here, the inventors have presented disulfide-stabilized and functionally empty HLA-A*<NUM>:<NUM> 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*<NUM>:<NUM> 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*<NUM>:<NUM>-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 <NUM>. Analysis of an <NUM>-SV9 crystal structure, as well as an accompanying alanine scan by Cole et al. (<NUM>), revealed prominent interactions between the CDR3α region and the amino acids 4N and 5T of SLYNTVATL. This behavior seems to be conserved although a significant part of the CDR3α 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.

Claim 1:
A method for screening for a TCR-binding peptide ligand/MHC molecule complex (pMHC), comprising the steps of:
a) providing a suitably stabilized MHC I molecule, which is a HLA-A protein, wherein said MHC molecule comprises at least one artificially introduced covalent bridge:
between two amino acids of the alpha1 domain of said stabilized MHC molecule by mutating an amino acid corresponding to position <NUM> of SEQ ID NO: <NUM> and an amino acid corresponding to position <NUM> of SEQ ID NO: <NUM>,
b) contacting said suitably stabilized MHC I molecule with a multitude of peptide ligands thereof, to form peptide ligand/MHC (pMHC) molecule complexes, and
c) screening said pMHC molecule complexes for TCR-binding.