Assays and Reagents for Characterizing MHCII Peptide Binding

Abstract

Described herein are reagents and methods for high throughput screening of antigens that bind to MHCII alleles.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international stage application claiming benefit of priority to U.S. Provisional Application No. 63 / 238,728, filed August 30, 2021, the entirety of which is incorporated herein by reference.

[0002] Sequence Listing The material in the attached sequence listing is incorporated herein by reference in its entirety. The attached file named "048893-534001WO_SL_ST26.xml" was created on August 29, 2022 and is 42,603 ​​bytes. This file can be accessed using Microsoft Word on a computer using the Windows OS. [Background technology]

[0003] background Major histocompatibility complex II (MHCII) is a group of cell surface proteins located on the extracellular surface of antigen-presenting cells (APCs) and certain immune cells. MHCII proteins are encoded by the human leukocyte antigen (HLA) locus by the classical alleles HLA-DP, -DQ, and -DR, and the nonclassical alleles HLA-DM and -DO. MHCII proteins are heterodimers of α and β chains that undergo a multi-step maturation process to reach their final antigen-presenting form. The α and β chains are translated as single-pass transmembrane proteins in the endoplasmic reticulum and then associate with the invariant chain (Ii) to form a complex. After cycling between the Golgi apparatus, the plasma membrane, endosomes, and finally intraluminal vesicles of multivesicular bodies, the invariant chain is processed by cathespins into smaller peptides, namely class II-associated invariant chain peptides (CLIPs), which are bound within the peptide-binding groove of the MHCIIα / β heterodimer. In a subsequent step, CLIP is exchanged with the antigenic peptide via a chaperone process, and the MHCII / antigenic peptide complex is returned to the APC cell surface for presentation to CD4(+) T cells.

[0004] Antigen-presenting cells regulate the adaptive immune response, which is responsible for maintaining memory of pathogenic attacks, vaccinations and other non-self antigens, and for training helper or CD4(+) T cells to control various types of attacks in a specific manner. In recent years, there has been great interest in generating non-self antigens for targeted therapeutics against cancer, novel viral, bacterial and fungal infections, as well as certain autoimmune diseases. Indeed, the ability to generate and select antigens or neoantigens in vitro will accelerate and expand our ability to address these disease threats. Summary of the Invention

[0005] overview The present technology relates generally to reagents and methods for high throughput screening of peptides against MHCII complexes.

[0006] In one embodiment, provided herein is a composition comprising a test peptide and a major histocompatibility complex class II (MHCII)-ligand complex, the MHCII-ligand complex containing (i) an MHC molecule containing an α chain and a β chain, and (ii) a ligand associated with the α chain and the β chain.

[0007] In one embodiment, provided herein is a major histocompatibility complex class II (MHCII)-ligand complex comprising (i) an MHCII molecule comprising an α chain and a β chain, and (ii) a ligand associated with said MHCII molecule. The MHCII-ligand complex further comprises a ligand having an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:3.

[0008] In one embodiment, provided herein is a method of detecting binding of a major histocompatibility complex class II (MHCII) molecule to a peptide, the method comprising: providing a first composition containing a peptide and an MHCII-ligand complex containing (i) an MHCII molecule containing an α chain and a β chain, and (ii) a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; subjecting the first composition to conditions that cause cleavage of the cleavable linker; incubating the first composition for a period of time sufficient to form a second composition, the second composition containing the α chain, the β chain, the ligand, free peptide, and / or an MHCII-peptide complex comprising the MHCII molecule and the peptide non-covalently bound to the MHCII molecule; and determining whether the MHCII molecule is bound to the peptide.

[0009] In one embodiment, provided herein is a method for detecting affinity of a test peptide for a major histocompatibility complex class II (MHCII) molecule, the method comprising: providing a first composition containing the test peptide, a tagged MHCII binding peptide (tagged peptide), and (i) the MHCII molecule containing an α chain and a β chain, and (ii) an MHCII-ligand complex containing a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; subjecting the first composition to conditions that cause cleavage of the cleavable linker; incubating the first composition for a period of time sufficient to form a second composition, the second composition containing the α chain, the β chain, the ligand, free test peptide, free tagged peptide, MHCII-tagged peptide complex containing the tagged peptide associated with the MHCII molecule, and / or MHCII-test peptide complex containing the test peptide associated with the MHCII molecule; and determining whether the MHCII molecule is bound to the test peptide.

[0010] In one embodiment, provided herein is a method for multiple epitope mapping to major histocompatibility complex class II (MHCII) alleles, comprising: providing a first composition comprising a plurality of test peptides and a plurality of MHCII-ligand complexes, each MHCII-ligand complex comprising (i) an MHCII molecule comprising an α chain and a β chain, and (ii) a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; subjecting the first composition to conditions that cause cleavage of the cleavable linker; incubating the first composition for a period of time sufficient to form a second composition, the second composition containing the α chain, the β chain, the ligand, free test peptide, and / or a plurality of MHCII-test peptide complexes, each MHCII-test peptide complex comprising the MHCII molecule and a test peptide non-covalently bound thereto; and determining whether one or more of the plurality of test peptides are bound to the MHCII molecule in the second composition.

[0011] In one embodiment, provided herein is a method for multiple epitope mapping to major histocompatibility complex class II (MHCII) alleles, comprising: providing a first composition containing a plurality of test peptides and a plurality of MHCII-ligand complexes, each MHCII-ligand complex containing (i) an MHCII molecule containing an α chain and a β chain, and (ii) a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; subjecting the first composition to conditions that cause cleavage of the cleavable linker; incubating the first composition for a period of time sufficient to form a second composition, the second composition containing the α chain, the β chain, the ligand, free test peptide, and / or a plurality of MHCII-test peptide complexes, each MHCII-test peptide complex containing the MHCII molecule and a test peptide non-covalently bound thereto; and determining whether one or more of the plurality of test peptides are bound to the MHCII molecule in the second composition.

[0012] In one aspect, provided herein is a peptide that binds at least one major histocompatibility complex class II (MHCII) molecule, the peptide comprising an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:16. [Brief description of the drawings]

[0013] [Figure 1]Figure 1 is a schematic diagram of a peptide exchange assay for MHCII protein complexes. MHCII α-chain, β-chain and a covalently attached ligand (e.g., CLIP ligand) are expressed and purified. The covalently attached ligand is removed via a peptide backbone cleavage reaction in the presence of an excess of test peptide. If the test peptide binds MHCII and effectively competes with the CLIP ligand, an MHCII-test peptide complex is formed. The MHCII / test peptide complex is separated by size exclusion chromatography and the MHCII / test peptide complex is analyzed by reversed-phase LC-MS.

[0014] [Diagram 2] FIG. 2 is a Coomassie stained SDS-PAGE gel run showing expression of MHC II proteins in HEK-293 and CHO cells.

[0015] [Figure 3A] Figures 3A and 3B provide an example of covalently bound high affinity CLIP (class II associated invariant chain peptide, sequence PVSQMRMATPLLMRP, SEQ ID NO: 4) enzymatic processing and subsequent exchange reaction with a test peptide (OVA peptide, sequence ISQVHAAHAEINEAGR, SEQ ID NO: 5) for MHCII heterodimers. Figure 3A shows that the CLIP peptide is removed by processing with thrombin or TEV protease, with the enzymatic cleavage site located within the linker sequence between the CLIP peptide and the N-terminus of the MHCII β chain. The exchange reaction is initiated by overnight incubation of the MHCII / cleaved CLIP peptide complex in the presence of a 20-fold concentration of the test peptide (OVA) in acetate buffer at pH 5.2 and 37°C. The reaction mixture was separated by size exclusion chromatography and analyzed by RP LC-MS. FIG. 3B shows that the LC-MS chromatogram obtained from the MHCII / peptide complex size-exclusion fraction shows the amount of MHCII / OVA peptide (5.2 min) versus MHCII / CLIP peptide (7 min). [Figure 3B] Same as above.

[0016] [Figure 4A] Figures 4A and 4B provide an example of covalently bound low affinity CLIP (class II associated invariant chain peptide, sequence PVSQARMATGALARP, SEQ ID NO: 6) enzymatic processing and subsequent exchange reaction with a test peptide (OVA peptide, sequence ISQVHAAHAEINEAGR, SEQ ID NO: 5) for MHCII heterodimers. Figure 4A shows that the CLIP peptide is removed by processing with thrombin or TEV protease, with the enzymatic cleavage site located within the linker sequence between the CLIP peptide and the N-terminus of the MHCII β-chain. The exchange reaction is initiated by overnight incubation of the MHCII / cleaved CLIP peptide complex in the presence of a 20-fold concentration of the test peptide (OVA) in acetate buffer at pH 5.2 and 37°C. The reaction mixture was separated by size exclusion chromatography and analyzed by RP LC-MS. FIG. 4B shows that the LC-MS chromatogram obtained from the MHCII / peptide complex size-exclusion fraction shows the abundance of the MHCII / OVA peptide (5.2 min) versus the MHCII / CLIP peptide (approximately 6.8 min). [Figure 4B] Same as above.

[0017] [Figure 5A] Figures 5A and 5B show the SEC chromatogram from the first dimension of 2D LC-MS (Figure 5A) and the extracted ion chromatogram from the LC-MS second dimension detection (Figure 5B) showing the corresponding counts versus acquisition time window illustrating the difference in the results of three separate exchange assays for the HLA-DRB1*01:01 complex with three different CLIP peptide sequences and the same test peptide sequence (pp65, LPLKMLNIPSINVH, SEQ ID NO: 7). [Figure 5B] Same as above.

[0018] [Figure 6A]Figures 6A and 6B show LC-MS chromatograms (Figure 6A) and corresponding counts versus acquisition time window (Figure 6B) showing the difference in three separate exchange assay results for HLA-DRB1*03:01 complex with three different CLIP peptide sequences and the same test peptide sequence (SP41, HTYTIDWTKDAVTWS, SEQ ID NO:8). [Figure 6B] Same as above.

[0019] [Figure 7A] Figures 7A and 7B show LC-MS chromatograms (Figure 7A) and corresponding counts versus acquisition time window (Figure 7B) showing the difference in three separate exchange assay results for HLA-DRB1*04:01 complex with three different CLIP peptide sequences and the same test peptide sequence (HA, PKYVKQNTLKLAT, SEQ ID NO:9). [Figure 7B] Same as above.

[0020] [Figure 8A] Figures 8A and 8B show LC-MS chromatograms (Figure 8A) and corresponding counts versus acquisition time window (Figure 8B) showing the difference in three separate exchange assay results for HLA-DRB1*07:01 complex with three different CLIP peptide sequences and the same test peptide sequence (pp65, EPDVYYTSAFVFPTK SEQ ID NO: 10). [Figure 8B] Same as above.

[0021] [Figure 9A] Figures 9A and 9B show LC-MS chromatograms (Figure 9A) and corresponding counts versus acquisition time window (Figure 9B) showing the difference in three separate exchange assay results for HLA-DRB1*07:01 complex with three different CLIP peptide sequences and the same test peptide sequence (HA, PKYVKQNTLKLAT, SEQ ID NO:9). [Figure 9B] Same as above.

[0022] [Figure 10]FIG. 10 is a table of CLIP variants (or wild-type CLIP) identified for HLA-DR alleles.

[0023] [Figure 11A] Figures 11A-11C show a table of test peptides for MHCII binding (Figure 11A), an illustration of a complex containing fluorescent avidin tetramers of biotinylated MHCII molecules associated with test peptides for use in screening mouse T cells and subsequent flow cytometry experiments + / - CD44 T cell screening (Figure 11B). Figure 11C is an interferon gamma ELISPOT assay showing activation of T cells against a shorter mutant peptide identified as the only binder from peptide exchange. The identified short peptide gives a comparable ELISPOT response to the long synthetic peptide (M112_7788-5). [Figure 11B] Same as above. [Figure 11C] Same as above.

[0024] [Figure 12] FIG. 12 shows multiplexed analysis of peptide binding of a 150 peptide library from Humira and Remicade variable domains in a single exchange.

[0025] [Figure 13] Figure 13 is a schematic diagram of a competitive peptide exchange assay. A low affinity-containing peptide is covalently attached to an MHCII molecule via a cleavable linker. The linker is cleaved in the presence of a labeled high affinity peptide and a pool of test peptides, and the low affinity peptide is exchanged. Three outcomes are illustrated: the MHCII molecule binds the high affinity peptide and is detected, the MHCII molecule binds the test peptide and is not detected, and / or the MHCII complex is no longer able to bind peptide and is not detected.

[0026] [Figure 14A]Figures 14A-14C show an exemplary workflow for screening MHCII and peptide complexes via ELISA. The illustration in Figure 14A shows a time course of competitive peptide exchange for a pool containing MHCII molecules and labeled high affinity peptides (e.g., biotinylated) and a range of test peptide concentrations. The peptide exchange reaction mix is ​​incubated for a fixed time and at a fixed pH (e.g., 60-72 hours at pH 5) and then applied to an ELISA plate coated with anti-MHC antibodies. The loaded ELISA plate is developed by adding a color development reagent (e.g., streptavidin-horseradish peroxidase). The resulting signal is plotted against concentration, the resulting data points are fitted to a binding model, and the binding affinity is calculated for the test peptides. Figure 14B is a plot showing the binding curves and affinity ranking of eight high affinity peptides for MHCII. Figure 14C is a table containing the IC50 values ​​(binding affinity) of each peptide for MHCII along with the R2 values ​​of the model fit. [Figure 14B] Same as above. [Figure 14C] Same as above.

[0027] [Figure 15A]Figures 15A-15G show normalized signal vs. log concentration plots of HLA-DRB1 allele / low affinity ligand peptide complexes, a labeled high affinity peptide (biotinylated huCLIP, 50 nM) and eight different test peptides at various concentration ranges. All results are measured by ELISA. Figure 15A shows log plots of competitive binding for DRB1*01:01 and peptides mCLIP, LCMV RNAP, LASV RNAP, VETF (275-289), LCMV NP (201-215), VV HP (23-37), MOG (35-55) and OVA (323-339). Figure 15B is a logarithmic plot of competitive binding for DRB1*08:01 and peptides mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339). Figure 15C is a logarithmic plot of competitive binding for DRB1*15:01 and peptides mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339). Figure 15D is a logarithmic plot of competitive binding for DRB1*04:01 and peptides mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339). Figure 15E is a logarithmic plot of competitive binding for DRB1*09:01 and peptides mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339). FIG. 15F is a logarithmic plot of competitive binding for DRB1*11:01 and peptides mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339).See Figure 15G pp65-biotinylated DRB1*01:01. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 15G] Same as above.

[0028] [Figure 16] Figures 16A and 16B show saturation binding curves of normalized signal versus peptide concentration for eight different HLA DRB1 allele samples / low affinity ligand peptide complexes (100 nM) and one high affinity peptide (biotinylated huCLIP). The concentration range of the high affinity CLIP peptide is 40,000 nM to 2.4 nM. The peptide exchange time course was followed for 70 hours at 37°C, pH 5 (Figure 16A). Figure 16B is a table of binding affinity values ​​for eight different HLA DRB1 allele samples and the high affinity peptide (biotinylated huCLIP). The data was fitted to a specific binding model with a Hill slope, where Bmax is the maximum specific binding, Kd is the half-maximal ligand binding at equilibrium, h is the Hill slope, and the R-squared value is how well the model fits the data.

[0029] [Figure 17A]Figures 17A-17C are the determination of optimal biotinylated huCLIP (huCLIP-bio) peptide concentrations for allele-specific probes in binding competition assays against mCLIP as the test peptide. Peptide exchange assays were performed for the six DRB1 alleles and a reference allele (pp65-DRB1*01:01). Figure 17A shows a plot of the relative signal for 50 nM huCLIP-bio versus the logarithm of the mCLIP concentration range (40,000-0.38 nM), and Figure 17B shows a plot of the relative signal for 25 nM huCLIP-bio versus the logarithm of the mCLIP concentration range (40,000-0.38 nM). Figure 17C is a table of calculated Kd values. [Figure 17B] Same as above. [Figure 17C] Same as above.

[0030] [Figure 18A]Figures 18A-18F show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio as the high affinity peptide against DRB1*01:01, DRB1*08:01 and DRB1*11:01. Figures 18A and 18D are plots of 50 nM and 25 nM hCLIP-bio used to compete with mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339) test peptides for DRB1*08:01 binding. Figures 18B and 18E are plots of 50 nM and 25 nM hCLIP-bio used to compete with mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339) test peptides for DRB1*11:01 binding. Figures 18C and 18F are plots of 50 nM and 25 nM hCLIP-bio used to compete with mCLIP, LCMV RNAP, LASV RNAP, VETF(275-289), LCMV NP(201-215), VV HP(23-37), MOG(35-55) and OVA(323-339) test peptides for DRB1*11:01 binding. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 18D] Same as above. [Figure 18E] Same as above. [Figure 18F] Same as above.

[0031] [Figure 19A]Figures 19A-19D show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio against DRB1*04:01 and DRB1*15:01. Figures 19A and 19B are plots of 50 nM hCLIP-bio used to compete with mCLIP, LCMV RNAP, LASV RNAP, VETF (275-289), LCMV NP (201-215), VV HP (23-37), MOG (35-55), and OVA (323-339) test peptides for DRB1*04:01 and DRB1*15:01 binding, respectively. Figures 19C and 19D are plots of 25 nM hCLIP-bio used to compete with mCLIP, LCMV RNAP, LASV RNAP, VETF (275-289), LCMV NP (201-215), VV HP (23-37), MOG (35-55) and OVA (323-339) test peptides for DRB1*04:01 and DRB1*15:01 binding, respectively. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above.

[0032] [Figure 20A] Figures 20A and 20B show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio against DRB1*09:01. Plots of 50 nM (Figure 20A) and 25 nM (Figure 20B) hCLIP-bio used to compete with mCLIP, LCMV RNAP, LASV RNAP, VETF (275-289), LCMV NP (201-215), VV HP (23-37), MOG (35-55) and OVA (323-339) test peptides for DRB1*09:01 binding. [Figure 20B] Same as above.

[0033] [Figure 21] FIG. 21 is an example of a workflow diagram for a peptide exchange assay.

[0034] [Figure 22] FIG. 22 is a table of test peptide sequences.

[0035] [Figure 23A] Figures 23A-23E show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio (Figures 23A and 23B), an overlay of the SEC chromatogram for the completed peptide exchange reaction showing the MHCII / peptide complex peak at 2.5 minutes (Figure 23C), an overlay of the counts vs. acquisition time for each SEC peak showing the amount of test peptide exchanged with the low affinity CLIP (Figure 23D), and a table of test peptide sequences and the resulting exchange profile for each test peptide compared to the low affinity CLIP peptide for the MHCII HLA allele DRB1*01:01 (Figure 23E). The concentration of MHCII was 16 μM and the concentration of test peptide was 400 μM. The test peptide sequences are as shown in Figure 22. [Figure 23B] Same as above. [Figure 23C] Same as above. [Figure 23D] Same as above. [Figure 23E] Same as above.

[0036] [Figure 24A]Figures 24A-24E show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio (Figures 24A and 24B), an overlay of the SEC chromatogram for the completed peptide exchange reaction showing the MHCII / peptide complex peak at 2.5 minutes (Figure 24C), an overlay of counts vs. acquisition time for each SEC peak showing the amount of test peptide exchanged with low affinity CLIP (Figure 24D), and a table of test peptide sequences (Figure 24E) and the resulting exchange profile for each test peptide compared to the low affinity CLIP peptide for the MHCII HLA allele DRB1*04:01. The concentration of MHCII was 16 μM and the concentration of test peptide was 400 μM. The test peptide sequences are as shown in Figure 22. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 24E] Same as above.

[0037] [Figure 25A] Figures 25A-25E show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio (Figures 25A and 25B), an overlay of the SEC chromatogram for the completed peptide exchange reaction showing the MHCII / peptide complex peak at 2.5 minutes (Figure 25C), an overlay of counts vs. acquisition time for each SEC peak showing the amount of test peptide exchanged with low affinity CLIP (Figure 25D), and a table of test peptide sequences (Figure 25E) and the resulting exchange profile for each test peptide compared to the low affinity CLIP peptide for the MHCII HLA allele DRB1*08:01. The concentration of MHCII was 16 μM and the concentration of test peptide was 400 μM. The test peptide sequences are as shown in Figure 22. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above.

[0038] [Figure 26A] Figures 26A-26D show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio (Figures 26A and 26B), an overlay of the SEC chromatogram for the completed peptide exchange reaction showing the MHCII / peptide complex peak at 2.5 minutes (Figure 26C), and an overlay of counts vs. acquisition time for each SEC peak showing the amount of test peptide exchanged with the low affinity CLIP (Figure 26D). The concentration of MHCII was 16 μM and the concentration of test peptide was 400 μM. The test peptide sequences are as shown in Figure 22. [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 26D] Same as above.

[0039] [Figure 27A] Figures 27A-27E show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio (Figures 27A and 27B), an overlay of the SEC chromatogram for the completed peptide exchange reaction showing the MHCII / peptide complex peak at 2.5 minutes (Figure 27C), an overlay of counts vs. acquisition time for each SEC peak showing the amount of test peptide exchanged with low affinity CLIP (Figure 27D), and a table of test peptide sequences (Figure 27E) and the resulting exchange profile for each test peptide compared to the low affinity CLIP peptide for the MHCII HLA allele DRB1*11:01. The concentration of MHCII was 16 μM and the concentration of test peptide was 400 μM. The test peptide sequences are as shown in Figure 22. [Figure 27B] Same as above. [Figure 27C] Same as above. [Figure 27D] Same as above. [Figure 27E] Same as above.

[0040] [Figure 28A] Figures 28A-28B show plots of peptide exchange assays performed with either 50 nM or 25 nM huCLIP-bio (Figures 28A and 28B), an overlay of the SEC chromatogram for the completed peptide exchange reaction showing the MHCII / peptide complex peak at 2.5 minutes (Figure 28C), an overlay of counts vs. acquisition time for each SEC peak showing the amount of test peptide exchanged with low affinity CLIP (Figure 28D), and a table of test peptide sequences (Figure 28E) and the resulting exchange profile for each test peptide compared to the low affinity CLIP peptide for the MHCII HLA allele DRB1*15:01. The concentration of MHCII was 16 μM and the concentration of test peptide was 400 μM. The test peptide sequences are as shown in Figure 22. [Figure 28B] Same as above. [Figure 28C] Same as above. [Figure 28D] Same as above. [Figure 28E] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description After reading this description, it will be clear to a person skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present invention are described herein. It will be understood that the embodiments presented herein are presented by way of example only, and not by way of limitation. Therefore, this detailed description of various alternative embodiments should not be interpreted as limiting the scope or breadth of the present disclosure described herein.

[0042] Before disclosing and describing the present technology, it is to be understood that the embodiments described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0043] The present application provides compositions and methods for assaying MHCII peptides for use in a wide range of immunotherapies.

[0044] In one aspect, provided herein is a major histocompatibility complex class II (MHCII) protein complex comprising an alpha chain, a beta chain and a ligand, wherein the linker is associated with the alpha chain and the beta chain.

[0045] In one aspect, provided herein is a major histocompatibility complex class II (MHCII)-ligand complex comprising an MHCII α chain and an MHCII β chain, and a ligand associated with an MHCII molecule, wherein the ligand is a test peptide.

[0046] In one aspect, provided herein is a method for detecting binding of a major histocompatibility complex class II (MHCII) molecule to a peptide, comprising: providing a first composition comprising a peptide and an MHCII-ligand complex, the MHCII-ligand complex comprising an α chain and a β chain and a ligand, the ligand being linked to the α chain or the β chain via a cleavable linker; subjecting the first composition to conditions that cause cleavage of the cleavable linker; and incubating the first composition for a period of time sufficient to form a second composition, the second composition comprising the α chain, the β chain, the ligand, free peptide, and / or an MHCII-peptide complex comprising the MHCII molecule and the peptide non-covalently bound to the MHCII molecule. In some embodiments, the method comprises determining whether an MHCII molecule is bound to the peptide.

[0047] In one embodiment, provided herein is a method for detecting affinity of a test peptide for a major histocompatibility complex class II (MHCII) molecule, the method comprising: providing a first composition comprising the test peptide, a tagged MHCII binding peptide (tagged peptide), and an MHCII-ligand complex comprising (i) the MHCII molecule comprising an α chain and a β chain, and (ii) a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; subjecting the first composition to conditions that cause cleavage of the cleavable linker; and incubating the first composition for a period of time sufficient to form a second composition, the second composition comprising the α chain, the β chain, the ligand, free test peptide, free tagged peptide, an MHCII-tagged peptide complex comprising the tagged peptide associated with the MHCII molecule, and / or an MHCII-test peptide complex comprising the test peptide associated with the MHCII molecule.

[0048] In some embodiments, the method further comprises determining whether the MHC II molecule is bound to the test peptide.

[0049] The detailed description is divided into various sections solely for the convenience of the reader, and disclosures found in any section may be combined with those of another section. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the disclosure.

[0050] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and the claims that follow, reference will be made to certain terms that shall be defined to have the following meanings.

[0051] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0052] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0053] The term "about," when used prior to a numerical designation including a range, e.g., temperature, time, amount, concentration, etc., indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about," when used in reference to an amount, means that the amount may vary by ±10%.

[0054] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the described purpose. Thus, a composition consisting essentially of the elements defined in the present invention does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0055] As used herein, the term "cancer" refers to all types of cancer, neoplasms or malignant tumors found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma and sarcoma.Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease and non-Hodgkin's lymphoma.Exemplary cancers that can be treated with the compounds or methods provided herein include thyroid, endocrine system, brain, breast, cervix, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach and uterus cancer. Further examples include thyroid cancer, bile duct carcinoma, pancreatic adenocarcinoma, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, endocrine or exocrine pancreatic neoplasms, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

[0056] "Selective" or "selectivity" and the like with respect to a compound refer to the ability of a compound to distinguish between molecular targets (eg, a compound has selectivity for HMT SUV39H1 and / or HMT G9a).

[0057] "Specific," "specifically," "specificity," and the like with respect to a compound refer to the ability of the compound to cause a particular effect, e.g., inhibition, on a particular molecular target, with minimal or no effect on other proteins within the cell (e.g., a compound with specificity for HMT SUV39H1 and / or HMT G9a exhibits inhibition of the activity of those HMTs, while the same compound exhibits little or no inhibition of other HMTs, e.g., DOT1, EZH1, EZH2, GLP, MLL1, MLL2, MLL3, MLL4, NSD2, SET1b, SET7 / 9, SET8, SETMAR, SMYD2, SUV39H2).

[0058] Terms such as "immune response" refer in their normal accustomed sense to a response by an organism that protects against disease. The response can be initiated by the innate or adaptive immune system, as is well known in the art.

[0059] Terms such as "modulating an immune response" refer to a change in a subject's immune response as a result of administration of an agent, such as a compound disclosed herein, including embodiments thereof. Thus, the immune response can be activated or deactivated as a result of administration of an agent, such as a compound disclosed herein, including embodiments thereof.

[0060] "B cell" or "B lymphocyte" refer to their standard usage in the art. B cells are lymphocytes, a type of white blood cell (leukocyte), that develop into antibody-producing plasma cells ("mature B cells"). "Immature B cells" are cells that can develop into mature B cells. Generally, pro-B cells undergo immunoglobulin heavy chain rearrangement to become pro-B pre-B cells, which further undergo immunoglobulin light chain rearrangement to become immature B cells. Immature B cells include T1 and T2 B cells.

[0061] "T cells" or "T lymphocytes", as used herein, are a type of lymphocyte (a subtype of white blood cells) that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, e.g., B cells and natural killer cells, by the presence of T cell receptors on the cell surface. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTLs), regulatory T (Treg) cells, and helper T cells. Different types of T cells can be distinguished by the use of T cell detection agents.

[0062] "Memory T cells" are T cells that have previously encountered and responded to their cognate antigen during a previous infection, cancer encounter, or previous vaccination. Upon a second encounter with its cognate antigen, memory T cells can regenerate (divide) to mount a faster and stronger immune response than when the immune system first responded to the pathogen.

[0063] "Regulatory T cells" or "suppressor T cells" are lymphocytes that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease.

[0064] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly accepted one-letter codes.

[0065] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which may be linked to moieties not composed of amino acids. These terms apply not only to naturally occurring and non-naturally occurring amino acid polymers, but also to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0066] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that modify, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" that result in the replacement of the amino acid with a chemically similar amino acid. Lists of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present disclosure.

[0067] The "position" of an amino acid or nucleotide base is designated by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc., which must be taken into account when determining the optimal alignment, in general, the amino acid residue number in the test sequence, determined by simply counting from the N-terminus, will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in the case where a variant has a deletion compared to the aligned reference sequence, the variant will not have an amino acid at the site of the deletion that corresponds to the position in the reference sequence. In the case where an insertion is present in the aligned reference sequence, the insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions, there may be stretches of amino acids in either the reference sequence or the aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0068] The terms "numbered with reference to" or "corresponding to," when used in the context of numbering a given amino acid or polynucleotide sequence, refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0069] The term "amino acid side chain" refers to a functional substituent contained in an amino acid. For example, the amino acid side chain can be the side chain of a naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic code (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine), as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In some embodiments, the amino acid side chain can be an unnatural amino acid side chain.

[0070] The term "unnatural amino acid side chain" refers to a functional substituent of a compound having the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, allylalanine, 2-aminoisobutyric acid. Unnatural amino acids are non-proteinogenic amino acids that are either naturally occurring or chemically synthesized. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.

[0071] The term "MHCII" or "Major Histocompatibility Complex Class II" or "Major Histocompatibility Complex II" as provided herein includes any recombinant or naturally occurring form of Major Histocompatibility Complex II (MHCII) or a variant or homolog thereof that maintains MHC II activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to MHCII). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150 or 200 contiguous amino acids) compared to a naturally occurring MHCII polypeptide. In several embodiments, MHCII is a heterodimer of two non-covalently associated proteins, an alpha (α) chain and a beta chain (β), homologs or functional fragments thereof. In some embodiments, the MHCII comprises a peptide ligand.

[0072] The term "HLA" or "human leukocyte antigen" refers to a group of proteins encoded by the MHC gene complex. Specifically, but not exclusively, the MHCII gene complex encodes the proteins of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ and HLA-DR groups.

[0073] The term "ligand" refers to a molecule that forms a complex with a biomolecule to perform a biological function. Binding can occur between, but is not limited to, proteins, peptides, RNA, DNA, nucleic acids, nucleic acid derivatives, non-natural nucleic acids, amino acids, amino acid derivatives, non-natural amino acids, carbohydrates, monosaccharides, disaccharides, oligosaccharides, oligonucleotides, metals, metal complexes, drugs, lipids, fatty acids, metabolites, inorganic molecules, organic molecules, biopolymers and polymers. Ligand complexes can be formed by ionic bonds, covalent bonds, van der Waals interactions and / or hydrogen bonds. Ligands for MHCII are generally peptides.

[0074] The terms "class II-associated invariant chain peptide" and "CLIP" refer to a portion of the invariant chain (Ii) protein sequence that binds within the MHCII peptide-binding groove during the affinity maturation process.

[0075] The terms "bond" and "bonded" as used herein are used according to their obvious and ordinary meaning and refer to an association between atoms or molecules. The association may be direct or indirect. For example, the bonded atoms or molecules may be direct, for example, by a covalent bond or linker (e.g., a first linker or a second linker), or indirect, for example, by a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interactions, etc.).

[0076] The term "antibody" refers to a polypeptide encoded by immunoglobulin genes or functional fragments thereof that specifically binds and recognizes an antigen. Commonly recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.

[0077] When referring to a protein or peptide, the phrases "specifically (or selectively) bind" to an antibody or "specifically (or selectively) immunoreactive with" often refer to a binding reaction that determines the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a specified antibody will bind to a particular protein at least twice background, and more typically more than 10-100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with a selected antigen and not with other proteins. This selection can be accomplished by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0078] The term "denaturing" refers to a process in which the three-dimensional structure of a protein, polypeptide, DNA, RNA, or other biopolymer is disrupted by chemical or mechanical means, or by heating or cooling.

[0079] The term "peptide linker" refers to an amino acid sequence that links one peptide to another. The peptide linker can be a cleavable linker. The cleavable linker can be any cleavable linker. For example, but not limited to, the cleavable linker can be a UV-cleavable linker, an enzyme-cleavable linker, a pH-dependent linker, a salt-dependent linker, etc. Preferably, the peptide linker or cleavable linker that links the peptide to the MHCII subunit does not result in the peptide binding to MHCII more strongly than in the absence of the linker. The cleavable linker may be made of any suitable molecule and is not limited to a peptide linker.

[0080] It is to be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0081] MHCII composition In one aspect, provided herein is a major histocompatibility complex class II (MHCII) / ligand complex comprising an MHCII molecule comprising an alpha chain, a beta chain, and a ligand, wherein the ligand is a peptide associated with the alpha chain and the beta chain.

[0082] In some embodiments, the MHCII / ligand complex contains an alpha chain and a beta chain. In some embodiments, the alpha chain and the beta chain are associated in a heterodimer. In some embodiments, the alpha chain contains a first dimerization tag. In some embodiments, the beta chain contains a second dimerization tag. In some embodiments, the alpha chain containing a first dimerization tag and the beta chain containing a second dimerization tag are associated.

[0083] In some embodiments, the MHCII / ligand complex contains an alpha chain and a beta chain that are more stable as a heterodimer. In some embodiments, an alpha chain containing a first dimerization tag and a beta chain containing a second dimerization tag associate in a stable complex. In some embodiments, an alpha chain containing a first dimerization tag and a beta chain containing a second dimerization tag associate in a more stable complex than the complex formed by the same alpha and beta chain sequences without the first and second dimerization tags.

[0084] In some embodiments, the MHCII / ligand complex contains an alpha chain containing a first dimerization tag and a beta chain containing a second dimerization tag, the dimerization tag being selected from a leucine zipper, Fos / Jun, coiled-coil heterodimerization tag, knobs-in-holes or other heterodimeric Fc, or an iDimerize pair.

[0085] In embodiments, the MHCII / ligand complex contains an α chain and a β chain and a ligand. In embodiments, the MHCII / ligand complex contains an α chain and a β chain and a ligand, and the ligand is a peptide. In embodiments, the peptide ligand is covalently linked to the α chain. In embodiments, the peptide ligand is covalently linked to the α chain via a peptide linker sequence. In embodiments, the peptide ligand is covalently linked to the β chain. In embodiments, the peptide ligand is covalently linked to the β chain via a peptide linker sequence. In embodiments, the peptide linker sequence contains an enzyme cleavage site. In some embodiments, the enzyme cleavage site is selected from thrombin, enterokinase, factor Xa, small ubiquitin-like modifier (SUMO) protease, tobacco etch virus (TEV) protease, PreScission™ protease, rhinovirus 3C protease, carboxypeptidase A, carboxypeptidase B, dipeptidyl aminopeptidase (DAP), tobacco vein mottling virus protease (TVMV), and any variants thereof. In some embodiments, the peptide is fused to the N-terminus of the alpha chain with a flexible peptide linker. In some embodiments, the linker contains the thrombin recognition sequence GGGGSLVPRGSGGGGS (SEQ ID NO: 17). In some embodiments, the linker contains the TEV protease recognition sequence GGGGSENLYFQGGGGGS (SEQ ID NO: 18).

[0086] In some embodiments, the MHCII / ligand complex contains an α-chain and a β-chain and a ligand, the ligand being a peptide, and the ligand peptide being covalently linked to the α-chain or the β-chain via a peptide linker sequence. In some embodiments, the peptide linker sequence contains an unnatural amino acid. In some embodiments, the unnatural amino acid is UV-cleavable. In some embodiments, the unnatural amino acid is selected from 2-nitrophenylglycine (NPG), expanded o-nitrobenzyl linker, o-nitrobenzyl caged phenol, o-nitrobenzyl caged thiol, 32 nitroveratryloxycarbonyl (NVOC) caged aniline, o-nitrobenzyl caged selenide, bis-azobenzene, coumarin, cinnamyl, spiropyran, 2-nitrophenylalanine (2-nF), and 3-amino-3-(2-nitrophenyl)propionic acid (ANP) amino acid analogs. In some embodiments, the unnatural amino acid is 3-amino-3-(2-nitrophenyl)propionic acid (ANP).

[0087] In some embodiments, the MHCII complex contains an α chain and a β chain and a tagged peptide. In some embodiments, the tagged peptide selectively recognizes and associates with the MHCII complex without a ligand. In some embodiments, the tagged peptide associates with an additional molecule to form a detectable complex.

[0088] In some embodiments, the MHCII / ligand complex contains an α-chain and a β-chain, a ligand, and a tagged peptide. In some embodiments, the tagged peptide selectively recognizes and associates with the MHCII complex / ligand complex. In some embodiments, the tagged peptide associates with an additional molecule to form a detectable complex.

[0089] In some embodiments, the tagged peptide is capable of binding a molecule to generate a detectable label. In some embodiments, the tagged peptide contains a fluorescent compound or a bioluminescent compound. In some embodiments, the tagged peptide is tagged with a multivalent platform. In some embodiments, the tagged peptide is tagged with biotin and the additional molecule is streptavidin. In some embodiments, the tagged peptide is tagged with a subunit of a tetrameric assembly.

[0090] In some embodiments, the MHCII / ligand complex contains an α and β chain, a ligand, and an antibody. In some embodiments, the antibody binds to the MHCII molecule. In some embodiments, the antibody is immobilized on a surface.

[0091] In some embodiments, the MHCII / ligand complex has affinity for a peptide ligand. In some embodiments, the peptide ligand contains a CLIP sequence. In some embodiments, the peptide ligand meets one or more of the following criteria: a) are produced in usable amounts during expression in MHC II chains; b) is detected at high levels in 2D LCMS (after cleavage of the linker); and / or c) In an exchange reaction with a peptide known to have high affinity for the allele, it has a peptide exchange yield of about 20% to 100%.

[0092] In embodiments, the peptide ligand meets two or more of the above criteria, hi embodiments, the peptide ligand meets all three of the above criteria.

[0093] In some embodiments, the usable amount of the expressed MHCII / ligand complex is greater than 1 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is between about 1 mg / L and about 300 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is between about 1 mg / L and about 250 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is between about 1 mg / L and about 200 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is between about 1 mg / L and about 190 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is between about 1 mg / L and about 180 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is between about 1 mg / L and about 170 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is between about 1 mg / L and about 160 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 150 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 140 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 130 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 120 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 110 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 100 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 90 mg / L. In some embodiments, the usable amount of the expressed MHCII / ligand complex is about 1 mg / L to about 80 mg / L. In embodiments, the usable amount of expressed MHCII / ligand complex is from about 1 mg / L to about 70 mg / L.In some embodiments, the usable amount of expressed MHCII / ligand complex is about 1 mg / L to about 60 mg / L. In some embodiments, the usable amount of expressed MHCII / ligand complex is about 1 mg / L to about 50 mg / L. In some embodiments, the usable amount of expressed MHCII / ligand complex is about 1 mg / L to about 40 mg / L. In some embodiments, the usable amount of expressed MHCII / ligand complex is about 1 mg / L to about 30 mg / L. In some embodiments, the usable amount of expressed MHCII / ligand complex is about 1 mg / L to about 20 mg / L. In some embodiments, the usable amount of expressed MHCII / ligand complex is about 1 mg / L to about 10 mg / L. The amount can be any value or subrange within the recited range, including the endpoints.

[0094] In some embodiments, the signal range of the MHCII / ligand complex detected by 2D LC-MS is about 103-105 counts in the extracted ion chromatogram (EIC) of the peptide ligand. In some embodiments, the signal is about 103 counts. In some embodiments, the signal is about 104 counts. In some embodiments, the signal is about 105 counts.

[0095] In some embodiments, the peptide ligand has a peptide exchange yield of about 20% to about 100% in an exchange reaction with a peptide known to have high affinity for the allele. In some embodiments, the peptide ligand has a peptide exchange yield of about 40% to about 100% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of about 50% to about 100% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 20% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 30% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 40% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 50% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 60% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 70% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 80% in an exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of at least about 90% in the exchange reaction. In some embodiments, the peptide ligand has a peptide exchange yield of about 100% in the exchange reaction. The values ​​can be any value or subrange within the recited range, including the endpoints.

[0096] In embodiments, the MHCII complex has a higher affinity / IC50 for the tagged peptide than for the CLIP sequence. In some embodiments, the MHCII complex has a higher affinity for the tagged peptide than for the CLIP sequence.

[0097] In embodiments, the MHCII / ligand complex contains an alpha chain, the alpha chain being encoded by any one of the following loci: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ and HLA-DR alleles. In some embodiments, the alpha chain is encoded by the HLA-DP locus. In some embodiments, the alpha chain is encoded by the HLA-DM locus. In some embodiments, the alpha chain is encoded by the HLA-DOA locus. In some embodiments, the alpha chain is encoded by the HLA-DOB locus. In some embodiments, the alpha chain is encoded by the HLA-DQ locus. In some embodiments, the alpha chain is encoded by the HLA-DR locus.

[0098] In embodiments, the MHCII / ligand complex contains a β chain, the β chain being encoded by any one of the following loci: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ and HLA-DR alleles. In some embodiments, the β chain is encoded by the HLA-DP locus. In some embodiments, the β chain is encoded by the HLA-DM locus. In some embodiments, the β chain is encoded by the HLA-DOA locus. In some embodiments, the β chain is encoded by the HLA-DOB locus. In some embodiments, the β chain is encoded by the HLA-DQ locus. In some embodiments, the β chain is encoded by the HLA-DR locus.

[0099] In some embodiments, the MHCII / ligand complex is encoded by an HLA-DR allele and the ligand is selected from the list of HLA-DR and ligand sequences. In some embodiments, the HLA allele is HLA-DRB1*01:01 and the peptide ligand is PVSKARMATGALAQA (SEQ ID NO:1). In some embodiments, the HLA allele is HLA-DRB1*03:01 and the peptide ligand is PVSKMRMATGALAQA (SEQ ID NO:2). In some embodiments, the HLA allele is HLA-DRB1*04:01 and the peptide ligand is PVSKMRMATGALAQA (SEQ ID NO:2). In some embodiments, the HLA allele is HLA-DRB1*07:01 and the peptide ligand is PVSKMRMATGALAQA (SEQ ID NO:2). In some embodiments, the HLA allele is HLA-DRB1*08:01 and the peptide ligand is PVSKMRMATPLLMQA (SEQ ID NO:3). In some embodiments, the HLA allele is HLA-DRB1*11:01 and the peptide ligand is PVSKMRMATGALAQA (SEQ ID NO:2). In some embodiments, the HLA allele is HLA-DRB1*13:01 and the peptide ligand is PVSKMRMATPLLMQA (SEQ ID NO:3). In some embodiments, the HLA allele is HLA-DRB1*15:01 and the peptide ligand is PVSKARMATGALAQA (SEQ ID NO:1). In some embodiments, the HLA allele is HLA-DRB1*11:04 and the peptide ligand is PVSKMRMATGALAQA (SEQ ID NO:2).

[0100] In embodiments, the MHCII complex comprises an MHC molecule comprising an alpha and a beta chain, and a tagged peptide associated with the alpha and beta chain.

[0101] In some aspects, a composition is provided that includes an MHCII complex (an MHC molecule containing an α-chain and a β-chain and a peptide ligand associated with the α-chain and the β-chain) and a test peptide. In some embodiments, the MHCII complex contains an MHC molecule containing an α-chain and a β-chain, a peptide ligand, a test peptide, and a tagged peptide associated with the α-chain and the β-chain. In some embodiments, the MHCII complex contains an MHC molecule containing an α-chain and a β-chain, a peptide ligand, two or more test peptides, and a tagged peptide associated with the α-chain and the β-chain. In some embodiments, the MHCII complex also contains a molecule that forms a detectable complex with the tagged peptide.

[0102] In some embodiments, the major histocompatibility complex class II (MHCII) / ligand complex comprises an MHCII molecule comprising an α chain and a β chain, and a ligand associated with the MHCII molecule. In some embodiments, the ligand is a peptide. In some embodiments, the ligand is a tagged peptide. In some embodiments, the ligand is a test peptide.

[0103] In some embodiments, the MHCII complex comprises an MHCII molecule comprising an α chain and a β chain, and a test peptide associated with the α chain and the β chain. In some embodiments, the test peptide is a tumor antigen. In some embodiments, the test peptide is an autoantigen. In some embodiments, the test peptide is a neoantigen.

[0104] In embodiments, the test peptide is 7-30 amino acid residues long. The length can be any value or subrange within the recited range, including the endpoints. In some embodiments, the test peptide is 7 amino acid residues long. In some embodiments, the test peptide is 8 amino acid residues long. In some embodiments, the test peptide is 9 amino acid residues long. In some embodiments, the test peptide is 10 amino acid residues long. In some embodiments, the test peptide is 11 amino acid residues long. In some embodiments, the test peptide is 12 amino acid residues long. In some embodiments, the test peptide is 13 amino acid residues long. In some embodiments, the test peptide is 14 amino acid residues long. In some embodiments, the test peptide is 15 amino acid residues long. In some embodiments, the test peptide is 16 amino acid residues long. In some embodiments, the test peptide is 17 amino acid residues long. In some embodiments, the test peptide is 18 amino acid residues long. In some embodiments, the test peptide is 19 amino acid residues long. In some embodiments, the test peptide is 20 amino acid residues long. In some embodiments, the test peptide is 21 amino acid residues long. In some embodiments, the test peptide is 22 amino acid residues long. In some embodiments, the test peptide is 23 amino acid residues long. In some embodiments, the test peptide is 24 amino acid residues long. In some embodiments, the test peptide is 25 amino acid residues long. In some embodiments, the test peptide is 26 amino acid residues long. In some embodiments, the test peptide is 27 amino acid residues long. In some embodiments, the test peptide is 28 amino acid residues long. In some embodiments, the test peptide is 29 amino acid residues long. In some embodiments, the test peptide is 30 amino acid residues long.

[0105] In some embodiments, the test peptide comprises an amino acid sequence selected from SEQ ID NO: 1 through SEQ ID NO: 16. In some embodiments, the test peptide comprises the amino acid sequence of SEQ ID NO:1.

[0106] Methods for detecting MHCII / ligand complexes In one embodiment, provided herein is a method of detecting binding of a major histocompatibility complex class II (MHCII) molecule to a test peptide, the method comprising: (a) providing a first composition comprising a peptide and an MHCII-ligand complex comprising (i) the MHCII molecule comprising an α chain and a β chain, and (ii) a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; (b) subjecting the first composition to conditions that cause cleavage of the cleavable linker; (c) incubating the first composition for a period of time sufficient to form a second composition, the second composition comprising the α chain, the β chain, the ligand, free peptide, and / or an MHCII-peptide complex comprising the MHCII molecule and the peptide non-covalently bound to the MHCII molecule; and (d) determining whether the MHCII molecule is bound to the peptide.

[0107] In some embodiments, whether the MHCII molecule binds to the test peptide is determined by measuring the level of MHCII / peptide complexes in the second composition. In some embodiments, the level of MHCII / peptide complexes is measured by two-dimensional liquid chromatography-mass spectrometry (2D LC / MS) of the second composition. In some embodiments, a combination of HPLC and MS and / or 2D LC / MS is used to distinguish between the MHCII molecule and the test peptide. In some embodiments, the free test peptide and / or ligand is removed from the second composition prior to 2D LC / MS. In some embodiments, the free test peptide is removed from the second composition prior to 2D LC / MS. In some embodiments, the free test peptide is removed from the second composition via size exclusion chromatography (SEC). In some embodiments, the free test peptide is removed from the second composition via ion exchange.

[0108] In some embodiments, the presence of the test peptide as determined by HPLC and MS indicates that the MHCII molecule is capable of binding to the test peptide, hi some embodiments, the presence of the test peptide as determined by HPLC and MS indicates that the test peptide has a higher affinity for the MHCII molecule compared to the ligand peptide.

[0109] The methods described herein may be performed multiplexedly. In some embodiments, the first composition contains a plurality of MHCII / ligand complexes and at least two different test peptides. In some embodiments, the first composition contains 2 to about 1000 different test peptides. In some embodiments, the first composition contains 10 to about 500 different test peptides. In some embodiments, the first composition contains 50 to about 500 different test peptides. In some embodiments, the first composition contains 100 to about 500 different test peptides. In some embodiments, the first composition contains 10 to about 250 different test peptides. The number of test peptides may be any value or subrange within the recited ranges, including the endpoints. In some embodiments, the test peptides are distinguished from one another via mass spectrometry.

[0110] In some embodiments, the test peptide is present in the first composition at a ratio of test peptide to MHCII / ligand complex of about 1000:1 to about 1:1000. In some embodiments, the test peptide is present in the first composition at a ratio of test peptide to MHCII / ligand complex of about 1000:1 to about 1:10. In some embodiments, the test peptide is present in the first composition at a ratio of test peptide to MHCII / ligand complex of about 1000:1 to about 1:1. In some embodiments, the test peptide is present in the first composition at a ratio of test peptide to MHCII / ligand complex of about 1000:1 to about 10:1. In some embodiments, the test peptide is present in the first composition at a ratio of test peptide to MHCII / ligand complex of about 1000:1 to about 100:1. The ratio can be any value or subrange within the recited range.

[0111] In some embodiments, the MHCII molecule contains an HLA peptide encoded by an HLA-DP, HLA-DM, HLA-DO, HLA-DQ, or HLA-DR allele. In some embodiments, the HLA allele is HLA-DP. In some embodiments, the HLA allele is HLA-DM. In some embodiments, the HLA allele is HLA-DO. In some embodiments, the HLA allele is HLA-DQ. In some embodiments, the HLA allele is HLA-DR.

[0112] In some embodiments, the MHCII molecule contains an HLA peptide encoded by an HLA-DR allele, and the ligand is a peptide. In some embodiments, the HLA-DR allele is HLA-DRB. In some embodiments, the HLA-DR allele is HLA-DRB1. In some embodiments, the HLA-DRB allele is selected from HLA-DRB1*01:01, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*11:01, HLA-DRB1*11:01, HLA-DRB1*13:01, or HLA-DRB1*15:01. In some embodiments, the allele is HLA-DRB1*01:01. In some embodiments, the allele is HLA-DRB1*03:01. In some embodiments, the allele is HLA-DRB1*04:01. In some embodiments, the allele is HLA-DRB1*07:01. In some embodiments, the allele is HLA-DRB1*08:01. In some embodiments, the allele is HLA-DRB1*11:01. In some embodiments, the allele is HLA-DRB1*13:01. In some embodiments, the allele is HLA-DRB1*15:01. In some embodiments, the HLA allele is HLA-DRB1*11:04.

[0113] In embodiments, the MHCII molecule / ligand complex contains a ligand that is a peptide. In embodiments, the ligand peptide is selected from: PVSKARMATGALAQA (SEQ ID NO: 1), PVSKMRMATGALAQA (SEQ ID NO: 2), or PVSKMRMATPLLMQA (SEQ ID NO: 3). In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO: 1). In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2). In some embodiments, the ligand peptide is PVSKMRMATPLLMQA (SEQ ID NO: 3).

[0114] In some embodiments, the MHCII molecule / ligand complex contains a ligand that is a peptide, and the MHCII molecule is encoded by an HLA-DR allele. In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO: 1) and the HLA-DR allele is HLA-DRB1*01:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2) and the HLA-DR allele is HLA-DRB1*01:01. In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO: 1) and the HLA-DR allele is HLA-DRB1*03:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2) and the HLA-DR allele is HLA-DRB1*04:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2) and the HLA-DR allele is HLA-DRB1*07:01. In some embodiments, the ligand peptide is PVSKMRMATPLLMQA (SEQ ID NO:3) and the HLA-DR allele is HLA-DRB1*08:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO:2) and the HLA-DR allele is HLA-DRB1*11:01. In some embodiments, the ligand peptide is PVSKMRMATPLLMQA (SEQ ID NO:3) and the HLA-DR allele is HLA-DRB1*13:01. In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO:1) and the HLA-DR allele is HLA-DRB1*15:01. In some embodiments, the HLA allele is HLA-DRB1*11:04 and the peptide ligand is PVSKMRMATGALAQA (SEQ ID NO:2).

[0115] In embodiments, the test peptide contains a sequence derived from an antigen, an autoantigen, or a neoantigen. In some embodiments, the test peptide contains a sequence derived from an antigen. In some embodiments, the test peptide contains a sequence derived from a neoantigen. In some embodiments, the test peptide contains a sequence derived from an autoantigen.

[0116] In some embodiments, the test peptides are 7-30 amino acid residues in length, as described above.

[0117] In some embodiments, the test peptide comprises an amino acid sequence selected from SEQ ID NO:1-SEQ ID NO:16.

[0118] In some embodiments, a method for detecting affinity of a test peptide for a major histocompatibility complex class II (MHCII) molecule includes: (a) providing a first composition comprising the test peptide, a tagged MHCII binding peptide (tagged peptide), and an MHCII-ligand complex comprising (i) the MHCII molecule comprising an α chain and a β chain, and (ii) a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; and (b) subjecting the first composition to conditions that cause cleavage of the cleavable linker. and (c) incubating the first composition for a period of time sufficient to form a second composition, the second composition comprising the α chain, the β chain, the ligand, free test peptide, free tagged peptide, an MHCII-tagged peptide complex comprising the tagged peptide associated with the MHCII molecule, and / or an MHCII-test peptide complex comprising the test peptide associated with the MHCII molecule; and (d) determining whether the MHCII molecule is bound to the test peptide.

[0119] In some embodiments, the first composition is incubated for at least 24 hours to 96 hours after initiation of linker cleavage. In some embodiments, the first composition is incubated for 24 hours after initiation of linker cleavage. In some embodiments, the first composition is incubated for about 24 hours after initiation of linker cleavage. In some embodiments, the first composition is incubated for about 48 hours after initiation of linker cleavage. In some embodiments, the first composition is incubated for about 72 hours after initiation of linker cleavage. In some embodiments, the first composition is incubated for about 96 hours after initiation of linker cleavage. The incubation time can be any value or subrange within the recited range, including the endpoints.

[0120] In some embodiments, the second composition is contacted with an antibody that binds to the MHCII molecule. In some embodiments, the antibody binds to the MHCII molecule. In some embodiments, the antibody is immobilized on a solid surface.

[0121] In some embodiments, a second composition is contacted with an antibody that binds to the MHCII molecule, and the second composition contains a tagged peptide. In some embodiments, the tagged peptide is associated with the MHCII molecule.

[0122] In some embodiments, the tagged peptide contains a label. In some embodiments, the tagged peptide contains a fluorescent or bioluminescent label. In some embodiments, the tagged peptide contains a biotin label.

[0123] In some embodiments, the second composition is contacted with an antibody that binds to the MHCII molecule. In some embodiments, the second composition contains an MHCII molecule and a tagged peptide. In some embodiments, the tagged peptide contains a fluorescent label or a bioluminescent label. In some embodiments, the tagged peptide contains a biotin label. In some embodiments, the antibody is tagged with a fluorescent label, a bioluminescent label, or a substrate. In some embodiments, the antibody is tagged with a streptavidin-horseradish peroxidase conjugate.

[0124] In some embodiments, the test peptide has a low affinity for the MHCII allele when the MHCII-tagged peptide complex is bound to the antibody.

[0125] In some embodiments, the test peptide has high affinity for the MHCII allele when the MHCII-tagged peptide complex is not bound to the antibody.

[0126] In some embodiments, the affinity of the test peptide for the MHCII molecule is confirmed by a peptide exchange assay. In some embodiments, the affinity of the test peptide for the MHCII molecule is determined in comparison to the affinity of a known peptide for the MHCII molecule.

[0127] In some embodiments, the tagged peptide contains a human CLIP sequence.

[0128] In some embodiments, the cleavable linker is a UV-cleavable linker or an enzyme-cleavable linker. In some embodiments, the peptide linker sequence contains an enzyme cleavage site. In some embodiments, the enzyme cleavage site is selected from thrombin, enterokinase, factor Xa, small ubiquitin-like modifier (SUMO) protease, tobacco etch virus (TEV) protease, PreScission™ protease, rhinovirus 3C protease, carboxypeptidase A, carboxypeptidase B, dipeptidyl aminopeptidase (DAP), tobacco vein mottling virus protease (TVMV), and any variants thereof. In some embodiments, the peptide linker sequence contains a non-natural amino acid. In some embodiments, the non-natural amino acid is UV-cleavable. In some embodiments, the unnatural amino acid is selected from 2-nitrophenylglycine (NPG), expanded o-nitrobenzyl linker, o-nitrobenzyl caged phenol, o-nitrobenzyl caged thiol, 32 nitroveratryloxycarbonyl (NVOC) caged aniline, o-nitrobenzyl caged selenide, bis-azobenzene, coumarin, cinnamyl, spiropyran, 2-nitrophenylalanine (2-nF), and 3-amino-3-(2-nitrophenyl)propionic acid (ANP) amino acid analogs. In some embodiments, the unnatural amino acid is 3-amino-3-(2-nitrophenyl)propionic acid (ANP).

[0129] In embodiments, the peptide ligand meets one or more of the following criteria: a) are produced in usable amounts during expression in MHC II chains; b) is detected at high levels in 2D LCMS (after cleavage of the linker); and / or c) In an exchange reaction with a peptide known to have high affinity for the allele, it has a peptide exchange yield of about 20% to 100%.

[0130] In some embodiments, the test peptide is an antigen. In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen is a tumor-associated antigen. In some embodiments, the antigen is a neo-antigen. In some embodiments, the antigen is a self-antigen.

[0131] In some embodiments, the MHCII molecule comprises an HLA peptide encoded by an HLA-DP allele, an HLA-DM allele, an HLA-DOA allele, an HLA-DOB allele, an HLA-DQ allele, or an HLA-DR allele. In some embodiments, the HLA allele is HLA-DP. In some embodiments, the HLA allele is HLA-DM. In some embodiments, the HLA allele is HLA-DO. In some embodiments, the HLA allele is HLA-DQ. In some embodiments, the HLA allele is HLA-DR.

[0132] In some embodiments, the MHCII molecule contains an HLA peptide encoded by an HLA-DR allele, and the ligand is a peptide. In some embodiments, the HLA-DR allele is HLA-DRB. In some embodiments, the HLA-DR allele is HLA-DRB1. In some embodiments, the HLA-DRB allele is selected from HLA-DRB1*01:01, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*11:01, HLA-DRB1*11:04, HLA-DRB1*13:01, or HLA-DRB1*15:01. In some embodiments, the allele is HLA-DRB1*01:01. In some embodiments, the allele is HLA-DRB1*03:01. In some embodiments, the allele is HLA-DRB1*04:01. In some embodiments, the allele is HLA-DRB1*07:01. In some embodiments, the allele is HLA-DRB1*08:01. In some embodiments, the allele is HLA-DRB1*11:01. In some embodiments, the allele is HLA-DRB1*13:01. In some embodiments, the allele is HLA-DRB1*15:01.

[0133] In embodiments, the MHCII molecule / ligand complex contains a ligand that is a peptide. In embodiments, the ligand peptide is selected from: PVSKARMATGALAQA (SEQ ID NO: 1), PVSKMRMATGALAQA (SEQ ID NO: 2), or PVSKMRMATPLLMQA (SEQ ID NO: 3). In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO: 1). In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2). In some embodiments, the ligand peptide is PVSKMRMATPLLMQA (SEQ ID NO: 3).

[0134] In some embodiments, the MHCII molecule / ligand complex contains a ligand that is a peptide, and the MHCII molecule is encoded by an HLA-DR allele. In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO: 1) and the HLA-DR allele is HLA-DRB1*01:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2) and the HLA-DR allele is HLA-DRB1*01:01. In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO: 1) and the HLA-DR allele is HLA-DRB1*03:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2) and the HLA-DR allele is HLA-DRB1*04:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO: 2) and the HLA-DR allele is HLA-DRB1*07:01. In some embodiments, the ligand peptide is PVSKMRMATPLLMQA (SEQ ID NO:3) and the HLA-DR allele is HLA-DRB1*08:01. In some embodiments, the ligand peptide is PVSKMRMATGALAQA (SEQ ID NO:2) and the HLA-DR allele is HLA-DRB1*11:01. In some embodiments, the ligand peptide is PVSKMRMATPLLMQA (SEQ ID NO:3) and the HLA-DR allele is HLA-DRB1*13:01. In some embodiments, the ligand peptide is PVSKARMATGALAQA (SEQ ID NO:1) and the HLA-DR allele is HLA-DRB1*15:01. In some embodiments, the HLA allele is HLA-DRB1*11:04 and the peptide ligand is PVSKMRMATGALAQA (SEQ ID NO:2).

[0135] Multiple epitope mapping Multiple epitope mapping can be used to determine the peptides from a given antigen that are most likely to bind to MHCII alleles (e.g., most likely to bind in vivo). For example, a neo-antigen containing mutations (point mutations, indels, etc.) can be presented by MHCII as any of a number of peptides. Each possible peptide has a different affinity for a given MHCII allele. The methods described herein allow for competitive binding and analysis in a multiplexed manner to map long mutant peptides in a single run. Different peptides compete for binding of MHCII molecules, allowing the determination of the "best binder" (the peptide with the highest affinity to the MHCII allele under the conditions used). The relative binding affinity of various peptides can also be determined. The best binder has a high probability of being presented by MHCII at the surface of a cell. As will be appreciated by those skilled in the art, this method can use any of the compositions, MHCII molecules, MHCII-ligand complexes and methods described elsewhere in this disclosure.

[0136] In one aspect, provided herein is a method for multiple epitope mapping to major histocompatibility complex class II (MHCII) alleles. In embodiments, the method comprises: a) providing a first composition comprising a plurality of test peptides and a plurality of MHCII-ligand complexes, each MHCII-ligand complex having (i) an MHCII molecule comprising an α chain and a β chain, and (ii) a ligand, wherein the ligand is linked to the α chain or the β chain via a cleavable linker; b) subjecting said first composition to conditions that cause cleavage of said cleavable linker; c) incubating the first composition for a period of time sufficient to form a second composition, the second composition comprising the α chain, the β chain, the ligand, a free test peptide and / or a plurality of MHCII-test peptide complexes, each MHCII-test peptide complex comprising the MHCII molecule and a test peptide non-covalently bound thereto; d) determining whether one or more of said plurality of test peptides binds to said MHC II molecule in said second composition; Includes.

[0137] In some embodiments, each test peptide in the plurality of test peptides is present in a substoichiometric amount compared to the amount of MHCII molecules in the first composition.Without being bound by theory, it is expected that this allows kinetic competition between the test peptides, but not thermodynamic competition.It is expected that this allows the determination of which test peptides can bind, but not necessarily the test peptide that is the best binder.

[0138] In some embodiments, each test peptide in the plurality of test peptides is present in excess relative to the amount of MHCII molecules in the first composition. Without being bound by theory, it is expected that this allows for the determination of the test peptide that best binds (e.g., with the highest affinity) to the MHCII molecule.

[0139] In some embodiments, the plurality of test peptides includes overlapping peptides of an antigen. In some embodiments, the plurality of test peptides contains contiguous peptides from a single protein. In some embodiments, the antigen is a neo-antigen, a tumor-associated antigen, or an auto-antigen. In some embodiments, the antigen is a neo-antigen. In some embodiments, the antigen is a tumor-associated antigen. In some embodiments, the antigen is an auto-antigen. As used herein, the phrase "overlapping peptides of an antigen" refers to peptides that correspond to a portion of an antigen or potential antigen, where each peptide (or a subset of peptides) corresponds to a slightly different portion of the antigen. For example, if an antigen includes the sequence ABCDEFGH, overlapping peptides of the antigen can include ABC, BCD, CDE, DEF, EFG, FGH and / or ABCD, BCDE, CDEF, DEFG, EFGH, etc.

[0140] In embodiments, each test peptide is between 7 amino acids and 30 amino acids in length. The length can be any value or subrange within the recited range, including the endpoints. In some embodiments, the test peptide is 7 amino acid residues in length. In some embodiments, the test peptide is 8 amino acid residues in length. In some embodiments, the test peptide is 9 amino acid residues in length. In some embodiments, the test peptide is 10 amino acid residues in length. In some embodiments, the test peptide is 11 amino acid residues in length. In some embodiments, the test peptide is 12 amino acid residues in length. In some embodiments, the test peptide is 13 amino acid residues in length. In some embodiments, the test peptide is 14 amino acid residues in length. In some embodiments, the test peptide is 15 amino acid residues in length. In some embodiments, the test peptide is 16 amino acid residues in length. In some embodiments, the test peptide is 17 amino acid residues in length. In some embodiments, the test peptide is 18 amino acid residues in length. In some embodiments, the test peptide is 19 amino acid residues in length. In some embodiments, the test peptide is 20 amino acid residues in length. In some embodiments, the test peptide is 21 amino acid residues in length. In some embodiments, the test peptide is 22 amino acid residues long. In some embodiments, the test peptide is 23 amino acid residues long. In some embodiments, the test peptide is 24 amino acid residues long. In some embodiments, the test peptide is 25 amino acid residues long. In some embodiments, the test peptide is 26 amino acid residues long. In some embodiments, the test peptide is 27 amino acid residues long. In some embodiments, the test peptide is 28 amino acid residues long. In some embodiments, the test peptide is 29 amino acid residues long. In some embodiments, the test peptide is 30 amino acid residues long.

[0141] In some embodiments, MHCII molecule binding to one or more of the plurality of test peptides is determined by measuring the level of each test peptide bound to MHCII molecules in the second composition, hi some embodiments, the level of each test peptide bound to MHCII molecules is measured by two-dimensional liquid chromatography-mass spectrometry (2D LC / MS) of the second composition.

[0142] In some embodiments, the method further comprises performing high performance liquid chromatography (HPLC) and mass spectrometry (MS) to distinguish the MHC II molecule from the test peptides. In some embodiments, the test peptides are distinguished from one another by mass spectrometry based on the mass of each test peptide.

[0143] In some embodiments, the 2D LC / MS includes removing the free test peptide and / or ligand from the second composition, hi some embodiments, the free test peptide is removed from the second composition by size exclusion or ion exchange chromatography.

[0144] In some embodiments, the presence of a given test peptide as determined by HPLC and MS indicates that the given test peptide can bind to an MHCII molecule. In some embodiments, the relative level of the test peptide bound to the MHCII molecule indicates the relative affinity of the test peptide to the MHCII molecule. In some embodiments, a lower level of the first test peptide bound to the MHCII molecule compared to the second test peptide indicates that the first test peptide has a lower likelihood of binding an MHCII molecule on the surface of a cell than the second test peptide. In some embodiments, a higher level of the second test peptide bound to the MHCII molecule compared to the first test peptide indicates that the second test peptide has a higher likelihood of binding an MHCII molecule on the surface of a cell than the first test peptide.

[0145] In some embodiments, one or more of the test peptides determined to have a higher affinity than at least one other test peptide are further analyzed. For example, one or more test peptides can be analyzed.

[0146] In some embodiments, the peptide binds at least one major histocompatibility complex class II (MHCII) molecule, the peptide comprising an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:16. EXAMPLES

[0147] Those skilled in the art will appreciate that the descriptions of making and using the particles described herein are for illustrative purposes only, and that the disclosure is not limited by these examples.

[0148] Example 1. Effect of CLIP variants on peptide exchange A genetically encoded variant of CLIP peptide fused to the N-terminus of HLA-DR β chain was identified that allows robust expression and peptide exchange across a panel of HLA-DR alleles. CLIP peptide can be released by cleavage using either thrombin or TEV cleavage site, and under appropriate conditions, CLIP peptide can be released and exchanged by candidate antigen peptide. The extent and validation of peptide exchange can be determined by either competitive ELISA assay or 2D-LCMS assay described in the second component of the present invention.

[0149] HLA-CLIP peptide fusion proteins were expressed in HEK-293 or CHO cells. The expressed HLA fusion proteins containing protein-CLIP peptide linkers with thrombin recognition sites were enzymatically processed using thrombin-agarose digest. Briefly, approximately 3 mg / mL of uncleaved pMHCII fusion protein complexes were buffer exchanged into 25 mM Tris, pH 8.0, 2 mM NaN3 using a pre-equilibrated Zeba™ spin column. The exchanged complexes were incubated in the presence of thrombin-agarose at room temperature with mixing until the majority of the pMHCII fusion protein was cleaved as determined by LC / MS.

[0150] Expressed HLA fusion proteins containing a protein-CLIP peptide linker with a TEV (Tobacco Etch Virus) protease recognition site were enzymatically processed using a TEV digest. Briefly, approximately 3 mg / mL of uncleaved pMHCII fusion protein complex at pH 6-8 was incubated at room temperature with approximately 30 µg of TEV protease. The pMHCII fusion protein was digested until it was largely cleaved as determined by LC / MS.

[0151] The pMHCII fusion protein complex was then purified by size-exclusion chromatography (SEC). Briefly, sodium chloride (NaCl) was added to the pMHCII fusion protein complex solution to a final concentration of 300 mM. The solution was concentrated to 0.5-1.0 mg / mL and injected into an FPLC equipped with an S-200 SEC column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS), 150 mM NaCl, 2 mM NaN3. The column was run at 0.5 mL / min, and the protein was collected after determining its concentration in the eluate by UV-Vis spectroscopy (Imlen NanoPhotometer). The eluted purified protein was further concentrated by spin filters (Amicon, Millipore) to a concentration of less than approximately 5 mg / mL.

[0152] The pMHCII fusion protein complexes were exchanged with test peptides. Briefly, a master mix of approximately 18 μM CLIP-MHCII in 40 mM sodium acetate, pH 5.0, 150 mM NaCl, 4 mM EDTA, 2 mM NaN3 was prepared (for cysteine-containing peptides, 0.5 mM TCEP was added). For a 50 μL reaction, 5 μL of 4 mM peptide was added to 45 μL of master mix, mixed, and incubated at 37° C. for 60 min. The exchange reaction was quenched by the addition of an equal volume of 50 mM Tris pH 8, 150 mM NaCl (final pMHCII concentration=8 μM). The peptide exchange mixture was centrifuged at 14,000×g for 2 min to remove insoluble peptides or protein aggregates. Before further analysis, the mixture was stored at 4° C. and protected from light.

[0153] Although the use of lower affinity peptides for MHCII:HLA-DRB1*01:01 has been reported, novel CLIP peptide variants can be used that are superior to several HLA-DR alleles that could not be identified without experimental validation. Furthermore, these variants allow for robust protein expression while also allowing for peptide exchange.

[0154] Example 2 Biotinylated Tetramer Formation The pMHCII fusion protein complex was biotinylated via BirA biotin protein ligase. Briefly, a reaction mixture was prepared containing 100 mM magnesium acetate, 10 mM ATP, 5 mM biotin and approximately 3 mg / mL pMHCII. The reaction was initiated by adding 1.3 mg / mL FLAG-BirA and incubated overnight at room temperature.

[0155] The biotinylated pMHCII fusion protein complex was then purified by size-exclusion chromatography (SEC). Briefly, sodium chloride (NaCl) was added to the biotinylated pMHCII fusion protein complex solution to a final concentration of 300 mM. The solution was concentrated to 0.5-1.0 mg / mL and injected into an FPLC equipped with an S-200 SEC column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS), 150 mM NaCl, 2 mM NaN3. The column was run at 0.5 mL / min, and the protein was collected after determining its concentration in the eluate by UV-Vis spectroscopy (Imlen NanoPhotometer). The eluted purified protein was further concentrated by spin filters (Amicon, Millipore) to a concentration of less than approximately 5 mg / mL.

[0156] Biotinylated pMHCII fusion protein complexes were exchanged with test peptides. Briefly, a master mix of approximately 18 μM CLIP-MHCII in 40 mM sodium acetate, pH 5.0, 150 mM NaCl, 4 mM EDTA, 2 mM NaN3 was prepared (for cysteine-containing peptides, 0.5 mM TCEP was added). For a 50 μL reaction, 5 μL of 4 mM peptide was added to 45 μL of master mix, mixed, and incubated at 37° C. for 60 min. The exchange reaction was quenched by the addition of an equal volume of 50 mM Tris pH 8, 150 mM NaCl (final pMHCII concentration=8 μM). The peptide exchange mixture was centrifuged at 14,000×g for 2 min to remove insoluble peptides or protein aggregates. Purified, peptide-exchanged, biotinylated pMHCII was added to streptavidin-fluorophore (2:1 pMHCII to streptavidin-phyrocoerythrin) and mixed thoroughly. The mixture was stored at 4° C. and protected from light prior to analysis by analytical SEC.

[0157] Purified, biotinylated, peptide-exchanged pMHCII fusion protein-streptavidin complexes were evaluated to determine the extent of biotinylation in each mixture. Briefly, the following mixtures were analyzed per injection via analytical SEC: 2x pMHCII: 20 μL 8 μM pMHCI-biotin, 10 μL PBS, 2x pMHCII, 1x PE-SAv: 20 μL 8 μM pMHCII-biotin, 5.5 μL 0.2 mg / mL streptavidin-phyrocoerythrin (SAv-PE, BioLegend), 4.5 μL PBS, and 1x PE-SAv: 10 μL 8 μM pMHCII-biotin, 5.5 μL 0.2 mg / mL SAv-PE, 14.5 μL PBS. The mixture was measured on an HPLC equipped with an analytical SEC column (TSKgel G3000SWXL) equilibrated with 1×PBS, run at a flow rate of 0.65 mL / min.

[0158] Example 3 2D LC-MS assay to identify MHCII peptide binders: This assay identifies peptide binders after a peptide exchange assay on MHCII complexes. Mass spectrometry-based analysis of the peptide exchange process relies on first separating the MHCII complexes in solution from the free peptides prior to analysis. Here, we describe a 2D LC-MS analysis method in which the sample is first run on a SEC column and then only the peaks corresponding to the MHCII complexes are injected onto a second HPLC column for mass analysis. This allows for the complete analysis of MHCII reagents in a single step. This process can be used to identify a single peptide binder at a time or to identify multiple peptides within a larger pool of peptides.

[0159] The first dimension of the 2D LC-MS analysis is size exclusion chromatography. The setup used here was an HPLC (Agilent 1200 series) equipped with a Zenix SEC-100, 3 μm, 100 Å, 4.6 × 50 mm column, run at 30 °C with a flow rate of 0.4 mL / min. The column was equilibrated and run in 25 mM Tris pH 8, 150 mM NaCl, 2 mM NaN3. The collected eluate was eluted from 2.43 to 2.88 min using a heartstring cutoff and subsequently transferred to the second dimension of analysis.

[0160] The second dimension of 2D LC-MS analysis is reversed-phase chromatography. The configuration used here was an HPLC equipped with a PLRP-S, 8 μm, 1000 Å, 2.1 × 50 mm reversed-phase column (Agilent) equilibrated with 5% buffer B, running at a flow rate of 0.55 mL / min with a column temperature of 80 °C. In addition, the cycle modulation time was 8 min, the loop volume was 180 μL, and the diverter valve switch time was 2 min. A gradient was used to elute the injected peptides and proteins, where buffer A was 0.05% trifluoroacetic acid in water and buffer B was 0.05% trifluoroacetic acid in acetonitrile. The gradient was as follows: 0.00 min to 5.0% buffer B, 2.00 min to 5.0% buffer B, 6.70 min to 50.0% buffer B, 6.71 min to 95.0% buffer B, 7.60 min to 95% buffer B, and 7.61 min to 5.0% buffer B.

[0161] The eluate was injected into an Agilent Technologies 6224 TOF LC / MS with extended dynamic range and dual ESI ion source. Briefly, mass spectrometer settings were positive ion polarity, carrier gas temperature, 350°C, drying gas flow rate, 13 L / min, nebulizer pressure, 45 psi, skimmer voltage, 65 V, OCT 1RF VPP, 750 V, and Vcap, 4000 V. The acquisition mass range was minimum 100 m / z and maximum 3000 m / z, and the acquisition speed was 1.03 spectra / sec, 970.9 ms / spectrum, and 9679 transients / spectrum.

[0162] Sample types and resulting absorbance / count ranges were as follows: Briefly, 20 μL of 8 μM (~0.5 mg / mL) peptide-MHCII was injected per LC-MS run. When injecting 20 μL of unexchanged 8 μM CLIP-MHCII, the absorbance at 280 nm in the first dimension was in the range of 150-200 mAU, and in the second dimension the EICs for CLIP (M+H, M+2H, M+3H) were ~10^5 counts. For 20 μL of >95% exchanged peptide-MHCII injected, the EICs for CLIP were ~10^3 counts. Partial exchange lowered the EICs to 10^4-10^3 counts depending on the amount of protein recovered from the peptide exchange reaction after centrifugation. Low CLIP-MHCII samples were injected at concentrations of 16 μM MHCII and 400 μM peptide.

[0163] Example 4 Identification of mutation-specific CD4 T cells. Mutations identified from the MC38 colorectal carcinoma mouse cell line (Yadav M. et al., Nature 2014) were screened for their ability to induce mutation-specific T cell responses upon vaccination in animal studies using 24 amino acid (AA) long mutated peptides (Capietto AH et al., JEM, 2020 and unpublished data). A peptide library from the immunogenic mutations was then designed and synthesized (Genscript) using four overlapping 15 amino acid peptides containing the mutations. Each peptide was tested for binding to the IAb molecule using the 2D-LCMS method described above. To confirm specific T cell recognition of the bound peptides, healthy WT C57Bl / 6 mice (Jackson Laboratory) were vaccinated on days 0 and 10 with the 24 AA long peptide (n=3, 100 μg / mouse; intraperitoneal injection) and adjuvant (poly(IC), 100 μg / mouse, Invitrogen and anti-CD40 antibody, 50 μg / mouse, Genentech). Spleens were harvested 5 days after the last vaccination and processed to obtain single cell suspensions. T cell responses were then determined using a mouse IFN-gamma ELISpot assay (Biotechne) with total splenocytes (5 10^5 cells) incubated overnight with each 15AA peptide (1 μg / mL), 24AA peptide (10 μg / mL), or without peptide as a control, in complete RPMI1640 medium (10% FBS, 1% penicillin / streptomycin, 1% glutamate). IFNg spots were revealed according to the manufacturer's instructions. Finally, tetramers were formed using peptides that showed both binding to IAb molecules (2D-LCMS) and immunogenicity (ELISpot) and tested for staining by flow cytometry on splenocytes from vaccinated mice and non-vaccinated mice as controls (20 μg / mL in 100 μL of FACS buffer, 1 h in the dark at room temperature (RT)).

[0164] Results obtained from vaccination of mice from mutation 112 from MC38 tumor cells are shown as an example in Figures 11A-11C. Figure 11A shows the overlapping peptide library. Figure 11B shows the tetramer schematic with peptide 7788-5 as ligand with PE fluorophore label, and the resulting flow cytometry (FACS) analysis. Splenocytes from whole spleens of vaccinated mice were stained with pIAb(7788-5) tetramer in FACS buffer (20 μg / mL, 100 μL) for 1 h at RT in the dark, washed twice with 200 μL of FACS buffer, and stained with anti-PE microbeads (Miltenyi) according to the manufacturer's instructions. Tetramer-positive cells were then enriched by magnetic LS columns (Miltenyi) and stained for surface antibodies and live / dead staining for 20 min at 4 °C. The cells were then washed twice with 200 μL and acquired on a BD-Symphony flow cytometer to detect activated (CD44+) tetramer+CD4 T cells. Total splenocytes from unvaccinated mice were used as a negative control. Figure 11C shows the ELISpot assay obtained for in vivo vaccination in mice of peptides M112_7788-5, M112_7788-6, M112_7788-7, M112_7788-8 and M112_24mer. Only peptide 7788-5 and 24AA peptide showed IFN-γ spots after recognition by specific CD4 T cells from vaccinated mice in the ELISpot assay.

[0165] Example 5 Multiplexed epitope mapping This mass spectrometry-based assay identifies peptide binders after a peptide exchange assay on MHCII complexes. Here, an automated immunoprecipitation approach is described to enrich for exchanged peptides that complex with biotinylated recombinant MHCII proteins. Peptide-MHCII complexes are loaded onto Agilent AssayMAP streptavidin cartridges (cat G5496-60010, Agilent, Santa Clara, CA). Peptides are acid eluted from streptavidin-bound peptide-MHCII complexes using the Agilent Bravo AssayMAP system. The peptide eluate is loaded onto a reversed-phase C18 column followed by gradient elution directly into a Thermo tribrid orbitrap mass spectrometer for downstream LC-MS / MS analysis. Peaks from the mass spectrometry corresponding to m / z of interest are selected to confirm their presence and area under the curve values ​​are calculated to determine binding affinity compared to that of a control sample signal in which all peptides were present at a 1-1 molar ratio.

[0166] FIG. 12 shows multiplexed analysis of peptide binding of a 150 peptide library from Humira and Remicade variable domains in a single exchange.

[0167] Figure 13 is a schematic diagram of a competitive peptide exchange assay. A low affinity-containing peptide is covalently attached to an MHCII molecule via a cleavable linker. The linker is cleaved in the presence of a labeled high affinity peptide and a pool of test peptides, and the low affinity peptide is exchanged. Three outcomes are illustrated: the MHCII molecule binds the high affinity peptide and is detected, the MHCII molecule binds the test peptide and is not detected, and / or the MHCII complex is no longer able to bind peptide and is not detected.

[0168] Example 6 High-throughput ELISA competitive assay Peptide exchange: A master mix containing 110 nM MHCII / low affinity CLIP and 56 nM or 28 nM biotinylated high affinity huCLIP (huCLIP-bio) was prepared in exchange buffer (40 mM sodium acetate, 150 mM NaCl, 4 mM EDTA, pH 5.0). In a 384-well plate, a 4 mM peptide stock in 100% ethylene glycerol (EG) was first diluted to 400 μM, followed by a 3-fold, 11-point serial dilution in 100% ethylene glycerol on a BioMek system (Beckman Dickson, i7) to generate a 10× intermediate working solution with a concentration range of 400000 nM to 0.38 nM. Ethylene glycerol (no peptide) was used as a negative control. Peptide exchange was performed in 384-well deep well plates by transferring 90 μL of master mix and 10 μL of pre-titrated peptide to each well. The final peptide reaction mix contained 100 nM MHCII / low affinity CLIP, 50 or 25 nM huCLIP-bio and test peptides ranging from 40,000 nM to 0.038 nM. The plates were then sealed and incubated at 37 °C for 48-70 h to allow peptide exchange. After completing the incubation, the plates were spun down and the reaction mix was neutralized by transferring 50 μL / well of the reaction mix to wells of a new 384-well plate containing 50 μL / well of neutralization buffer (400 mM tris, 150 mM NaCl, pH 8.0).

[0169] Competitive ELISA: 384-well Maxisorp plates (Thermo Fisher, Nunc#464718) were coated with 25 μL / well of mouse IgG2a anti-HLA-DR1 L243 monoclonal antibody (Genentech, PUR85601) at 10 μg / mL in coating buffer (0.05M sodium carbonate, pH 9.6). After overnight incubation at 4° C. and washing the plate three times with wash buffer (PBS buffer with 0.05% Tween 20), 50 μL / well of blocking buffer (PBS, 0.5% BSA, 15 ppm Proclin) was added and incubated for 1 hour at room temperature (RT). The plate was then washed three times with wash buffer and 25 μL of neutralized peptide exchange reaction sample was transferred to the appropriate wells. The plate was incubated for 2 hours at room temperature and unbound components were removed by washing the plate six times with wash buffer. Bound MHCII / peptide complexes were then detected by adding 25 μL / well of horseradish peroxidase-conjugated streptavidin (HRP-SA) at 25 ng / mL in assay diluent (PBS 0.5% BSA, 0.05% Tween 20, 15 ppm Proclin, pH 7.4) and incubated for 1 h at room temperature. After six washes to remove HRP-SA, the enzymatic reaction was developed with the peroxidase substrate tetramethylbenzidine (TMB, Moss Inc. Cat. No. 1000) and incubated for 15 min at room temperature. The reaction was stopped with 25 μL / well of 1 M phosphate acid and absorbance was measured at 450 nm using a 630 nM reference in a Multiscan spectrophotometer (Thermo Fisher). Samples containing HLA-DRB1 / low affinity CLIP and biotinylated high affinity huCLIP without test peptides were used as negative controls for each allele. To rank the binding affinities of the peptides, the IC50 of each peptide was determined using the One-site-Fit logIC50 model in GraphPad Prism 8.4.3.

[0170] To optimize the concentration of huCLIP-bio across different DRB1 alleles, huCLIP-bio was titrated to yield a final concentration range of 20,000 nM to 0.02 nM in reaction mixes containing 100 nM of DRB1 allele with low-binding CLIP. Bmax and Kd were determined using a curve-fitting model of saturation specific binding with Hill slope in GraphPad Prism 8.4.3.

[0171] All publications mentioned in the above specification are incorporated herein by reference.Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.Although the invention has been described in connection with certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of biochemistry and biotechnology or related fields are intended to be within the scope of the claims.

[0172] array [Table 1] References 1. Yadav, M., et al. Predicting immunogenic tumor mutations by combining mass spectrometry and exome sequencing. Nature (2014) Nov 27;515(7528):572-6. 2.Capietto,AH,et al.Mutation position is an important determinant for predicting cancer neoantigens.J.Exp.Med.(2020)Apr 6;217(4):e20190179.

Claims

1. 1. A composition comprising a test peptide and a major histocompatibility complex class II (MHCII)-ligand complex, wherein the MHCII-ligand complex comprises (i) an MHC molecule comprising an α chain and a β chain, and (ii) a ligand associated with the α chain and the β chain, wherein the MHCII-ligand complex comprises an HLA peptide encoded by an HLA-DP, HLA-DM, HLA-DO, HLA-DQ, or HLA-DR allele, and wherein the HLA-DR allele and the ligand are: 【Table 1】 A composition selected from:

2. The composition of claim 1, wherein the alpha chain comprises a first dimerization tag and the beta chain comprises a second dimerization tag, and in particular the dimerization tags interact with each other to stabilize the association of the alpha chain and the beta chain.

3. 3. The composition of claim 2, wherein the first dimerization tag and the second dimerization tag comprise a dimerization pair selected from a leucine zipper, a Fos / Jun, an iDimerize pair, a coiled-coil heterodimerization tag, or a knob-in-hole or other heterodimeric Fc.

4. 2. The composition of claim 1, wherein the ligand is covalently attached to the α chain or the β chain via a cleavable linker, particularly wherein the cleavable linker is a UV-cleavable linker or an enzyme-cleavable linker.

5. The composition of claim 1, further comprising a tagged peptide capable of binding the MHCII molecule and comprising a tag.

6. The composition of claim 5 , further comprising a molecule that binds to the tag and comprises a detectable label, particularly wherein the detectable label comprises a chemiluminescent or fluorescent compound.

7. The composition of claim 5 , wherein the tag comprises biotin and the molecule that binds to the tag comprises streptavidin.

8. The composition of claim 5 , further comprising an antibody that binds to the MHCII molecule, particularly wherein the antibody is immobilized on a surface.

9. 2. The composition of claim 1, wherein the ligand is produced at a level of at least about 1 mg / L, is detectable by 2D LCMS after cleavage of the linker, and has a peptide exchange yield of about 20% to 100% in an exchange reaction with a peptide known to have high affinity for the MHCII molecule.

10. The composition of claim 5 , wherein the tagged peptide has an affinity for the MHCII molecule that is higher than the affinity of the ligand for the MHCII molecule.

11. 2. The composition of claim 1, further comprising an MHCII-test peptide complex comprising: (i) an MHC molecule comprising an α chain and a β chain; and (ii) a test peptide associated with the α chain and the β chain.

12. 2. The composition of claim 1, further comprising an MHCII-tagged peptide complex comprising: (i) an MHC molecule comprising an α chain and a β chain; and (ii) a tagged peptide associated with the α chain and the β chain.

13. The composition of claim 1 , further comprising one or more additional test peptides.

14. The composition of any one of claims 1 to 13, wherein the test peptide and / or the one or more further test peptides comprise a tumor antigen or a neoantigen.

15. The composition of any one of claims 1 to 13, wherein the test peptide and / or the one or more further test peptides are between 7 amino acids and 30 amino acids in length.

Images