WO2021048381A1

WO2021048381A1 - Method for identifying stable mhc binding peptides using mass spectrometry

Info

Publication number: WO2021048381A1
Application number: PCT/EP2020/075512
Authority: WO
Inventors: Jens KRINGELUM; Emma JAPPE; Christian Garde; Anthony Purcell; Sri RAMARATHINAM; Nathan Paul CROFT
Original assignee: Evaxion Biotech Aps; Monash University
Priority date: 2019-09-13
Filing date: 2020-09-11
Publication date: 2021-03-18

Abstract

Disclosed is an MS based method for identification of MHC binding peptides, where the binding capability is quantitatively assessed to allow distinction between stably binding peptides and peptides that are unlikely to be presented to T-cells. The method includes a step of time-course or thermostability testing of naturally processed peptides bound to MHC. Also disclosed are methods for preparation of immunogenic compositions.

Description

METHOD FOR IDENTIFYING STABLE MHC BINDING PEPTIDES USING MASS SPECTROMETRY

FIELD OF THE INVENTION

The present invention relates to the field of immunology, in particular to the identification of MHC binding peptides that are potential T-cell epitopes.

BACKGROUND OF THE INVENTION

A key component of effective immunotherapy involves T cell recognition of peptides bound to cell surface major histocompatibility complex (MHC) (Yewdell, Reits and Neefjes, 2003). Peptide immunogenicity is multifaceted, yet current algorithms incorporate only a limited number of features such as peptide-MHC ("pMHC" or "pMHC complex") binding affinity and antigen processing, offering poor predictive outcome (Mei et al., 2019), (Ko§aloglu-Yalgm et al., 2018; Gfeller et al., 2016). pMHC stability has been shown to be an important feature driving T cell responses (Stronen et al., 2016; Rasmussen et al., 2016). The stability of the pMHC complex is hypothesised to play an important role in the induction of an immune response, since more stable complexes can be presented on the cell surface for a prolonged period of time allowing more effective T cell receptor engagement with the pMHC (Tummino and Copeland, 2008). Several studies have indicated a correlation between pMHC stability and peptide immunogenicity (Stronen et al., 2016; Harndahl et a/., 2012; Blaha et al., 2019); however, current pMHC stability assays are biased and suffer experimental limitations in scale. Furthermore, prediction algorithms developed based on selected pMHC stability data have not demonstrated impressive results in predicting T cell epitopes when benchmarked against comparable pMHC affinity predictors (Rasmussen et al., 2016; Jorgensen and Buus, 2014). Thus, novel high-throughput assays are required to evaluate pMHC stability in a more global manner and determine its correlation with and impact on peptide immunogenicity.

Within recent years, the field of mass spectrometry (MS) and the application of MS to the identification of peptides bound to MHC molecules (the immunopeptidome) has undergone impressive development allowing detection of thousands of peptides in one MS run (cf. the detailed protocol presented in Purcell, Ramarathinam and Ternette, 2019). MS allows the study of peptides, which have been processed by the antigen processing machinery within cells and subsequently bound to an MHC molecule expressed on the cell surface; in other words, the peptides identified as MHC binders by this type of technology are the true products of antigen processing. In contrast, older methods used to identify MHC binding peptides often failed to identify naturally processed forms of these peptides. However, despite the advantages of using MS to study the immunopeptidome, many MS-based peptide identification assays typically detect MHC-bound peptides qualitatively, i.e. either the peptide is detected or it is not detected, and hence the current MS-based methods do not provide further information about the suitability of the peptide as an immunogen. Moreover, those methods that are in fact able to provide quantitative data on MHC bound peptides are not able to provide any further indications of the peptides' suitability as immunogens either.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide methods for analysis of the stability of complexes between MHC molecules (in humans termed HLA molecules) and naturally processed and presented peptides.

SUMMARY OF THE INVENTION

As detailed above, existing MS methods for identification of MHC (in humans termed HLA) binding peptides typically provide qualitative, but limited quantitative information about the binding properties of the identified peptides. In particular, the stability of the complex between the MHC molecule and the peptide is not determined. This is partly due to the fact that the methodology for preparing the peptides for MS detection in essence provides a "snapshot" of the repertoire of MHC-peptide complexes on the surfaces of the cells presenting the peptides (see Fig. 4 in Purcell, Ramarathinam and Ternette, 2019). In addition, Croft et al. (PLoS Pathogens 2014, Wu et al 2019) have shown that peptide abundance cannot be used as a sole measure to determine CTL responses. Thus, quantitative measurements of specific pMHC only indirectly provide an indication of stability as they are a product of the levels of the peptide precursor/antigen turnover and the affinity of the peptide for a given MHC. The inventors have hypothesised that it is possible that a relatively unstable pMHC could be abundant if there is a high level supply of the precursor to drive pMHC complex formation. Equally abundant pMHC complexes may on the other hand accumulate to high levels even with modest precursor supply if the complexes are stable. These two scenarios cannot be distinguished by simple prior art qualitative or quantitative MS-methods, which will only reveal the presence/abundance of what is bound to the MHC, irrespective of the reason.

The present inventors have concluded that the MHC-bound peptides identified from such a "snapshot" could include peptides that exhibit individual stabilities for their binding to the MHC molecules, and that this could subsequently be reflected in the probabilities of the MHC- peptide complexes being presented effectively to a T-cell. The reasoning is that when a peptide dissociates from the MHC molecule, the chance that the same peptide will subsequently associate with the same or a different MHC molecule is relatively low, in particular under the experimental conditions for isolated pMHC, because the MHC molecules, being heterodimers, require a peptide bound in the peptide binding groove in order to constitute stable complexes over time. The dissociation of peptide from MHC has the consequence that the MHC heterodimer will dissociate (into the individual a and b chains in the case of MHC Class II and into the a chain and 32-microglobulin in the case of MHC Class I). Based on the nature of the experimental protocol currently used to isolate peptides that have not dissociated from MHC (immunoaffinity capture and peptide isolation is carried out at 4°C) (Purcell et al 2019), the current "snapshot" of peptides presented by a cell's MHC repertoire, that can be obtained from such workflows, will inherently include peptides that exhibit low stability for MHC binding at physiological conditions. The consequence of this is that these peptides are not stably present on the cell and that such peptides would therefore stand small chances of being effective T-cell epitopes. Also, it was considered likely by the present inventors that some of the identified peptides would conversely exhibit a high degree of stability for their binding to the MHC molecule, which could be reflected in an increased chance that such peptides would be ultimately presented to T-cells. So if ways could be devised that would allow not only a qualitative determination of naturally processed MHC- binding peptides but also a quantitative measure of their stability as MHC binders in the natural context of the cell environment, this would in turn enable a rational selection of peptide sequences, e.g. for the purpose of rational vaccine preparation and design. At any rate, if information concerning the stability of the binding to MHC for different naturally processed peptides can be obtained, it is possible to rank the peptides accordingly, and thereby an excellent tool for decision making is obtained, because peptides will not be chosen at random for a desired purpose (that may require strong, intermediate or weak MHC binding stability) but rather based on the knowledge of their observed binding stability.

It was therefore decided by the inventors to investigate whether modifications of the experimental protocol set forth by Purcell, Ramarathinam and Ternette (Purcell et al 2019) could be used to differentiate the identified peptides with respect to their capability of binding stably to the MHC molecules. Thus, a small-scale approach was devised in order to simultaneously carry out multiple elutions of peptides with the same conceptual workflow as that described in (Purcell et al 2019), enabling the investigation of many conditions in one experimental setup rather than simply having a snapshot of the peptides bound to the surface MHC molecules at a given point in time. To this end, the protocol was modified to investigate the number of detectable MHC-bound peptides as a function of time between cell lysis and isolation of MHC-peptide complexes. In another set of experiments, the protocol was modified to investigate the influence of temperature conditions after cell lysis on the amount of detectable MHC-bound peptides.

It was from these experiments found by the present inventors that in the context of the immunopeptidome, mass spectrometry analysis (MS) can indeed be used to study the stability of the pMHC. By incubating cell lysates for longer periods of time or at different temperatures (or other entropy modifying conditions), the change in pMHC binding over time or temperature can be studied and directly applied to determine the stability of the individual pMHC complexes. So, rather than carrying out pMHC complex isolation (as described in Purcell 2019) immediately following cell lysis at 4°C, cell lysates can be incubated for different periods of time or different temperatures in order to study the change in binding of peptide to MHC over time or at various temperatures, which can be directly applied to determine the stability of the individual pMHC complexes.

So, in a 1^st aspect, the present invention relates to a method for quantitative determination of stability of binding between at least one peptide and an MHC molecule, comprising the subsequent steps of a) preparing a plurality of samples of cell lysates comprising complexes between MHC molecules and peptides, where the lysates are obtained from a plurality of MHC expressing cells (preferably human cells) that have naturally processed said peptides from protein antigens, b) subjecting the plurality of samples to the conditions of i) incubation at defined physicochemical conditions, where incubation time varies between the plurality of samples and where the physicochemical conditions are kept constant between the plurality of samples, or ii) incubation at defined physicochemical conditions, where the incubation time is kept constant between the plurality of samples and where the physicochemical conditions vary between the plurality of samples, c) isolating complexes between MHC molecules and peptides from the plurality of samples, d) determining, by mass spectrometric analysis, the at least one peptide's relative quantities in the plurality of samples after step c), and deriving at least one stability score for the at least one peptide based on the quantities determined in step d).

This and other aspects of the invention are partly founded in the realization that peptides identified as binders at the "conventional" lysis and incubation temperature of 4°C are not necessarily relevant as effective MHC ligands at physiological temperatures. It was concluded by the inventors that this finding has the consequence that a sample of pMHC complexes from cell lysates obtained from a "snapshot" established and/or incubated for a period of time at higher than conventional temperatures ( i.e . higher than 4°C) will results in a higher degree of dissociation of pMHC complexes in the lysates, in turn, resulting in a smaller number of peptides bound to MHC. Thus, in addition to the 1^st aspect of the invention, simplified versions of the stability determination can be constructed. These simplified approaches involve similar steps in cell lysate preparation, in which lysates are incubated at elevated temperature (higher than 4°C, for instance at room temperature or even at physiological temperature or above) and/or incubated at a defined temperature (or at an otherwise defined level that provides increased entropy) for a prolonged time after lysis. The resulting samples can be analysed using either a data-dependent acquisition (DDA) MS approach or a data-independent acquisition (DIA) approach. After a given pre-defined incubation condition, the result is that a lower number of peptides have to be sequenced/analyzed by LC-MS/MS in these samples; fewer peptide species in a sample renders the sample less complex, thus facilitating identification of individual peptide species using MS. These fewer peptide species are furthermore those that due to their stable binding to MHC exhibit a higher likelihood of being presented by APCs under physiological conditions. So it becomes possible to simplify the entire determination of stably MHC-bound peptides by concentrating solely on those peptide species that can be detected after the incubation rather than having to also determine their quantities as in the 1^st aspect of the invention even though it is of course possible to also test for quantity of the peptides.

Where the method of the 1^st aspect of the invention provides very detailed knowledge about pMHC stability for each tested peptide, a more simplified exploitation of e.g. the time or temperature dependence of peptide-MHC complex preservation can provide a convenient and comparably simple method for assessing pMHC stability in qualitative terms where the information derived e.g. is an answer to the question "is the peptide stably bound to MHC at physiological conditions?". Put in simpler words: peptides that it is possible to elute from MHC-peptide complexes after lysis/incubation under increased entropic conditions compared to the entropy at the 4°C lysis conditions must be considered "better MHC binders" because the increased entropy is less favourable for preservation of the peptide MHC complex. Likewise, prolonged incubation of such complexes under conditions that allow time- dependent dissociation of the peptide-MHC complexes will cause a loss of complexes due to the higher likelihood of dissociation over association until equilibrium has been reached; further this loss will be preferential of the complexes that are least stable.

So, in a 2^nd aspect the present invention relates to a method for (typically qualitative) determination of binding between at least one peptide and an MHC molecule, comprising the subsequent steps of I) preparing at least one sample of cell lysates comprising complexes between MHC molecules and peptides, where the lysates are obtained from a plurality of MHC expressing cells (preferably human cells) that have naturally processed said peptides from protein antigens, wherein the at least one sample of cell lysates is prepared at a temperature >4°C and/or wherein the at least one sample of cell lysates is/are incubated, for a period of time after obtaining the cell lysates, at defined physicochemical conditions at a temperature >0°C ,

II) determining, by mass spectrometric analysis, whether the at least one peptide is present as part of a complex in the at least one sample after step I).

In a related 3^rd aspect, the invention relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule by the method of the 1^st or 2^nd aspect of the invention and any embodiment thereof disclosed herein, and subsequently admixing one or more peptides, which are selected from peptides of the plurality that exhibit the characteristics 1) and/or 2):

1) the peptide has a quantitative stability score above the average quantitative stability score of the plurality of peptides as a whole (e.g. a smaller number of peptides, which constitute the top 1-5% of the peptides identified as good MHC binders) in the first aspect of the invention,

2) the peptide is selected from peptides that are found to be present as part of the complexes in step II of the method of the 2^nd aspect of the invention, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient. A peptide that exhibits any of characteristics 1 and 2, is termed "a stably MHC binding peptide" herein.

In a related 4^th aspect, the invention relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule by the method of the 1^st or 2^nd aspect of the invention and any embodiment thereof disclosed herein and subsequently preparing a polypeptide, which comprises amino acid sequences of one or more stably MHC binding peptides, and admixing the polypeptide with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

In a related 5^th aspect, the invention relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule by the method of the 1^st or 2^nd aspect of the invention and any embodiment thereof disclosed herein and subsequently admixing a nucleic acid, which is capable of expressing nucleotide sequences encoding one or more stably MHC binding peptides with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

In a related 6^th aspect, the invention relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule by the method of the 1^st or 2^nd aspect of the invention and any embodiment thereof disclosed herein and subsequently admixing a nucleic acid, which is capable of expressing a nucleotide sequence encoding a polypeptide comprising the amino acid sequences of one or more stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

In a related 7^th aspect, the invention relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule by the method of the 1^st or 2^nd aspect of the invention and any embodiment thereof disclosed herein and subsequently admixing a microorganism or virus, which is capable of expressing nucleotide sequences encoding one or more stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

In a related 8^th aspect, the invention relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule by the method of the 1^st or 2^nd aspect of the invention and any embodiment thereof disclosed herein and subsequently admixing a microorganism of virus, which is capable of expressing a nucleotide sequence encoding a polypeptide comprising the amino acid sequences of one or more stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

LEGENDS TO THE FIGURE

Fig. 1 : Schematic overview of an embodiment of the 1^st aspect of the invention.

Fig. 2: Example of peptide filtering in Skyline software (example peptide TLTHVIHNL).

A spectral library generated in PEAKS® software using 1% FDR provided 1696 peptides (8mers-llmers) was loaded into the Skyline software, and thereafter peptides were filtered and manually picked to ensure correct precursors and transitions. Fig. 3: Thermal stability curves for naturally processed peptides eluted from complexes between peptides and MHC molecules isolated from the cell line C1R-A*02:01.

X axis is incubation temperature (°C), Y axis is relative amounts of isolated peptide.

A: Curves for 12 A*02:01 binding peptides with measured T_m values (°C) ranging between 44.90 and 61.40.

B: Curves for 12 B*07:02 binding peptides with T_m values (°C) ranging between51.78 and 61.99.

C: Curves for 12 peptides binding both A*02:01 (circles) and B*07:02 (triangles) with T_m values (°C) for binding A*02:01 ranging between 45.71 and 58.96 and with T_m values (°C) for binding B*07:02 ranging between 46.85 and 59.81.

Fig. 4: Graphs showing the distribution of normalized T_m values for 491 peptides when compared to prior art determination of ligand binding via MS.

Fig. 5: Graphs showing comparison of T_m values determined according to the present invention and ligand rank score determined with netMHCpan4.0. a) results for HLA-A*02:01 ligands b) results for HLA-B*07:02 ligands

Fig. 6: Graph of peak area ratio relative to global standard in Skyline for peptide ALNELLQHV. Bar represents the peak area ratio of the peptides obtained after incubation of cell lysates at 37°C for 0, 0.5, 1, 1.5, 2, 3, 5 and 24 hours, respectively.

Fig. 7: Peak curves for peptide ALNELLQHV from 8 samples.

Peaks are shown from samples obtained after incubation of cell lysates at 37°C for 0, 0.5, 1, 1.5, 2, 3, 5 and 24 hours, respectively.

Fig. 8: Decay curves for 6 peptides subjected to incubation at 37°C for 0, 0.5, 1, 1.5, 2, 3, 5 and 24 hours, respectively.

Curves shown for peptides RLFDEPQLA, SLLESVQKL, FLFQEPRSI, ILLPEPSIRSV, TLITDGMRSV, and FLDENVHFF.

Fig. 9: Correlation between thermal melting point and half-life. DETAILED DISCLOSURE OF THE INVENTION

Definitions

A "peptide" is in the present context a polyamino acid having a length which allows it to fit into the binding groove of an MHC molecule. That is, if the MHC molecule is of class I, the peptides that can bind typically have lengths ranging between 8 and 11 amino acid residues, due to the physical form of the peptide binding cleft. If the MHC molecule is of class II, the peptide has, typically, a minimum length of 9-13 amino acids, but can be considerably longer because the peptide binding cleft in MHC Class II molecules allows for an "overhang".

An "MHC molecule" (major histocompatibility molecule) is a tissue antigen expressed by nucleated cells in vertebrates, which binds to peptide antigens and displays ("presents") the antigens to T-cells carrying T-cell receptors. MHC class I is expressed by all nucleated cells and primarily present proteolytically degraded protein fragments derived from proteins present in the cell. MHC class II is expressed by professional antigen presenting cells that typically take up extracellular protein, degrade it with lysosomal proteases, and present protein fragments on the surface. In humans, the MHC molecules are known as human leukocyte antigens (HLA), which in the present invention are the preferred MHC molecules to evaluate binding to.

A "T-cell epitope" is an MHC binding peptide, which is recognized as foreign (non-self) by a T- cell in a vertebrate due to specific binding between a T-cell receptor and the cell carrying the MHC-peptide complex on its surface. Hence, a peptide, which constitutes a T-cell epitope in one individual will not necessarily be a T-cell epitope in a different individual of the same species. First of all, two individuals having differing MHC molecules that bind different sets of peptides, do not necessarily present the same peptides complexed to MHC, and further, if a peptide is autologous in one of the individuals it may not be able to bind any T-cell receptor.

"Naturally processed peptides" are in the present context peptides that can be eluted from an MHC-carrying cell after the peptides have emerged as products of antigen processing by the MHC-carrying cell. Thus, a naturally processed peptide is not simply a peptide, which can form a complex with an MHC molecule. Rather, the naturally processed peptide is by nature a degradation product from the cell's antigen processing machinery. In most prior art methods where peptide-MHC complex formation is measured, peptides - often synthetic - are complexed directly with MHC. This approach can provide for useful insights into peptide-MHC binding, but it does not provide any indication that the MHC binding peptides would or could ever be presented in an MHC context in vivo after processing of a protein antigen (Rock, K. L, Reits, E, and Neefjes J. (2016); Neefjes, 1, Jongsma, Paul, P and Bakke, O (2011)).

Specific embodiments of the invention

As indicated above, the 1^st aspect of the invention relates to a method for quantitative determination of stability of binding between at least one peptide and an MHC molecule, comprising the subsequent steps of a) preparing a plurality of samples of cell lysates comprising complexes between MHC molecules and peptides, where the lysates are obtained from a plurality of MHC expressing cells (preferably human cells) that have naturally processed said peptides from protein antigens, b) subjecting the plurality of samples to the conditions of i) incubation at defined physicochemical conditions, where incubation time varies between the plurality of samples and where the physicochemical conditions are kept constant between the plurality of samples, or ii) incubation at defined physicochemical conditions, where the incubation time is kept constant between the plurality of samples and where the physicochemical conditions vary between the plurality of samples, c) isolating complexes between MHC molecules and peptides from the plurality of samples, d) determining, by mass spectrometric analysis, the at least one peptide's relative quantities in the plurality of samples after step c), and e) deriving at least one stability score for the at least one peptide based on the quantities determined in step d).

This inventive method has proven (cf. the Example section) to provide detailed information about peptides that are natural products of antigen processing in nucleated cells and in particular to provide a means for developers of e.g. peptide-based vaccines and diagnostics to focus on those peptides that are likely to be specifically presented to T-cells by antigen presenting cells for a prolonged period of time, thereby increasing the likelihood of recognition and binding. By subjecting the complexes to step b), it is determined for each complex how its binding properties are under near-physiological conditions over time or under varying entropy conditions, and - importantly - it thereby becomes possible to rationally select peptides for further development based on ranking of their binding properties. This also implies that the at least one peptide normally is a larger number of peptides that each obtain a stability score after being subjected to the method of the invention.

The cells that are initially used to provide the cell lysates in step a) are as a rule pelleted into pellets of 5xl0⁷-lxl0⁹ cells; however, the number of cells is not crucial, but merely has to be large enough to allow that the subsequent steps provides a sufficiently high number of samples of cell lysates so as to obtain the necessary information in step d. Post lysis of these large pellets, the lysate is divided into the desired number of replicates (each of the same number of cells), which are each subjected to conditions specified in step b). The large pellets can also be used in the protocol described in Purcell et ai. 2019 to provide a large spectral library of peptides which serves as reference for the MS analysis carried out in the method of the invention.

Typically, the MHC-expressing cells are mono-allelic for the MHC molecule; this allows for a definite mapping of peptide binding versus a given MHC molecule, in humans mapping of peptide binding versus a specific HLA molecule.

When the MHC molecule is an MHC class I molecule, it is preferably selected from HLA-A, HLA-B, and HLA-C. The frequencies of known HLA alleles is provided at www.allelefrequencies.net/hla6006a.asp and since the method of the invention is applicable to any HLA allele, it is e.g. of interest to carry out the present invention using the most relevant alleles for the population that is to be vaccinated with peptides.

When the MHC molecule is an MHC class II molecule, it is preferably HLA-DP, HLA-DQ, and HLA-DR.

In general, the inventive method of the 1^st aspect of the invention conceptually follows the general outline of steps for cell preparation/isolation, isolation of complexes, elution of peptides and MS analysis, which is detailed in Purcell et ai 2019. For instance, it is preferred that the plurality of MHC-expressing cells prior to step a) have been isolated/separated from other organic material by centrifugation and optionally have been frozen for storage prior to step a). Freezing the cells should be carried out at sufficiently low temperature to ensure that the cells, and thereby the MHC complexes with peptides, are not degraded - freezing in liquid nitrogen is preferred.

Also, in line with Purcell et ai. 2019, step c) preferably comprises isolation of the complexes by means of affinity purification specific for the MHC molecule; detailed protocols are set forth in the examples. I.e. the step utilises a reagent that detects/isolates the intact pMHC complex. This reagent can be an antibody or any molecule that has or mimics the binding properties of an antibody: antibody fragments and variants can be used and so can molecular imprinted polymers. Also here, it is important that the temperature is kept sufficiently low to ensure integrity of the MHC complexes with the peptides; somewhat unexpectedly, a sufficiently low temperature has proven to be room temperature. In the examples, two different procedures are reported for isolation of the complexes in the large-scale and small- scale experiments, respectively. In the large-scale experiment, the complexes are captured with cross-linked antibodies bound to a matrix in an affinity column and subsequently eluted, thus providing an eluate without capture antibody, whereas the small-scale experiment utilises capture antibody coupled to protein A, where the eluate comprises both the complexes and the antibodies, followed by filtration (to remove antibody). However, in practice the immunoprecipitation method used in the large-scale experiment could be used for the method of the first aspect of the invention, since it is possible to apply it on the lysates that have been subjected to step b. Thus, the exact separation method for isolation of the complexes is not essential to the invention.

Steps a) and b) constitute a deviation from/addition to the protocol in Purcell et ai 2019: the preparation of a plurality of samples (typically corresponding to the number of different physiochemical and/or time-course conditions applied in the next step), is novel and necessary in order to investigate the stability of binding between MHC and peptides under a set of different conditions. It is, however, often convenient to utilize the present method in combination with the protocol of Purcell et a/. 2019 because this will provide a large spectral peptide library against which the peptides examined in the presently presented method can be analysed.

Since each peptide examined in the later MS step d) cannot be directly quantitatively compared with the other peptides, it is according to the present invention advantageous to investigate the quantity of each peptide relative to its own quantity measured from one of the plurality of samples. In other words the quantities for a peptide determined in step d) are normalized relative to one single of the quantities determined for the peptide - this can e.g. be a median or average value of multiple measured values from peptides subjected to the same circumstances; typically, the quantities are normalized relative to the highest quantity measure for peptide, which for each peptide typically will be the quantity found in the sample subjected to either the shortest incubation time in step b)i) or the quantity determined for the condition that provides the lowest incubation entropy in step b)ii).

When the method of the 1^st aspect in step b) comprises subjecting the plurality of samples to conditions i), it is preferred that the stability score is in the form of a decay constant (A) for peptide binding to the MHC molecule, or any value being a strictly increasing or decreasing function of the decay constant such as the half-life (ti ₂) or the mean lifetime (T) of the peptide binding to the MHC molecule. As is well-known, the decay constant, half-life, and mean lifetime are related as follows: t_1/2

= tΐh (2).

In order to accurately determine a decay constant, the representing MHC-peptide complexes are conveniently fitted to a decay curve (cf. below), with incubation times represented on the x-axis and a quantity measure represented on the y-axis. It is for practical reasons preferred that data are sampled within 24-48 hours when incubation of cell lysates is made at physiological temperature (in the examples, incubation times range between 0 hours to 24 hours) but if selecting a different incubation temperature, the incubation times could be longer (for instance if the selected incubation temperature is low) or shorter (if the incubation temperature was selected to be increased). Also, some peptides have been observed by the inventors to remain stably bound at physiological conditions even after 48 hours, which is hence not a general limitation. In general, the incubation times can be reduced if the physicochemical constant conditions provide for relative high entropy and vice versa - however, the physicochemical conditions should not be destructive in the sense that they could irreversibly denature the MHC-peptide complexes by causing intramolecular degradation of the MHC sub-components.

When the method of the 1^st aspect of the invention comprises subjecting the plurality of samples to conditions ii), it is preferred that the stability score is in the form of a T_m value, or any strictly increasing or decreasing function thereof. Use of T_m as the stability score presupposes that the physicochemical condition that is varied in step b)ii) is temperature, which is also the preferred embodiment, but the invention is not limited to this embodiment. In practice, application of chaotropic agents such as urea, n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulphate, and thiourea in various concentrations would also be able to cause the dissociation of the binding between the MHC molecules and their bound peptides, but it would require a somewhat laborious setup (such as hollow fibres that have the complexes stably bound) in order to rapidly interrupt the contact between the complexes and such chaotropic agents so as to ensure that the plurality of samples are subjected to the chaotropic agent for the same period of time. At any rate, the important feature of step b)ii) is to be certain that the MHC-peptide complexes are subjected to conditions that provide different levels of entropy but for defined periods of time.

The duration of the constant incubation time in step b)ii) is not essential as long as it is sufficient to provide a measurable effect of the varying physicochemical conditions on the stability of the complexes. Also, the varied physicochemical condition (such as temperature) must be chosen so as to at least avoid denaturation of the individual polypeptides being part of the MHC complexes thereof - it goes without saying that subjecting pMHC to temperatures or other conditions that would lead to intramolecular destruction (i.e. irreversible denaturation) of protein structure will provide no meaningful results in terms of stability of binding between MHC and peptide. So, setting out at - and concentrating on obtaining measurements from - conditions where the stability of pMHC is (almost) exclusively governed by the dissociation of peptides from intact MHC is preferred. In experiments carried out by the inventors (data not shown) it was found that an incubation time in step b)ii) of 5-10 minutes (with about 10 minutes being preferred) provides excellent results. In the present examples, the varied physicochemical condition was temperature, which was varied between body temperature (37°C) and 73°C, which was effective in providing the necessary information for a melting curve and T_m values for individual pMHC complexes.

For both of conditions i) and ii) the choice of physicochemical conditions is preferably made in order to ensure 1) that variations in isolated peptides between conditions can be obtained and 2) that the conditions are not too destructive to provide meaningful results. Hence, the choice of different temperatures is typically made within the interval 1-90°C - for instance, all incubation temperatures >0°C detailed under the second aspect of the invention are useful in the first aspect.

After having carried out step c), the method of the invention can again be carried out essentially as disclosed in Purcell et a/. 2019, meaning that it is preferred that step d) comprises tandem mass spectrometric analysis. For this purpose, step c) typically includes a further step of separating peptides from MHC molecules to allow the subsequent MS testing of the isolated peptides. This provides MS data that can subsequently be subjected to further analysis using state of the art software for peptide identification (such as the PEAKS® software) and for data independent acquisition quantitative methods (such as the Skyline software (Maclean et aL 2010) or DIA-NN (Demichev et al. 2020)).

An important feature of preferred embodiments of the method of the 1^st aspect of the invention is that step d) comprises that the amino acid sequence of the at least one peptide and a measure of its relative quantity is determined in step d) in each of the plurality of samples. As noted above, this provides the possibility to compare - for each peptide - its relative quantities (using as a reference point its own quantity in one sample or the mean or median of several quantities of the same peptide from samples subjected to identical conditions) in samples that have been subjected to different conditions in step b). When using the expression "relative quantity", it is meant that the data derived from the method of the invention at least have to provide information about the amount of each peptide subjected to one set of conditions relative to the same peptide subjected to a different set of conditions - this does not exclude that absolute values of quantity may be derived and useful, but in order to derive a stability score, it is not essential to derive an absolute measure of quantity.

The stability score of the at least one peptide is preferably derived by fitting its quantities determined in step d) to a decay curve against time if the plurality of samples have been subjected to conditions i) in step b) or to a sigmoid melting curve against temperature if the plurality of samples have been subjected to conditions ii) in step b).

In some embodiments of the method of the 1^st aspect of the invention at least two determinations are made of stability of binding between at least one peptide and an MHC molecule, wherein one determination comprises subjecting a first plurality of samples to conditions i) in step b) and another determination comprises subjecting a second plurality of samples to conditions ii) in step b). Therefore, in some embodiments of the invention, at least two stability scores are derived for the at least one peptide in step d), such as a stability scores detailed above. It is however relatively time- and resource-consuming to carry out both types of experiments, and since both sets of conditions will provide the necessary information on the stability between peptides and MHC, it is normally only relevant to carry out one of the two of which the thermostability (or stability towards other variations in entropy) condition testing has turned out to be the least time-consuming. It is to be noted that the inventors have demonstrated (cf. Fig. 9) that the stability measures obtained from time-course and thermostability studies, respectively, correlate, so that each can be used as a surrogate for the other.

Apart from merely providing information about naturally processed peptides, the method of the 1^st aspect of the invention can in a variety of embodiments be utilised to "map" the MHC binding peptides that are derived from antigen-presenting cells that have been subjected to controlled conditions such as infections, mutations (e.g. cancer related) etc.

So an embodiment of the method of the 1^st aspect of the invention comprises that the MHC expressing cells are nucleated cells that are obtained by infecting a sample of nucleated cells, preferably in vitro, with a an intracellular infectious agent (such as a virus, intracellular bacterium, and intracellular protozoans) and allowing the nucleated cells to subject the protein(s) of the intracellular infectious agent (or expression products produced by the infected cell when virally infected) to antigen processing and present peptide fragments from these proteins of the intracellular infectious agent bound to MHC molecules on their surface. By using this approach, naturally processed peptides that 1) match the proteome of infectious agent and 2) exhibit a desired stability (typically intermediate or high) for MHC binding can be identified and subsequently used as components in vaccines and diagnostics. Likewise, the method of the 1^st aspect of the invention include embodiments, wherein the MHC expressing cells are malignant cells obtained by recovering a sample of neoplastic cells from malignant tumour tissue or from a cell line derived from neoplastic cells. By using this approach, naturally processed peptides that 1) do not match the proteome of non-malignant cells (or are overexpressed or modified compared to the proteome of non-malignant cells) and 2) exhibit a desired stability (typically intermediate or high) for MHC binding can be identified and subsequently used as components in vaccines and diagnostics.

Furthermore, the method of the 1^st aspect of the present invention can entail that the MHC expressing cells are professional antigen presenting cells that are obtained by contacting a sample of professional antigen presenting cells with extracellular protein or an extracellular infectious agent and allowing the professional antigen presenting cells to take up protein, subject the protein to antigen processing and present peptide fragments from the protein bound to MHC molecules on their surface, and subsequently recovering the professional antigen presenting cells. By using this approach, naturally processed peptides that 1) match the proteome of infectious agent or the sequence(s) of the extracellular protein and 2) exhibit a desired stability (typically intermediate or high) for MHC binding can be identified and subsequently used as components in vaccines and diagnostics. Contacting the cells with the extracellular protein or extracellular infectious agent can be achieved by several methods - for instance, co-culture is one possibility, but also various steps for admixing the cells with the extracellular material are possibilities.

2^nd aspect of the invention

As mentioned above, the 2^nd aspect of the present invention relates to a method for (normally qualitative) determination of binding between at least one peptide and an MHC molecule, comprising the subsequent steps of

I) preparing at least one sample of cell lysates comprising complexes between MHC molecules and peptides, where the lysates are obtained from a plurality of MHC expressing cells (preferably human cells) that have naturally processed said peptides from protein antigens, wherein the at least one sample of cell lysates is prepared at a temperature >4°C and/or wherein the at least one sample of cell lysates is/are incubated for a period of time after obtaining the cell lysates at defined physicochemical conditions at a temperature >0°C ,

II) determining, by mass spectrometric analysis, whether the at least one peptide is present as part of a complex in the at least one sample after step I). The findings made in relation to the 1^st aspect of the invention can be summarized by concluding that naturally processed peptides that are isolated from MHC complexes have different stabilities for binding to the MHC and that those having high stability are more likely to be presented to T-cells by APCs. The method of the 2^nd aspect of the invention enables exploitation of this finding in a slightly simpler manner than by necessarily determining a stability score derived from multiple measurements of pMHC abundance as in the 1^st aspect of the invention.

For instance, one set of embodiments of the 2^nd aspect of the invention compares the results after step II between samples of peptide-MHC which have been subjected to different levels of entropy, typically different temperature levels or between samples that have been incubated at physicochemical conditions that allow an appreciable irreversible dissociation of peptide MHC complexes. Peptides that are not detected beyond a detection threshold at higher entropy levels (or after prolonged incubation) will be considered absent as part of a complex in the sample at these entropy levels or after the incubation period. The end result is ideally that from the original pool of binding peptides present on the MHC expressing cells (which can be considered the reference sample that defines the maximum number of potentially relevant peptides bound to MHC, a fraction thereof will be present as part of a complex in the sample at all entropy levels tested or after even the longest incubation times. These peptides are to be considered "generally stable binders". It is of note that the highest entropy levels that the complexes are exposed to will not result in denaturation of the MHC structure in the sense that the individual components of the MHC molecule remains largely intact - as mentioned under the discussion of the 1^st aspect of the invention, an entropy level that will render association between MHC and peptide impossible due to extensive destruction of the intramolecular structure of MHC will not provide any meaningful results. In practice, this means that temperatures exceeding 75°C should largely be avoided.

An even more simplified version uses only one single determination, preferably at an entropy level close to or higher than the entropy level found at physiological conditions but still at an entropy level that does not result in denaturing of MHC.

Typically, the determination of binding in the 2^nd aspect is "qualitative" in the sense that only presence or absence of a given peptide is determined. However, it is possible to employ any available quantitative MS determination methods in the 2^nd aspect of the invention, and if such quantitative determination methods are employed, the outcome of the method will be a quantitative determination of the peptide. This emphasizes that the exact choice of MS approach is of limited importance whereas it is essential that the peptides whose presence is determined have been subjected to entropy conditions and/or incubation times that allow for conclusions to be drawn with respect to their stability for binding to MHC molecules. In embodiments of the 2^nd aspect of the invention the temperature >4°C is selected from a temperature of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84 about 85, about 86, about 87, about 88, about 89, and about 90°C. Likewise, in embodiments the temperature >0°C is selected from a temperature of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84 about 85, about 86, about 87, about 88, about 89, and about 90°C.

In embodiments where incubation for a period of time is employed, the period of time is preferably at least or about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 120, about 240, about 480, about 720, about 960, about 1440, about or 1920, about 2160, about 2880, about 3600, about 4320, about 5040, about 5760, about 6480, about 7200, about 7920, about 8640, about 9360, about 10080, about 10800, or about 11520 minutes. The incubation period is, however, relative to the entropy conditions. If selecting to incubate at relatively low temperatures (<10°C) or other low entropy levels, incubation times of weeks or even months can be relevant. On the other hand, if selecting to incubate at high entropy levels, very short incubation times can be useful, e.g. as short at 30 seconds, 1 minute, 2 minutes, 3 minutes, and 4 minutes.

In general, the method steps described in detail for the 1^st aspect of the invention can where relevant be applied mutatis mutandis to the second aspect, i.e. all details pertaining to the provision and preparation of MHC expressing cells, MHC molecules, complex isolation, peptides isolation and MS procedures. In particular, step II preferably comprises the steps of isolating complexes of MHC and peptides, preferably by means of affinity purification specific for the MHC molecule, separating peptides from the complexes and subjecting separated peptides to MS.

In some embodiments a plurality of samples is prepared wherein lysis conditions and/or incubation conditions favour the preservation of complexes between MHC and peptides to different degrees across the samples. This provides for several different MS "fingerprints" of the samples, one for each condition, where peptides are determined to be present or absent in step II. With increasing temperature/entropy levels (or prolonged incubation), a decreasing number of peptides found in step II will be observed - and this allows selection of those peptides that are sufficiently stable by simply selecting those that appear at the higher (preferably all) selected entropy conditions. While this approach does not necessarily provide any indication of the abundance of the stable peptides, it nevertheless provides a simple method for screening of peptides that are not stable. It is understood, however, that the exact choice of MS determination method will dictate whether an indication of abundance can be arrived at or not.

Hence, in an embodiment of the 2^nd aspect, the at least one sample is subjected to one single set of lysis and incubation conditions; this set of conditions is preferably one that reflects physiologic conditions in the sense that the physicochemical conditions and the incubation time will effectively screen off those peptides that would not be stably MHC binding peptides in vivo.

As is the case for the 1^st aspect of the invention, the MHC expressing cells can be those discussed above in respect of that aspect, and the same is the case for the MHC molecules.

Further, and also as described under the first aspect, the plurality of MHC expressing cells may prior to step I) have been isolated from other organic material by centrifugation and optionally have been frozen for prolonged storage prior to step a); and/or step II) comprises tandem mass spectrometric analysis; and/or the MHC expressing cells are human cells; and/or the MHC expressing cells are nucleated cells that are obtained by infecting a sample of nucleated cells, preferably in vitro, with a an intracellular infectious agent and allowing the nucleated cells to subject the protein of the intracellular infectious agent to antigen processing and present peptide fragments from protein of the intracellular infectious agent bound to MHC molecules on their surface; and/or the MHC expressing cells are malignant cells obtained by recovering a sample of neoplastic cells from malignant tumour tissue or from a cell line derived from neoplastic cells; and/or the MHC expressing cells are professional antigen presenting cells that are obtained by contacting a sample of professional antigen presenting cells with extracellular protein or an extracellular infectious agent and allowing the professional antigen presenting cells to take up protein, subject the protein to antigen processing and present peptide fragments from the protein bound to MHC molecules on their surface, and subsequently recovering the professional antigen presenting cells.

Further aspects of the invention

The 3^rd aspect of the invention relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of the 1^st or 2^nd aspect and subsequently admixing one or more peptides, which are selected from peptides of the plurality that exhibit the characteristics 1) and/or 2):

2) the peptide is selected from peptides that are found to be present as part of the complexes in step II of the method of the 2^nd aspect of the invention, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient, see below. This aspect thus takes advantage of the findings made in the method of the 1^st or 2^nd aspects of the invention, and provides as a product an immunogenic composition "cocktail" such as a vaccine, which is produced by methods known per se.

A peptide that exhibits the characteristics in groups 1) and/or 2) above is termed "a stably MHC binding peptide" herein.

The 4^th aspect relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of the 1^st aspect or 2^nd aspect and subsequently preparing a polypeptide, which comprises amino acid sequences of one or more peptides, which are selected from stably MHC binding peptides of the plurality and admixing the polypeptide with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient. This aspect thus takes advantage of the findings made in the method of the 1^st or 2^nd aspect of the invention, and provides as a product a multi-epitope protein construct-based immunogenic composition such as a vaccine, which is produced by methods known per se. Vaccines prepared according to the invention typically comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid(s), usually in combination with "pharmaceutically acceptable carriers", which include any carrier that does not itself induce immune responses harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.

Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immune stimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogen, cf. the description of immunogenic carriers supra.

The 5th aspect relates to a method of preparing an immunogenic composition, comprising quantitative/qualitative determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of the 1^st or 2^nd aspect and subsequently admixing a nucleic acid (such as a plasmid), which is capable of expressing nucleotide sequences encoding one or more peptides, which are selected from stably binding MHC peptides of the plurality. This aspect corresponds to the 3rd aspect, but in a nucleic acid vaccine format.

The 6th aspect relates to a method of preparing an immunogenic composition, comprising quantitative determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of the 1^st or 2^nd aspect and subsequently admixing a nucleic acid (such as a plasmid), which is capable of expressing a nucleotide sequence encoding a polypeptide comprising the amino acid sequences of one or more peptides, which are selected from stably MHC binding peptides of the plurality. This aspect is analogous to the 4th aspect, but in a nucleic acid vaccine format.

Thus the 5th and 6th aspects constitute an alternative to protein-based vaccines, such as for instance DNA vaccination (also termed nucleic acid vaccination or gene vaccination) (cf. e.g. Robinson & Torres (1997) Seminars in Immunol 9: 271-283; Donnelly et at. (1997) Annu Rev Immunol 15 : 617-648). Also, RNA vaccination is possible. When administering such formats, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA or RNA constructs in the individual to whom it is administered.

For DNA vaccine preparation, the nucleic acid is typically integrated in a vector, such as an expression plasmid. Vectors of the invention may be used in a host cell to produce a polypeptide of the invention that may subsequently be purified for administration to a subject or the vector may be purified for direct administration to a subject for expression of the protein in the subject (as is the case when administering a nucleic acid vaccine).

Suitable expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in the vaccinated host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

1. Promoters and Enhancers

A "promoter" is a control sequence. The promoter is typically a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural state. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference). Naturally, it may be important to employ a promoter and/or enhancer that effectively direct(s) the expression of the DNA segment in the vaccinated individual. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression (see Sambrook et al, 2001, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, or inducible and in certain embodiments may direct high level expression of the introduced DNA segment.

Examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus, include but are not limited to Immunoglobulin Heavy Chain, Immunoglobulin Light Chain, T Cell Receptor, HLA DQa and/or DQ3, b- Interferon, Interleukin-2, Interleukin-2 Receptor, MHC Class II 5, MHC Class II HLA-DRa, b- Actin, Muscle Creatine Kinase (MCK), Prealbumin (Transthyretin), Elastase I, Metallothionein (MTII), Collagenase, Albumin, a-Fetoprotein, y-Globin, b-Globin, c-fos, c-HA-ras, Insulin, Neural Cell Adhesion Molecule (NCAM), al-Antitrypain, H2B (TH2B) Histone, Mouse and/or Type I Collagen, Glucose-Regulated Proteins (GRP94 and GRP78), Rat Growth Hormone, Human Serum Amyloid A (SAA), Troponin I (TN I), Platelet-Derived Growth Factor (PDGF), Duchenne Muscular Dystrophy, SV40, Polyoma, Retroviruses, Papilloma Virus, Hepatitis B Virus, Human Immunodeficiency Virus, Cytomegalovirus (CMV) IE, and Gibbon Ape Leukemia Virus.

Inducible Elements include MT II - Phorbol Ester (TFA)/Heavy metals; MMTV (mouse mammary tumor virus) - Glucocorticoids; b-Interferon - poly(rl)x/poly(rc); Adenovirus 5 E2 - EIA; Collagenase - Phorbol Ester (TPA); Stromelysin - Phorbol Ester (TPA); SV40 - Phorbol Ester (TPA); Murine MX Gene - Interferon, Newcastle Disease Virus; GRP78 Gene - A23187; a-2-Macroglobulin - IL-6; Vimentin - Serum; MHC Class I Gene H-2xb - Interferon; HSP70 - E1A/SV40 Large T Antigen; Proliferin - Phorbol Ester/TPA; Tumor Necrosis Factor - PMA; and Thyroid Stimulating Hormonea Gene - Thyroid Hormone.

Also contemplated as useful in the present invention are the dectin-1 and dectin-2 promoters. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression.

The particular promoter that is employed to control the expression of peptide or protein encoding polynucleotide of the invention is not believed to be critical, so long as it is capable of expressing the polynucleotide in the vaccinated individual. Where a human cell is targeted, it is preferable to position the polynucleotide coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a bacterial, human or viral promoter. In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high level expression of a related polynucleotide to this invention. The use of other viral or mammalian cellular or bacterial phage promoters, which are well known in the art, to achieve expression of polynucleotides is contemplated as well.

It is contemplated that a desirable promoter for use with the vector is one that is not down- regulated by cytokines or one that is strong enough that even if down-regulated, it produces an effective amount of the protein/ polypeptide of the current invention in a subject to elicit an immune response. Non-limiting examples of these are CMV IE and RSV LTR. In other embodiments, a promoter that is up-regulated in the presence of cytokines is employed. The MHC I promoter increases expression in the presence of IFN-y.

Tissue specific promoters can be used, particularly if expression is in cells in which expression of an antigen is desirable, such as dendritic cells or macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters. 2. Initiation Signals and Internal Ribosome Binding Sites (IRES)

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided.

One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic and may be operable in bacteria or mammalian cells. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well an IRES from a mammalian message. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference). 2. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. (See Carbonelli et al, 1999, Levenson et al, 1998, and Cocea, 1997, incorporated herein by reference). Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

3. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. If relevant in the context of vectors of the present invention, vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al, 1997, incorporated herein by reference).

4. Termination Signals

The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

The terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (poly A) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently in vertebrates. Thus, in other embodiments involving vertebrates such as humans, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message.

Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the bovine growth hormone terminator or viral termination sequences, such as the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation. 5. Polvadenylation Signals

One will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

6. Origins of Replication

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "on"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively, an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

7. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acid construct may be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. When transcribed and translated, a marker confers an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin or histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP for colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers that can be used in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a protein of the invention. Further examples of selectable and screenable markers are well known to one of skill in the art. As an alternative, RNA vectors encoding the immunogenic peptide or polypeptide can be used. A review of the most recent advances using this vaccine format is provided in Pardi N et al. 2018, Nat Rev Drug Discov 17(4): 261-279.

The 7th aspect relates to a method of preparing an immunogenic composition, comprising quantitative determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of the 1^st or 2^nd aspect and subsequently admixing a microorganism or virus (preferably non-pathogenic), which is capable of expressing nucleotide sequences encoding one or more peptides, which are selected from peptides of the plurality that exhibit a stability score above the average stability score of the plurality of peptides as a whole, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient. This aspect is analogous to the 3rd and 5th aspects but in a "live vaccine" format.

The 8th aspect relates to a method of preparing an immunogenic composition, comprising quantitative determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of the 1^st aspect and subsequently admixing a microorganism of virus (preferably attenuated and /or non-pathogenic), which is capable of expressing a nucleotide sequence encoding a polypeptide comprising the amino acid sequences of one or more peptides, which are selected from peptides of the plurality that exhibit a stability score above the average stability score of the plurality of peptides as a whole, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient. This aspect is analogous to the 4th and 6th aspects but in a "live vaccine" format.

Live vaccine vectors and viral vaccines are well known in the art and include attenuated and/or non-pathogenic bacteria (such as mycobacteria, such a M. bovis BCG) and virus (such as poxvirus vaccine vectors, including MVA).

In embodiments of any of aspects 2-7, it is preferred to also admix with an immunological adjuvant.

The compositions prepared according to the invention thus typically contain an immunological adjuvant, which is commonly an aluminium based adjuvant or one of the other adjuvants described in the following:

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to : (1) aluminium salts (alum), such as aluminium hydroxide, aluminium phosphate, aluminium sulphate, etc; (2) oil-in-water emulsion formulations (with or without other specific immune stimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell 8i Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE, although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM) ; (3) saponin adjuvants such as Stimulon™ (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immune stimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) other substances that act as immune stimulating agents to enhance the effectiveness of the composition.

As mentioned above, muramyl peptides include, but are not limited to, N -acetyl- mu ramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl- L-alanine-2"-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

The immunogenic compositions (e.g. the immunising antigen or immunogen or polypeptide or protein or nucleic acid, pharmaceutically acceptable carrier (and/or diluent and/or vehicle), and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Pharmaceutical compositions can thus contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the relevant immunogen, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individuals to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies or generally mount an immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount of immunogen will fall in a relatively broad range that can be determined through routine trials. However, for the purposes of protein vaccination, the amount administered per immunization is typically in the range between 0.5 pg and 500 mg (however, often not higher than 5,000 pg), and very often in the range between 10 and 200 pg.

The immunogenic compositions are conventionally administered parenterally, e.g, by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (cf. e.g. W0 98/20734). Additional formulations suitable for other modes of administration include oral, pulmonary and nasal formulations, suppositories, and transdermal applications. In the case of nucleic acid vaccination and antibody treatment, also the intravenous or intraarterial routes may be applicable.

Dosage treatment may be a single dose schedule or a multiple dose schedule, for instance in a prime-boost dosage regimen or in a burst regimen. The vaccine may be administered in conjunction with other immunoregulatory agents as may be convenient or desired. PREAMBLE TO EXAMPLES

The present experiments were carried out using and expanding on the protocol of Purcell,

Ramarathinam and Ternette, 2019 with certain modifications. The outline of the experiments performed below in the illustrative examples is set forth in the following and is also shown in schematic form in Fig. 1:

1) A mono-allelic cell line was prepared, cultured, and pelleted (in this case C1R cells were transfected to be mono-allelic for HLA-A*02:01).

2) Large scale immunoprecipitation/elution (cell pellet size ~ 8x10^s cells) was performed to create an MS spectral library as described in detail in the protocol of Purcell, Ramarathinam and Ternette, 2019.

3) Data dependent acquisition (DDA) mass spectrometry (MS) was performed using a Q Exactive (Thermo) on the large elution.

4) The PEAKS® software package was used to create a spectral library from DDA data for the MHC allele (in this case, the HLA allele) of interest.

5) Small-scale immunoprecipitation/elution (cell pellet sizes ~ 2xl0⁷-5xl0⁷) was performed on samples in triplicates or quadruplicates for each time/temperature point (cf. below) to create stability curves (time-course, exponential decay curves or thermal, sigmoidal curves)

6) Data independent acquisition (DIA) mass spectrometry (MS) was performed using a Q Exactive (Thermo) on all stability data replicates.

7) The Skyline software package was used to analyse and visualise peak areas of stability data replicates using the PEAKS®-generated spectral library to identify precursor and product ions.

8) MS peak areas (from step 6) of 8-mer - 11-mer peptides were normalised based on peak areas of iRT peptides spiked into samples.

9) Peptides were filtered based on Skyline confidence threshold (dotP 0.85) with peak areas changed to 0 if peak confidence was less than the set threshold. 10) Peptides were filtered based on sequences from background peptides and unusual sequences.

11) Points were outlier corrected by calculating median of time/temperature point and neighbouring time/temperature points and taking mean of these median values.

12) Median intensity values for each peptide were fitted to a sigmoidal curve (for thermal stability measurements) or exponential decay curves (time course stability measurements) and, subsequently, thermal melting points (T_m) or half-life values (A) were calculated, respectively.

EXAMPLE 1

Large scale and small scale immunoprecipitation/elution

A mono-allelic cell line (C1R cells, mono-allelic for HLA-A*02:01) was grown, pelleted and stored at -80°C for maximum 1 month.

The large scale protocol steps entailed

1) Crosslinking of antibody specific to the MHC molecule that it was desired to isolate (in the following experiments, antibody W6/32 was used) to Protein A Sepharose resin,

2) Grinding of large cell pellet (~ 8x 10^s cells) and clearing of lysate with centrifugation,

3) Addition of lysate to immune affinity column to isolate pMHC molecules of interest using w6/32 antibody,

4) Elution of pMHC molecules with 10% acetic acid,

5) Fractionation of sample and separation of peptides from the MHC molecule and 32m using HPLC (C18 clean-up of sample in preparation for MS),

6) MS analysis in DDA mode.

The small scale protocol steps entailed

1) Incubation of antibody specific to the MHC molecule that it was desired to isolate (in the following experiments, antibody w6/32 was used) to allow binding to Protein A Sepharose resin (1 hour incubation),

2) Grinding of large cell pellet (~ 8x 10^s cells) and separation of the lysate into sample replicates (~ 5x l0⁷ cells) for thermal or time course treatment,

3) Treatment of cell lysate with desired measure (heat or time) and subsequent cooling of the sample on ice for a few minutes, 4) Isolation of pMHC complexes using small columns (Mobispin®) with prepared antibody- resin,

5) Elution of pMHC complexes and antibody from resin using 10% acetic acid,

6) Separation of peptides from the larger molecules (MHC, 32m and antibody) using 5kDa cut-off filters,

7) C18 clean-up of samples using zip tips, elution of sample in 0.1% formic acid, 30% acetonitrile, and

8) MS analysis on samples in DIA mode.

Reagents and equipment for antibody cross-linking used in large scale immunoprecipitation/elution

• Meneclenal antibedy W6/32 (www.atcc.erg/preducts/all/HB-95.aspx) specific fcr HLA- A*02:01 was used, either purified cr from hybridcma supernatant. 10 mg pf purified antibedy per 1 ml pf resin is needed, cr approximately 1 litre cf supernatant per 1 ml cf resin depending cn the hybrid. The isctype cf the antibedy was checked tc determine whether it bcund tc protein A cr G. Purified antibedy or tissue culture supernatant were used to bind the resin. The amount of antibody in the supernatant generally ranged between 5-30 pg/ml depending on the B-cell hybrid.

Prior to doing a 10 mg coupling it was confirmed that DMP cross linking would not affect the binding capacity of the antibody. This was done using a small scale immunoprecipitation with antibody loaded beads with and without cross linking (as detailed at the end of this method). If this prevented the antibody from working a different resin such as NHS activated Sepharose® (the anti-HLA antibodies W6/32, L243, BB7.1, Rm5112, SPV-L3, B721, Y-3, 10.2.16 and 28.8.6s have previously been confirmed to work with DMP). For reference, antibody isotype and their affinities towards protein A and G are provided in the following table:

• Poly-Prep Chromatography column (BioRad), yellow top and bottom caps.

• Tubing and syringes.

• 10% acetic acid in fresh MilliQ (mass spec grade acid in new glassware that has not been washed with detergent but rinsed with MilliQ).

• Protein A Sepharose® Fast Flow (PAS; Amersham).

• PBS (phosphate-buffered saline), filtered.

• Borate Buffer: o Solution "A": 0.1 M Boric acid/0.1 M KCI Per 100 ml

• boric acid: (Mw 61.83 g): 0.62 g.

• KCI (Mw 74.56 g) 0.75 g. o Solution B: 0.1 M NaOH (Mw 40.00 g) 0.4 g/100 ml. o For 100 ml of Borate buffer pH 8: o 50 ml A + 3.97 ml B + 46.03 ml fresh MQ, checked for pH = 8 with pH paper, and filtered through 0.2 mM PES filter.

• Tris: 0.2 M, pH 8, filtered, kept cold.

• Citrate: 0.1 M, pH 3, filtered.

• Triethanolamine (TeO; stock density 1.125 g/ml, Mw 149.19 ie 7.54 M) o Viscous solution, use cut blue tip. o 1.326 ml TeO per 50 ml, adjust pH to 8.2 with HCI, not filterered. o Dimethyl pimelimidate (DMP; Sigma D8388): 40 mM in 0.2 M Triethanolamine. DMP is prepared by dissolving 250 mg (1 vial) DMP-2HCI in 22 ml 0.2 M Triethanolamine pH 8.2. pH is adjusted to 8.3 with NaOH, and brought to 24.1 ml, without filtering. DMP solutions should be prepared and used on the same day. Generally one 250 mg vial is used per 2 ml resin.

Retort stand

Procedure for antibody cross-linking in large scale protocol

1. A cap was placed on the bottom of the column and filled with 10% acetic acid and allowed to sit for 20 min at room temperature. The cap was removed, and the column allowed to flow through, rinsed with a further 10 ml of acid, and then thoroughly with milliQ water in order to extract any non-adhering polymer.

2. PAS was fully resuspended in a bottle and the required amount removed using a 1 ml tip. The protein A Sepharose® (PAS) was supplied as a ~50% slurry (confirmed by visual inspection before resuspending), therefore for every 1 ml of bed volume, 2 ml of slurry was required (the calculation was adjusted if slurry deviated from 50% resin).

3. PAS was added to a column with bottom cap in place and allowed to settle, and subsequently washed with 10 column volumes (CV) PBS.

Flowrate through the column was if necessary increased by attaching thoroughly cleaned tubing to the top of the column and the other end to the barrel of a 50 ml syringe secured as high as practical above the column, filling the syringe with PBS, removing the bottom cap, and washing the resin by gravity flow with 10 CV PBS. Alternatively, the flowrate was increased by attaching tubing to the top of the column and the other end to the barrel of a 50 ml syringe, and slowly depressing the plunger to create back pressure on the column to ensure that the drop rate through the column did not exceed 1 drop per second.

4. 10 mg of antibody was bound per 1 ml resin by batch i.e. using a 1 ml pipette, the PBS washed resin was removed from the column and placed in 50 ml tube. The purified antibody was purified to ~15 ml with PBS, added to the resin and rotated end over end in a cold room for 30-60 min. 5. The resin was loaded back into the column at room temperature using borate buffer to wash out the interior of the 50 ml tube and recover all the resin. If antibody containing supernatant was used, it was after step 3 loaded straight onto a washed column in the cold room (after supernatant was loaded, the procedures typically proceeded at RT). When using antibody-containing supernatant, it was also determined how much antibody the relevant hybrid was secreting, and it was tested that the supernatant contained specific antibody. If the secretion turned out to be low (less than 5 pg/ml) the hybrids were re-cloned.

(If purified antibody was used, a sample taken from the starting material added to the resin in step 5 and a sample of the flow through (i.e. step 6) (25 pi sample + 25 mI sample buffer) were compared to make sure the flow through was fairly well depleted).

6. A wash with 10 CV borate buffer pH 8 was carried out.

For testing: After washing, 25 mI aliquot of beads were placed into Eppendorf tubes by resuspending the beads at the top of the column and adding 25 mI reducing SDS sample buffer. At this point the antibody were not covalently bound to the beads, so when the sample was boiled in reducing sample buffer, the antibody dissociated from the beads and the heavy and light chains became clearly visible by Coomassie staining (approx. 50 kDa and 25 kDa).

7. A wash with 10 CV freshly made 0.2 M triethanolamine, pH 8.2 was carried out to equilibrate the column. The use of triethanolamine ensures that no free amines are present in the buffer system as this could interfere with the efficiency of crosslinking by DMP to primary amines in the protein A bound antibody.

8. Cross-linking was carried out by passing ~25 ml of 40 mM DMP in 0.2 M triethanolamine over the column, halting the flow leaving a meniscus covering the resin, then leaving at room temperature for 1 hr. This amount of DMP is sufficient for at least 20 mg of antibody and can stretch to 30 mg.

9. The cross-linking reaction was terminated by flowing over 10 CV of ice-cold 0.2 M Tris pH 8.

10. A wash was carried out with 10 CV 0.1 M citrate buffer pH 3 and collect flow through. The citrate wash will strip any antibody that has not been covalently linked. For testing: After washing in citrate, 25 pi aliquot2 of beads were mixed with 25 mI reducing SDS sample buffer. As the antibody was covalently cross-linked it remained attached to the beads even after boiling in SDS sample buffer (although generally there a small amount of leeching was observed).

11. A wash was carried out with 10 CV 0.1 M borate buffer (or PBS) with 0.02% NaN , pH 8, for incubation at 4°C.

12. The flow through from step 9 was concentrated down to 500 mI using a 15-30 kDa cut off Millipore concentrator. To monitor the crosslinking reaction, a 12% SDS PAGE gel was run for Coommassie staining as follows:

1. unstained protein ladder

2. 25 mI beads (step 5) + 25mI reducing SDS SB; boil, run 20 mI.

3. 25 mI beads (step 9) + 25mI reducing SDS SB; boil, run 20 mI.

4. 25 mI cone, flow through (step 11) + 25mI reducing SDS SB; boil, run 20mI.

This demonstrated the presence of antibody in sample 2 (before cross-linking) but not in sample 3 (after cross-linking, although there may be a small amount) and no or only a little in sample 4 (concentrated citric acid strip post cross- linking).

Reagents for large scale immunoprecipitation

10% IGEPAL 630 (Sigma) stock in MilliQ (protected from light)

1 M Tris pH 8

2 M NaCI

10% acetic acid (mass spec grade).

Total protease inhibitor cocktail (Roche): 1 tablet is enough for 50 ml buffer, if less than 50 ml is required, make 25X stock by dissolving 1 tablet in 2 ml fresh MilliQ water, aliquot and store at -20°C up to 4 months.

• Protein A Affinity Resin

• Poly-Prep Chromatography column (BioRad) for preparation of pre-column (if the column has not previously been used for peptide elution, place cap on bottom of column and fill with 10% acetic acid, sit for 20 min at RT, remove cap and allow to flow through, rinse with a further 10 ml of acid followed by MilliQ).

• For preparation of IX lysis buffer (for small cell pellets < 4xl0⁸ cells): o 0.5% IGEPAL 630 o 50 mM Tris, pH 8.0 o 150 mM NaCI o IX total protease inhibitor cocktail o MilliQ water (make sure this is freshly drawn)

• For preparation of 2X lysis buffer (for large cell pellets > 4xl0⁸ cells): o 1% IGEPAL 630 o 100 mM Tris, pH 8.0 o 300 mM NaCI o 2X total protease inhibitor cocktail o MilliQ water (make sure this is freshly drawn)

This buffer was adjusted to IX after cell grinding to accommodate the volume of the cells.

• Ultracentrifuge tubes (only required for cell pellets > 4xl0⁸ cells); polycarbonate 26.3ml capacity.

• Washbuffer 1: o 0.005% IGEPAL o 50 mM Tris, pH 8.0 o 150 mM NaCI o 5 mM EDTA o 100 mM PMSF (0.1 M stock in Abs EtOH; stored at -20°C) o 1 pg/ml Pepstatin A (1 mg/ml stock in isopropanol; stored at -20°C) o In MilliQ H₂0 o Filtered through 0.2 mM syringe filter, keep on ice.

• Washbuffer 2: o 50 mM Tris, pH 8.0 o 150 mM NaCI o in MilliQ H₂0 o Filter through 0.2uM syringe filter, keep on ice.

• Washbuffer 3: o 50mM Tris, pH 8.0 o 450mM NaCI o in MilliQ H₂0 o Filter through 0.2uM syringe filter, keep on ice.

• Washbuffer 4: o 50mM Tris, pH 8.0 o in MilliQ H₂0 o Filter through 0.2uM syringe filter, keep on ice.

• Retort stand When small cell pellets were prepared, IX lysis buffer was used and the ultracentrifugation step was replaced with centrifugation of lysates in a microcentrifuge at 13000 rpm for 20 min at 4°C. Column loading, washing and elution should be performed in cold room.

1. Cells were lysed in lysis buffer at approx.. 1.25x10^s cells per ml.

2. The frozen cell pellets were in each case ground in a cryogenic mill according to the following procedure:

• A foam dewar was filled with liquid nitrogen in a fumehood

• A 10 ml container was pre-cooled with one 10 mm ball in nitrogen bath.

• The Cell pellet was dislodged from the base of the tube by tapping on the workbench.

• If the pellet was large, it was dissected into pea sized pieces with a scalpel blade.

• 1-2 pieces of the pellet were placed in the 10 ml container with ball, placed back in nitrogen to cool again and then placed in the cryogenic mill ensuring the other side was balanced with a second 10 ml container in the same position.

• The cells were ground 30 Hz for 1 min, removed and checked to ensure that the material appeared like a fine powder, scraped out and placed directly into a tube containing cooled lysis buffer.

3. Step 2 was repeated with remaining pieces.

4. When all material was transferred to the lysis buffer, the volume was adjusted to IX using fresh MilliQ water and incubated in the cold room rotating end over end for 45 min.

5. While sample was lysing, columns were prepared in cold room:

• A 0.5 ml pre-column was prepared by placing 1 ml protein A slurry into a Poly- Prep column, washed with 10 CV of 50 mM Tris pH8 (wash buffer 4) to remove ethanol, then equilibrated with 10 CV of wash buffer 1, and capped at the bottom.

• The affinity column was set up, equilibrated with 10 CV wash buffer 1, and capped in the bottom.

6. Lysate was centrifuged for 10 min at 4000 rpm at 4°C to remove nuclei. 7. Supernatant was transferred into a pre-chilled ultracentrifuge tube filled almost to the top (if necessary with addition of further lysis buffer) and centrifuged in a Ti70 rotor for 45 min at 40,000 rpm, 4°C.

8. Supernatant was collected into pre-cooled 50 ml tubes. The supernatant should be clear, but if there remained layer of lipid on the top, this was removed carefully with a 1 ml filter tip and kept on ice in a separate tube.

9. Supernatant was run over the pre-column and collected in a 50 ml tube and then transferred onto the affinity column or the columns were set up in tandem to let the flow-through drip directly from the pre-column to the affinity column. The lysate was put over the affinity column without tubing for the first pass to ensure a slow passage over the column. The flow-through was collected.

10. The lysate was run over the affinity column two more times by attaching clean tubing to the top of the column and loading from a good height above the column from a 50 ml tube to ensure a quicker flow and allowing the lysate to be passed over multiple times.

11. The column was washed with 20 CV of cold wash buffer, with 20 CV of cold wash buffer 2 (to remove detergent), with 20 CV of cold wash buffer 3 (to remove non- specifically bound material), and finally with 20 CV of cold wash buffer 4 (to remove salt to prevent crystal formation).

12. It was ensured that the meniscus was just above the resin. All tubing was removed, and the column was eluted using 5 CV of 10% acetic acid by using either a 1 ml filter tip or a clean glass 10 ml cylinder. The eluate was collected into a clean 25 ml glass beaker or into as 2 ml low-bind Eppendorf tubes.

Reagents and equipment for MS ligand small scale experiment with stability testing (time scale varied and temperature varied)

• Cells that had been pelleted at snap-frozen, cf. above (cell pellet size 2xl0⁷-5xl0⁷)

• Protein A Sepharose Fast Flow (PAS; Amersham)

• Monoclonal antibody either purified or supernatant. Need 2 mg of purified antibody per 1 ml of Protein A resin

• 20% IGEPAL 630 (Sigma) stock in MilliQ (protect from light)

• 1 M Tris pH 8 • 5 M NaCI

• 10% acetic acid (mass spec grade, tested for purity)

• Total protease inhibitor cocktail (Roche): 1 tablet is enough for 25 ml buffer, if less than 25 ml is required, make stock by dissolving 1 tablet in 2 ml MS grade water, aliquot and store at -20°C for up to 4 months.

• For preparation of IX lysis buffer (25 ml total volume), 500 pi lysis buffer needed for lysis of 5e7 cells: o 0.5% IGEPAL 630 (0.625 ml 20% IGEPAL630) o 50 mM Tris, pH 8.0 (1.25 ml 1M Tris, pH 8.0) o 150 mM NaCI (0.75 ml 5M NaCI) o IX total protease inhibitor cocktail (2 ml of 25X stock (1 tablet dissolved in 2 ml MS grade water) o MS grade water (20.375 ml to make up total of 25 ml)

Filtered 1XPBS

MobiSpin® columns (www.mobitec.com/cms/products/bio/10 lab suppl/mobicols2.html) Low-bind 2 ml Eppendorf tubes Centrifugal filter units (Merck Millipore)

Pipettes and tips (Eppendorf)

Beaker for chemical waste

Heat block

Timer

Fridge at 4°C

Ice to keep lysis buffer and PBS cold

Sterile hood for preparation of resin and antibody

50 ml tubes for incubating Eppendorf tubes with samples

Sample roller at 4°C

Table-top centrifuge

Table-top ForceMini spinner to pulse-spin samples

Procedure ligand stability testing (time course and thermostability)

Day 1

1. lxPBS (sterile) was prepared from a lOx stock

2. lx affinity columns (MobiSpin®) were prepared for each sample:

• 2 ml Eppendorf tubes (one for each column) were prepared by clipping off the lid

(discard the lid) • All columns were uncapped and placed in Eppendorf tubes

• All columns were washed with 2x 550 pi of 10% acetic acid, the columns were sealed and pulse-spun 8-10 sec on ForceMini between washes, and the acetic acid was discarded

• All columns were washed with 2x 550 mI PBS, and spun (with the lid tightened) 8-10 sec between each wash rotein A resin was prepared in columns and antibody was coupled to protein A resin:

• Antibody was bound at a ratio of 400 pg to 200 mI (2 mg/ml in comparison to 10 mg/ml when performing large scale elutions) of Protein A resin

• 200 pL of Protein A resin was added to the affinity columns, which equates to 400 pL protein A-ethanol slurry (assuming 1: 1 ratio)

• After adding Protein A resin to each column, they were spun to remove ethanol (5-10 sec) and the ethanol discarded

• The columns were washed 3x with PBS (to max volume, ~500mI of PBS) and the PBS was discarded

• All columns were capped and ~150 pL PBS were added to the columns to avoid drying

• For the affinity column, antibody was under sterile conditions added to a 2 ml Eppendorf tube at the required volume to add 400 pg and used to transfer the washed resin from the column into the tube. It was ensured that all resin had been transferred by using additional PBS. The Eppendorf tubes were placed in a 50 ml tube and incubated at 4°C for at least lhr with gentle rotation

• The 'empty' affinity columns were left on ice (capped and with lid) ells were lysed as follows:

• The heat block was switched on at appropriate temperatures for incubation of lysate (37°C for the time scale experiments, a range of temperatures for the thermostability experiments).

• Set centrifuge (for 50 mL tubes) to 4°C (13,000 rpm, 10 mins) and centrifuge for Eppendorf tubes to 4°C (13,000 rpm, 10 mins)

• 500 uL lysis buffer per 5e7 cells was prepared and kept on ice:

• Grind cell pellet if >4e8 cells using cryogenic mill a. The foam dewar was filled with liquid nitrogen. The container was precooled with one 10 mm ball and on 7 mm ball in the nitrogen bath. b. The cell pellet was dislodged from the 50 mL tube and transferred to the pre cooled container. c. The container was balanced with a second container. The cell pellet was smashed at 30 Hz for the appropriate amount of time to make powder (5-90 mins), removed and checked during grinding. d. The ground cells were transferred to the appropriate amount of lysis buffer (in 50 mL tube). e. If pellets are small, lysis buffer is added directly to cell pellet(s); 500 uL lysis buffer per 5e7 cells and gently resuspend pellet with pipette until thawed/dissolved

• Leave to lyse at 4°C , 45 min, rolling

5. Lysate was centrifuged and the lysate supernatant was added to the affinity column

• Clear lysate by spinning for 10 mins at 13,000 rpm.

• Transfer lysates to 2 mL Eppendorf tubes to make up the desired number of sample replicates and spin for 10 mins at 13,000 rpm

• The lysate supernatant was added to a new 2 ml Eppendorf tube and placed on a heat block. For the time course stability experiment, the lysate was incubated at 37°C for either 0, 0.5, 1, 1.5, 2, 3, 5 or 24 hours (in desired number of replicates). For the thermal stability experiment the lysate was incubated for 10 mins at either 37°C, 40°C, 43°C, 46°C, 50°C, 53°C, 56°C, 60°C, 63°C, 66°C, 70°C, 73°C (in desired number of replicates).

• Upon completion of the incubation, the Eppendorf tubes were put straight on ice.

6. Treated lysate was added to affinity column with washed antibody-resin

• The antibody-resin mix was transferred back to the column and spun through the column. The antibody-resin column was then washed thoroughly, 3x with PBS (550 pi) and resuspended between washes. The columns were capped and a small volume of PBS was if necessary added to the Ab-resin beads to avoid them going dry.

• The lysate was added (300-400 mI at a time) to the washed Ab-resin mixture in the affinity mobispin column (capped), resuspended and transferred back to the lysate Eppendorf tube. Any residual resin beads in the column were transferred using additional PBS (100-200 mI). The Eppendorf tubes were each placed in a 50 ml tube to incubate and rotate at 4°C overnight.

Day 2

1. Centrifugal filter units (Merck Millipore) were prepared

• Filter units were washed with 500 mI of 10% acetic acid x2; spun at RT, 13,000 rpm for 60 mins after adding 10% acetic acid, and removal of the acid after spin 2. Antibody-bound molecules were eluted from protein A resin (after overnight incubation) according to the following steps:

• lxPBS (sterile) was prepared from lOxstock and kept on ice

• Resin with bound antibody and lysate was transferred from the overnight incubated Eppendorf tubes to the affinity columns saved from the day before

• Uncapped columns were pulse-spun for 8-10 s.

• The affinity column was washed with PBS (550 pi) x3 (up to x5), spun between each wash and the flow-through was discarded.

• New Eppendorf tubes were prepared (without cutting off the lid) for the eluate and elution was carried out using 10% acetic acid x4 rounds of 100 pi; in each round the elution was carried out for 5 mins for a slower flow-through, then the Eppendorf tubes were spun for 5-7 sec, and the flow-through was saved for each of the elution rounds (total 400 pi eluate).

• The eluate was heated to 70°C (~10 mins), then cooled to RT (~2-3 mins) before loading these onto the filter

3. Loading samples onto the filter units

• 1.5 ml Eppendorf tubes were prepared for the flow-through from the filter

• Once filter units had been washed, the lid was cut off and the bottom part was discarded while saving the filter and the lid

• The filter was placed in the new Eppendorf tubes, the pre-heated acetic acid eluate was added and the lid placed on the filter to ensure a tight closure

• Samples were spun at RT, 13,000 rpm, for at least 30 mins until all sample has passed through the filter

• Buffers were prepared in 50 ml tubes for zip tipping

• After the spin to filter the eluate, the filter was washed with 200 pi zip tip buffer A (0.1% formic acid) by spinning 13,000 rpm for approx. 30 mins (or more, ensuring that all of buffer A had passed through the filter) to allow for any remaining/additional peptides to come off the filter into a new Eppendorf.

4. Eppendorf tubes with flow-through from the filter units (peptides) are stored in the fridge until zip-tip protocol is carried out.

Zip-tip protocol for small scale samples

Reagents and materials:

• Buffer A: 0.1% formic acid in MS-grade water

For 1 ml: 999 pL water + 1 pL formic acid • Buffer B: 0.1% formic acid in 30% acetonitrile (v/v) in MS-grade water

For 1 ml: 300 mI acetonitrile, 699 water + 1 mI formic acid

• Eluted peptide samples

• iRT peptides (200 fmoles per sample) - www.biognosys.com/shop/irt-kit

• Low-bind Eppendorf tubes (1.5 ml)

• Zip tips (100 mI)

• 50 ml falcon tubes for buffers

• Pipettes and tips

• Beaker for chemical waste

• Speedy Vac

Procedure

• iRT peptides were taken from -80°C freezer and spiked in at 200 fmoles of iRTs per sample

• Zip-tip buffers were prepared

• 200 mI zip-tip was pre-wetted 3 times with 100 mI of buffer B

• Equilibration was carried out 3 times with 100 mI of buffer A

• Sample was bound by pipetting up 100-200 mI sample, transferring to new Eppendorf tubes, pipetting up and down several times until all sample has been bound

• 3 washes with 100 mI buffer A was carried out

• 3 time elution was carried out with 100 mI buffer B

• Samples were dried until almost completely dry on speedy vac (300 mI samples ~l-2 hours)

• Samples were reconstituted in 0.1% formic acid, 2% ACN, sonicated and spun down

• The desired volume (10-20 mI) was transferred to an MS vial

• MS samples were run

MS analysis of eluted peptides

The large scale eluted peptides were separated by means of RP-HPLC and subjected to LC- MS/MS analysis according to the protocol described in Purcell et aL 2019. The PEAKS® software package was used to create a spectral library from data dependent acquisition (DDA) data generated based on the large scale elution fractions for a specific HLA allele. The small scale samples which had been subjected to incubation at different temperatures/times as described in the protocol and cleaned up using the described zip tip protocol were subjected to LS-MS/MS in data independent acquisition (DIA) mode. In this case, both DDA and DIA MS were performed using a Q Exactive (Thermo). Subsequently, the Skyline software package was used to analyse and visualise peak areas of stability data replicates using the PEAKS®-generated spectral library to identify precursor and product ions (see Fig. 2 for an example).

All 8mer-llmer peptide peak areas were normalised based on iRT peptides spiked into the samples: a) The weighted iRT values were calculated: Each individual iRT values was divided by the mean of the iRT values for the given iRT peptide across replicates and 2) the mean value for each replicate was then calculated across all weighted iRT values for a given replicate:

Normalized value = Corrected ligand in tensity = Ligand intensity/Normalized value

Peptides were now filtered based on a Skyline confidence threshold (dotP 0.85) for the median value of the 37°C samples with peak areas set to 0 if peak confidence was less than the set threshold. Finally, the peptides were filtered based on sequences from background peptides (sequences from protein digests of the HeLa cell lines as well as sequence motifs indicating peptide binding to HLA-C*04:01 and HLA-B*35:05 which are naturally presented on parental C1R cells that have been transfected with the HLA of interest) and unusual contaminant sequences (often sequences with multiple prolines adjacent to one another). In the thermostability test series, this approach resulted in data for 491 peptides (8mers- llmers) from the different temperatures tested. In the time course experiment, the same approach provided data for 353 peptides (8mers-llmers). For the thermostability experiment, points were outlier corrected by calculating the median of a temperature point and neighbouring temperature points and selecting the mean of these median values. Then, the median intensity values fitted to a logistic sigmoidal curve

where s is proportional to the slope of the linear part of the fitted sigmoidal curve and T_m is the melting temperature.

Examples of melting point determinations from fitted sigmoidal curves are provided in Fig. 3.

For the time course stability experiment, the median intensity values were fitted to an exponential decay curve f{x ) = e ^Kx which indicates that the value of f(x) at the initial time point (time zero) is 1 and the exponential decay curve approaches the value f(x)=0 asymptotically. K is the rate constant from which the half-life of the complex can be calculated as follows

, _ ^ln (2) l - ~ϊG

Finally, the determined 491 melting points were subjected to linear normalization to arrive at melting point values arbitrarily set to values between 0.5-1.0 by calculating a normalized value for each of the T_m values:

T, mnormalised 0.5 0.5

These normalised T_m values allowed a simple ranking of the peptides with respect to their relative melting points. See Fig. 4, which in the left-hand panel shows - in a bar graph format - the distribution of the normalized T_m values and their frequencies, and which in the right-hand panel shows the information available if not performing a thermal stability determination. If solely relying on the data available in the right-hand graph, all 491 peptides would be considered equally useful ligands for HLA-A*02:01, whereas the left-hand panel bar graph demonstrate that only about 40% of the 491 peptides appeared in the group of peptides with high thermostability.

SUMMARY OF RESULTS

A novel assay was established. The assay combines thermal/time-course treatment of cell lysates with mass spectrometry, cf. Fig. 1.

Peptides were filtered using PEAKS® and Skyline software packages, with the latter software being used for peak picking, cf. Fig. 2.

The assay was successfully used to generate MS data that can be transformed into stability values for the HLA ligands present in the treated peptide samples from cells being mono- allelic for HLA, see Fig. 3, which depicts the thermal stability curves for a number of peptides identified and quantified according to the presently presented method. It was in addition investigated whether there is correlation between predicted ligand rank score (netMHCpan4.0, cf. www.cbs.dtu.dk/services/NetMHCpan/) and the determined thermal stability values for the HLA ligands, see Fig. 5. From this figure, which shows the paired results for HLA-A*02:01 and HLA-B*07:02 binding peptides, respectively, it is clear that a large number of high stability peptides are not predicted by the existing ligand rank score software and also that some peptides predicted in practice were demonstrated to be very poor ligands having low thermostability.

To summarize, the present inventive technology enables an enhanced MHC ligand determination, which in turn makes it possible to rationally design peptide based vaccines to 1) avoid inclusion of peptides, which - although they are ligands for MHC molecules - have too low stability to be relevant as T-cell immunogens, 2) allow inclusion of peptides which all exhibit the desired stability (typically high or intermediate) for MHC binding.

One important feature in this respect is that the method allows the stability of binding to be investigated at near-physiological temperatures, whereas previously applied methods for identifying naturally processed peptides have been carried out at non-physiologically low temperatures (in Purcell et ai. 2019, the complexes of MHC molecules and peptides are e.g. at no point subjected to temperatures >4°C, but the complexes were naturally presented by the cells at physiological conditions prior to the steps taken to isolation and elution). In particular, the present approach of applying a time-course treatment provides, when carried out at temperatures ~ 37°C, information about the stability (and in particular the lack of stability) of binding between peptides and MHC molecules that are found to be stably bound in vitro at low temperatures.

In addition, the fact that only peptides eluted from cells that have naturally processed proteins comprising the peptides means that the identified peptides are inherently verified as being products of antigen processing:

The assay assesses the 'true' off-rate, as peptides have already bound to the MHC complex within the cell as part of the natural antigen processing and presentation; the competition for binding to MHC between peptides in the natural cell environment is inherently part of the inventive assay, whereas traditional pMHC affinity assays gauge competition for MHC binding between a peptide and a labelled competitor in an isolated manner; processing of antigens via the antigen processing machinery is naturally incorporated; and the assay minimises bias as it does not require pre-selection of peptides for analysis - the cell has naturally selected the peptides via its intracellular machinery.

Furthermore, the method developed is readily applicable on all MHC expressing cells, in particular all mono-allelic cell lines and the method is not restricted by the ability to re-fold MHC heavy chain and 32m in vitro.

The natural cell setting that this method is built upon results in features such as affinity and antigen processing being anchored in the assay. Furthermore, the natural cell setting avoids the bias that other stability assays are prone to. Bias in other assays mainly results from the fact that many peptides are selected for synthesis based on prior knowledge from other studies that have investigated epitopes or based on affinity prediction models resulting in circular reasoning potentially becoming an issue. To our knowledge, this is the first assay that assesses the stability of pMHC complexes in a natural cell environment setting.

Applying MS to analyse the cell lysate allows for the generation of a high-throughput assay in which a full spectrum of peptides for the given MHC allele becomes the output. The half-life or thermal decay curve can therefore be determined for hundreds, even thousands, of peptides simultaneously.

LIST OF REFERENCES

1. Blaha, D. T. et at. (2019) 'High-Throughput Stability Screening of Neoantigen / HLA Complexes Improves Immunogenicity Predictions', Cancer Immunol Res 7(1): 50-62. doi: 10.1158/2326-6066.CIR- 18-0395.

2. Gfeller, D. et a/. (2016) 'Current tools for predicting cancer-specific T cell immunity', Oncolmmunology 5(7): 1-9. doi: 10.1080/2162402X.2016.1177691.

3. Harndahl, M. et al. (2012) 'Peptide-MHC class I stability is a better predictor than peptide affinity of CTL immunogenicity', Eur J Immunol 42(6): 1405-1416. doi:

10.1002/eji.201141774.

4. Jorgensen, K. W. and Buus, S. (2014) 'NetMHCstab - predicting stability of peptide - MHC-I complexes; impacts for cytotoxic T lymphocyte epitope discovery', Immunology 141(1): 18-26. doi: 10.1111/imm.12160.

5. Ko§aloglu-Yalgin, Z. et a/. (2018) 'Predicting T cell recognition of MHC class I restricted neoepitopes', Oncolmmunology 7(11): 1-15. doi: 10.1080/2162402X.2018.1492508. Mei, S. et a/. (2019) Ά comprehensive review and performance evaluation of bioinformatics tools for HLA class I peptide-binding prediction', Briefings in Bioinformatics: 1-17. doi: 10.1093/bib/bbz051 (Epub ahead of print). Purcell, A. W., Ramarathinam, S. H. and Ternette, N. (2019) 'Mass spectrometry-based identification of MHC-bound peptides for immunopeptidomics', Nature Protocols. 14(6): 1687-1707. doi: 10.1038/s41596-019-0133-y. Rasmussen, M. et a/. (2016) 'Pan-Specific Prediction of Peptide-MHC Class I Complex Stability, a Correlate of T Cell Immunogenicity', J Immunol 197(4): 1517-1524. Savitski, M. M. et a/. (2014) 'Tracking cancer drugs in living cells by thermal profiling of the proteome', Science 346(6205). doi: 10.1126/science.1255784. Strpnen, E. et a/. (2016) 'Targeting of cancer neoantigens with donor-derived T cell receptor repertoires', Science 352(6291): 1337-1341. doi: 10.1126/science. aaf2288. Tummino, P. J. and Copeland, R. A. (2008) 'Residence Time of Receptor - Ligand Complexes and Its Effect on Biological Function', Biochemistry 47(20): 5481-92. doi: 1021/bi8002023. Yewdell, J. W., Reits, E. and Neefjes, J. (2003) 'Making sense of mass destruction: Quantitating MHC class I antigen presentation', Nat Rev Immunol, 3(12): 952-961. doi: 10.1038/nril250. Rock, K. L., Reits, E, and Neefjes J. (2016), 'Present Yourself! By MHC Class I and MHC Class II Molecules', Trends in Immunology, 37(11): 724-737. Neefjes, J, Jongsma, Paul, P and Bakke, O (2011), 'Towards a systems understanding of MHC class I and MHC class II antigen presentation', Nature Reviews Immunology 11(12): 823-836.

Claims

1. A method for quantitative determination of stability of binding between at least one peptide and an MHC molecule, comprising the subsequent steps of a) preparing a plurality of samples of cell lysates comprising complexes between MHC molecules and peptides, where the lysates are obtained from a plurality of MHC expressing cells (preferably human cells) that have naturally processed said peptides from protein antigens, b) subjecting the plurality of samples to the conditions of i) incubation at defined physicochemical conditions, where incubation time varies between the plurality of samples and where the physicochemical conditions are kept constant between the plurality of samples, or ii) incubation at defined physicochemical conditions, where the incubation time is kept constant between the plurality of samples and where the physicochemical conditions vary between the plurality of samples, c) isolating complexes between MHC molecules and peptides from the plurality of samples, d) determining, by mass spectrometric analysis, the at least one peptide's relative quantities in the plurality of samples after step c), and deriving at least one stability score for the at least one peptide based on the quantities determined in step d).

2. The method according to claim 1, wherein the MHC expressing cells are mono-allelic for the MHC molecule.

3. The method according to claim 1 or 2, wherein the MHC molecule is an MHC Class I molecule.

4. The method according to claim 3, wherein the MHC Class I molecule is an HLA molecule selected from HLA-A, HLA-B, and HLA-C.

5. The method according to claim 1 or 2, wherein the MHC molecule is an MHC Class II molecule.

6. The method according to claim 5, wherein the MHC Class II molecule is an HLA molecule selected from HLA-DP, HLA-DQ, and HLA-DR.

7. The method according to any one of the preceding claims, wherein the plurality of MHC expressing cells prior to step a) have been isolated from other organic material by centrifugation and optionally have been frozen for prolonged storage prior to step a).

8. The method according to any one of the preceding claims, wherein step c) comprises isolation of the complexes by means of affinity purification specific for the MHC molecule.

9. The method according to any one of the preceding claims, wherein step c) after isolation of the complexes further comprises isolation of the peptides from the MHC molecules.

10. The method according to any one of the preceding claims, wherein the quantities for a peptide determined in step d) are normalized relative to one single of the quantities measured for the peptide.

11. The method according to claim 10, wherein, when the plurality of samples have been subjected to condition i), the quantities of the at least one peptide determined in step d) are normalized relative to the quantity determined for the condition of shortest incubation time.

12. The method according to claim 10, wherein, when the plurality of samples have been subjected to condition ii), the quantities of the at least one peptide determined in step d) are normalized relative to the quantity determined for the condition that provides the lowest entropy.

13. The method according to any one of the preceding claims, wherein step b) consists in subjecting the plurality of samples to conditions i).

14. The method according to claim 13, wherein the stability score is in the form of a decay constant for peptide binding to the MHC molecule, or any value being a strictly increasing or decreasing function of the decay constant such as the half-life or the mean lifetime of the peptide binding to the MHC molecule.

15. The method according to any one of claims 1-12, wherein step b) consists in subjecting the plurality of samples to conditions ii).

16. The method according to claim 15, wherein the stability score is in the form of a T_m value, or any strictly increasing or decreasing function thereof.

17. The method according to any one of the preceding claims, wherein step d) comprises tandem mass spectrometric analysis.

18. The method according to any one of the preceding claims wherein step d) comprises that the amino acid sequence of the at least one peptide and a measure of its quantity is determined in step d) in each of the plurality of samples.

19. The method according to any one of the preceding claims wherein the stability score of the at least one peptide is derived by fitting its quantities determined in step d) to

- a decay curve against time if the plurality of samples have been subjected to conditions i) in step b) or

- to a sigmoid melting curve against temperature if the plurality of samples have been subjected to conditions ii) in step b).

20. The method according to any one of the preceding claims, wherein the MHC expressing cells are human cells.

21. The method according to any one of the preceding claims, which comprises at least two determinations of stability of binding between at least one peptide and an MHC molecule, wherein one determination comprises subjecting a first plurality of samples to conditions i) in step b) and another determination comprises subjecting a second plurality of samples to conditions ii) in step b).

22. The method according to claim 21, wherein at least two stability scores are derived for the at least one peptide in step d), such as a stability score defined in claim 14 and a stability score as defined in claim 16.

23. The method according to any one of the preceding claims, wherein the MHC expressing cells are nucleated cells that are obtained by infecting a sample of nucleated cells, preferably in vitro, with a an intracellular infectious agent and allowing the nucleated cells to subject the protein of the intracellular infectious agent to antigen processing and present peptide fragments from protein of the intracellular infectious agent bound to MHC molecules on their surface.

24. The method according to any one of claims 1-22, wherein the MHC expressing cells are malignant cells obtained by recovering a sample of neoplastic cells from malignant tumour tissue or from a cell line derived from neoplastic cells.

25. The method according to any one of claims 1-22, wherein the MHC expressing cells are professional antigen presenting cells that are obtained by contacting a sample of professional antigen presenting cells with extracellular protein or an extracellular infectious agent and allowing the professional antigen presenting cells to take up protein, subject the protein to antigen processing and present peptide fragments from the protein bound to MHC molecules on their surface, and subsequently recovering the professional antigen presenting cells.

26. A method for determination of binding between at least one peptide and an MHC molecule, comprising the subsequent steps of

27. The method according to claim 26, wherein the temperature >4°C is selected from a temperature of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84 about 85, about 86, about 87, about 88, about 89, and about 90°C.

28. The method according to claim 26 or 27 wherein the temperature >0°C is selected from a temperature of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84 about 85, about 86, about 87, about 88, about 89, and about 90°C.

29. The method according to any one of claims 26-28, wherein the period of time is at least or about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 120, about 240, about 480, about 720, about 960, about 1440, about or 1920, about 2160, about 2880, about 3600, about 4320, about 5040, about 5760, about 6480, about 7200, about 7920, about 8640, about 9360, about 10080, about 10800, or about 11520 minutes.

30. The method according to any one of claims 26-29, wherein step II comprises the steps of isolating complexes of MHC and peptides, preferably by means of affinity purification specific for the MHC molecule, separating peptides from the complexes, and subjecting separated peptides to MS, preferably a data dependent acquisition (DDA) MS approach.

31. The method according to any one of claims 26-30, wherein a plurality of samples is prepared and wherein lysis conditions and/or incubation conditions favour the preservation of complexes between MHC and peptides to different degrees across the samples.

32. The method according to any one of claims 26-30, wherein the at least one sample is subjected to one single set of lysis and incubation conditions.

33. The method according to any one of claims 26-32, wherein the MHC expressing cells are as defined in claim 2 and/or wherein the MHC molecule is defined as in any one of claims 3-6.

34. The method according to any one of claims 26-33, wherein

- the plurality of MHC expressing cells prior to step I) have been isolated from other organic material by centrifugation and optionally have been frozen for prolonged storage prior to step a); and or

- step II) comprises tandem mass spectrometric analysis; and/or

- the MHC expressing cells are human cells; and/or

- the MHC expressing cells are nucleated cells that are obtained by infecting a sample of nucleated cells, preferably in vitro, with a an intracellular infectious agent and allowing the nucleated cells to subject the protein of the intracellular infectious agent to antigen processing and present peptide fragments from protein of the intracellular infectious agent bound to MHC molecules on their surface; and/or

- the MHC expressing cells are malignant cells obtained by recovering a sample of neoplastic cells from malignant tumour tissue or from a cell line derived from neoplastic cells; and/or

- the MHC expressing cells are professional antigen presenting cells that are obtained by contacting a sample of professional antigen presenting cells with extracellular protein or an extracellular infectious agent and allowing the professional antigen presenting cells to take up protein, subject the protein to antigen processing and present peptide fragments from the protein bound to MHC molecules on their surface, and subsequently recovering the professional antigen presenting cells.

35. A method of preparing an immunogenic composition, comprising determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of any one of the preceding claims and subsequently admixing one or more peptides, which are selected from peptides of the plurality that qualify as stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

36. A method of preparing an immunogenic composition, comprising determination of stability of binding between a plurality of peptides and an MHC molecule according to the method according to any one of claims 1-33 and subsequently preparing a polypeptide, which comprises amino acid sequences of one or more peptides, which are selected from peptides of the plurality that qualify as stably MHC binding peptides, and admixing the polypeptide with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

37. A method of preparing an immunogenic composition, comprising determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of any one of claims 1-33 and subsequently admixing a nucleic acid, which is capable of expressing nucleotide sequences encoding one or more peptides, which are selected from peptides of the plurality that qualify as stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

38. A method of preparing an immunogenic composition, comprising determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of any one of claims 1-33 and subsequently admixing a nucleic acid, which is capable of expressing a nucleotide sequence encoding a polypeptide comprising the amino acid sequences of one or more peptides, which are selected from peptides of the plurality that qualify as stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

39. The method according to claim 37 or 38, wherein the nucleic acid is a plasmid.

40. A method of preparing an immunogenic composition, comprising determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of any one of claims 1-33 and subsequently admixing a microorganism or virus, which is capable of expressing nucleotide sequences encoding one or more peptides, which are selected from peptides of the plurality that qualify as stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

41. A method of preparing an immunogenic composition, comprising determination of stability of binding between a plurality of peptides and an MHC molecule according to the method of any one of claims 1-33 and subsequently admixing a microorganism of virus, which is capable of expressing a nucleotide sequence encoding a polypeptide comprising the amino acid sequences of one or more peptides, which are selected from peptides of the plurality that qualify as stably MHC binding peptides, with a pharmaceutically acceptable carrier, diluent, vehicle, and/or excipient.

42. The method according to claim 40 or 41, wherein the microorganism or virus is non- pathogenic.

43. The method according to any one of claim 35-41, further comprising admixing with an immunological adjuvant.