Barcodeable interchangeable peptide-MHC polymer library

Abstract

MHC multimers are provided, in which peptide-loaded MHC monomers are covalently linked to multimerization domains through conjugation moieties on the monomers and multimerization domains.The multimers can further comprise oligonucleotide barcodes.Peptide exchange with multiple pMHC multimers can be performed to create pMHC multimer libraries.Methods for producing and using pMHC multimers and libraries are also provided.Peptide-loaded MHC class I and class II multimers and libraries thereof are provided.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 003,177, filed on 31 March 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background Identifying peptides recognized by individual T cells is crucial for understanding and treating immune-related diseases, as well as for vaccine development for disease prevention. Techniques for detecting antigen-responsive T cells leverage the interaction between a given TCR and its peptide-MHC (pMHC) recognition motif. The ability to prepare soluble MHC molecules allows for the preparation of soluble peptide-MHC complexes, which can then be formed into multimeric complexes. T cell detection using multimeric pMHC molecules has become a preferred method for detecting antigen-specific T cells in a wide variety of research and clinical settings.

[0003] Since Altman et al. (Science 274:94-96, 1996) demonstrated that tetramerization of peptide-loaded MHC class I (pMHCI) molecules provides sufficient stability for T cell receptor (TCR)-pMHC interactions, enabling the detection of fluorescently labeled MHC multimer-bound T cells using flow cytometry, MHC multimers have been used for the detection of antigen-responsive T cells. However, because MHC class I molecules are highly unstable when they are not part of a complex with a peptide, pMHCI-based techniques were initially limited by the cumbersome production of molecules, requiring individual folding and purification procedures for each peptide (Bakker et al., Curr. Opin. Immunol. 17:428-433, 2005).

[0004] More recently, various MHCI molecules containing covalently linked peptides have been reported (e.g., outlined by Goldberg et al., J. Cell. Mol. Med. 15:1822-1832, 2011). Several types of pMHCI microarray systems have also been developed, but most studies have focused on optimizing the supporting surface as well as modifying the conditions applied during binding and / or washing. The use of these systems is also limited due to insufficient detection limits and low reproducibility compared to existing cytometry-based analyses. For example, a common limitation to such array-based strategies is the tendency of a given T cell to track all possible pMHC interactions presented in a given array. Consequently, the frequency of antigen-responsive T cells in the cell preparation typically needs to be >0.1% to allow for robust readings.

[0005] MHCI multimers and their libraries are prepared using biotinylated peptide-MHCI monomers, which then associate with biotin-binding sites on streptavidin to form tetramers (see, e.g., Leisner et al., PLoS One 3(2):e1678, 2008). For the production of MHC class I libraries, methods have been described in which oligonucleotide barcode labels are conjugated to streptavidin. However, existing strategies involve complex and / or costly methods that limit the easy production of large libraries. For example, in one method, individual streptavidin precursors must be individually barcoded by overlap extension PCR before tetramerization of the biotinylated peptide-HLA monomer (Zhang et al., Nature Biotech. 2018; doi:10.1038.nbt.4282). In another approach, the expensive reagent streptavidin-conjugated dextran is used to create dextramers, which are then complexed with both biotinylated peptide-HLA monomers and biotinylated barcode oligonucleotides via streptavidin conjugated to the dextran backbone (Bentzen et al., Nature Biotech. 34:10: 1037-1045, 2016).

[0006] Similar to the method using pMHCI tetramers, soluble MHC class II molecules have also been used to prepare pMHCII tetramers, which have been used in studies of the antigen specificity of CD4+ helper T cells (as outlined, e.g., in Nepom et al. (2002) Arthrit. Rheumat. 46:5-12; Vollers and Stern (2008) Immunol. 123:305-313; Cecconi et al. (2008) Cytometry 73A:1010-1018). Typically, to prepare pMHCII multimers, soluble biotinylated MHCII α / β dimers are recombinantly expressed and then tetramerized by binding to streptavidin or avidin through their biotin-binding sites. Subsequently, fluorescent labeling of streptavidin or avidin allows for the isolation of T cells bound to pMHCII multimers by flow cytometry. Regarding antigenic peptide loading of MHCII molecules, one method involves covalent attachment of the peptide to the MHCII α / β dimer. Several groups have generated covalently bonded but cleavable "stuffer" peptide-loaded pMHCII that can be exchanged for the peptide of interest under acidic conditions (Day et al., J Clin Invest. 2003;112(6):831-842).

[0007] In alternative methods, "empty" MHCII α / β dimers are prepared and then loaded with soluble MHCII-binding peptides (see, e.g., Novak et al. (1999) J. Clin. Invest. 104:63-67; Nepom et al. (2002) Arthrit. Rheumat. 46:5-12; Macaubus et al. (2006) J. Immunol. 176:5069-5077). While this method allows for greater diversity in peptide loading on MHCII α / β dimers, its ability to recombinantly express stable "empty" MHCII α / β dimers is limited, thus again hindering the preparation of large-scale pMHCII multimer libraries. For example, the production of "empty" MHCII α / β dimers by refolding from E. coli inclusion bodies or by expression in insect or mammalian cells has been reported, but the yields are too low to support high-throughput methods (reviewed in Vollers and Stern (2008) Immunology 123: 305-313). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Altman et al. Science 274:94-96, 1996 [Overview of the Initiative] [Means for solving the problem]

[0009] Therefore, there remains a need for efficient and cost-effective methods to generate peptide-MHC libraries, including barcoded libraries, which can be used in various genome-wide methods, such as screening for T cell specificity for the analysis of T cell recognition, rather than analyses limited to the selection of model antigens. overview This disclosure provides a method for producing barcoded peptide-loaded MHC (pMHC) multimers (e.g., tetramers), including a library thereof. This method provides high protein yields of pMHC multimers within a short period of time using efficient reaction conditions that allow for easy peptide exchange and barcode labeling of the multimers, thereby enabling the efficient preparation of large pMHC multimer libraries. Accordingly, the compositions and methods described herein are suitable for routine laboratory studies, as well as large-scale industrial and clinical applications, in all cases where pMHC multimers are useful. In one embodiment, the pMHC multimer is a pMHC class I (pMHCI) multimer, which is useful for the analysis of CD8+ T cell antigen recognition. In another embodiment, the pMHC multimer is a pMHC class II (pMHCII) multimer, which is useful for the analysis of CD4+ T cell antigen recognition. The MHC polymer of the present invention comprises a covalent linkage between the MHC monomer and the polymerizing domain, thereby enabling the non-covalent site on the polymerizing domain to be easily used for barcode labeling.

[0010] In one embodiment, the disclosure provides a method for producing a peptide-loaded MHC (pMHC) multimer comprising two or more peptide-loaded MHC (pMHC) monomers, wherein each pMHC monomer is covalently linked to a multimerizing domain. In particular, the pMHC monomers are linked to the multimerizing domain through a chemical linkage other than biotin / streptavidin or biotin / avidin interactions, and this linkage is achieved in an efficient bulk chemical reaction. This chemical linkage is achieved by using a conjugation moiety and a multimerizing domain on the pMHC monomer, which then react to form a chemical linkage. Peptide exchange and oligonucleotide barcode labeling can then be readily performed on the pMHC multimer, enabling efficient large-scale pMHC library production.

[0011] Therefore, in one embodiment, the present disclosure is a method for producing major histocompatibility complex (MHC) multimers, (a) A step of providing two or more MHC monomers, each monomer comprising a conjugation moiety; (b) A step of providing a polymerizing domain, wherein each subunit of the polymerizing domain includes a conjugation moiety; (c) A step to produce an MHC polymer by combining the MHC monomer and the polymerizing domain under conditions sufficient for covalent conjugation between the MHC monomer and the polymerizing domain. This provides a method that includes [something].

[0012] In one embodiment, the MHC monomer is an MHC class I monomer. In another embodiment, the MHC monomer is an MHC class II monomer. In a particular embodiment, the MHC monomer is loaded with a placeholder peptide before being combined with the multimerization domain.

[0013] In one embodiment, the polymerizing domain includes a non-covalent binding site, and the method further comprises labeling the MHC polymer with an oligonucleotide barcode that binds to the non-covalent binding site of the polymerizing domain.

[0014] In one embodiment, the polymer is a tetramer. In one embodiment, the polymerizing domain is streptavidin. In one embodiment, the polymerizing domain is streptavidin, and the oligonucleotide barcode binds to the biotin-binding site of streptavidin.

[0015] Regarding the covalent linkage between the MHC monomer and the polymerizing domain, in one embodiment, each conjugation portion of the MHC monomer includes X, and each conjugation portion of the polymerizing domain includes Y. (i) X is a terminal alkyne, and Y is an azide; (ii) X is an azide, and Y is a terminal alkyne; (iii) X is a distorted alkyne, and Y is an azid; (iv) X is Azid, and Y is a distorted Alkyne; (v) X is a diene and Y is a dienophil; (vi) X is a dienophile and Y is a diene; (vii) X is a thiol and Y is an alkene; or (viii) X is an alkene and Y is a thiol.

[0016] In one embodiment, the azide is a copper chelated azide. In another embodiment, the copper chelated azide is a picolyl azide.

[0017] In one embodiment, the conjugation moiety of each MHC monomer and the conjugation moiety of each subunit of the polymerization domain include a sortag motif.

[0018] In one embodiment, the conjugation moiety of each MHC monomer and the conjugation moiety of each subunit of the polymerization domain include an intein sequence.

[0019] In one embodiment, the method further includes the step of replacing a placeholder peptide with a rescue peptide epitope that binds to an MHC monomer.

[0020] In another aspect, this disclosure is: (a) Two or more MHC monomers; (b) A multimerizing domain comprising two or more subunits and having at least one non-covalent site; and (c) Oligolinear barcode A barcode-labeled MHC polymer containing, Each MHC monomer is linked to a subunit of the polymerization domain via covalent linkage. The oligonucleotide barcode is attached to the multimerization domain by non-covalent bonding to a non-covalent bonding site on the multimerization domain. Related to MHC polymers

[0021] In one embodiment, the MHC polymer further comprises MHC-binding peptides loaded onto each MHC monomer of the polymer. In one embodiment, the MHC monomers are MHC class I monomers. In one embodiment, the MHC monomers are MHC class II monomers.

[0022] In one embodiment, the MHC polymer is a tetramer. In one embodiment, the polymerizing domain is streptavidin. In one embodiment, the oligonucleotide barcode is non-covalently bonded to the biotin-binding site on streptavidin.

[0023] In one embodiment of the MHC polymer, each MHC monomer includes a conjugation moiety X, and each subunit of the polymerization domain includes a conjugation moiety Y. (i) X is a terminal alkyne, and Y is an azide; (ii) X is an azide, and Y is a terminal alkyne; (iii) X is a distorted alkyne, and Y is an azid; (iv) X is Azid, and Y is a distorted Alkyne; (v) X is a diene and Y is a dienophil; (vi) X is a dienophile and Y is a diene; (vii) X is a thiol and Y is an alkene; or (viii) X is an alkene and Y is a thiol.

[0024] In one embodiment, the azide is a copper chelated azide. In another embodiment, the copper chelated azide is a picolyl azide.

[0025] In one embodiment of the MHC polymer, each MHC monomer and each subunit of the polymerizing domain includes a conjugation moiety, and each conjugation moiety includes a sortag motif.

[0026] In one embodiment of the MHC polymer, each MHC monomer and each subunit of the polymerizing domain includes a conjugation moiety, and each conjugation moiety includes an intein sequence.

[0027] In yet another embodiment, the present disclosure relates to a method for preparing MHC class I polymers. In one embodiment, a method for producing a pMHCI polymer is a method for producing a pMHCI polymer by loading two or more placeholder peptides, each comprising (a)(i) an MHCI heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a conjugation moiety, and (iv) a placeholder peptide bound in the peptide bond groove of each MHCI monomer. * (b) a step of providing a monomer; (c) a step of providing a polymerizing domain, wherein each subunit of the polymerizing domain includes a conjugation moiety; (d) two or more p * For covalent conjugation between the MHCI monomer and the polymerizing domain, p under sufficient conditions * By combining the MHCI monomer and the polymerizing domain, p * Steps to produce MHCI multimers; and (d)p * p in MHCI polymers * A method is provided which includes the step of producing a pMHCI polymer by replacing a placeholder peptide bound in each peptide bond groove of an MHCI monomer with a rescue peptide epitope. The method may further include the step of labeling the pMHCI polymer with an oligonucleotide barcode by reacting the pMHCI polymer with a barcoded oligonucleotide having a binding moiety that binds to, for example, the polymerization domain of the pMHCI polymer.

[0028] In another aspect, a method for producing barcoded peptide pMHCI multimers, comprising:[ (a) providing two or more placeholder peptide-loaded MHCIs (pMHCIs) each comprising (i) an MHC I heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a conjugation moiety, and (iv) a placeholder peptide bound in the peptide binding groove of each MHC monomer; * providing a multimerization domain, wherein each subunit of the multimerization domain comprises a conjugation moiety and the multimerization domain comprises at least one non-covalent binding site; (b) combining the pMHCIs and the multimerization domain under conditions sufficient for covalent conjugation between the two or more pMHCIs and the multimerization domain to produce a pMHC multimer; (c) replacing the placeholder peptide bound in the peptide binding groove of each pMHC monomer in the pMHC multimer with a rescue peptide epitope to produce a pMHCI multimer; and * (d) binding an oligonucleotide barcode to the non-covalent binding site on the multimerization domain. * A method is provided. * In a further aspect, a method for producing pMHCI multimers, comprising:[ (a) providing two or more placeholder peptide-loaded MHCIs (pMHCIs) each comprising (i) an MHC I heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a peptide linker comprising a conjugation moiety at the C-terminus of (i) or (ii), and (iv) a placeholder peptide bound in the peptide binding groove of each MHC monomer; * (b) providing a multimerization domain, wherein each subunit of the multimerization domain comprises a conjugation moiety and the multimerization domain comprises at least one non-covalent binding site; * (c) combining the pMHCIs and the multimerization domain under conditions sufficient for covalent conjugation between the two or more pMHCIs and the multimerization domain to produce a pMHC multimer; (d) replacing the placeholder peptide bound in the peptide binding groove of each pMHC monomer in the pMHC multimer with a rescue peptide epitope to produce a pMHCI multimer; and (e) binding an oligonucleotide barcode to the non-covalent binding site on the multimerization domain.

[0029] A method is provided. (a) providing two or more placeholder peptide-loaded MHCIs (pMHCIs) each comprising (i) an MHC I heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a peptide linker comprising a conjugation moiety at the C-terminus of (i) or (ii), and (iv) a placeholder peptide bound in the peptide binding groove of each MHC monomer;* Steps to provide MHCI monomers; (b) A step of providing a multimerizing domain that includes a peptide linker containing a conjugation moiety at the C-terminus of each subunit of the multimerizing domain; (c) Two or more p into the multimerization domain * For covalent conjugation between MHCI monomers, p under sufficient conditions * By combining the MHCI monomer and the polymerizing domain, p * A step of producing MHCI multimers; and (d)p * p in MHCI polymers * A step to produce pMHCI multimers by replacing the placeholder peptide bound in each peptide bond groove of the MHCI monomer with a rescue peptide epitope. A method is provided that includes this.

[0030] any suitable p * MHC monomers can be used in the methods described herein. In one embodiment, p * MHC monomers are of vertebrate origin. In another embodiment, p * The MHCI monomer comprises human MHCI heavy chain polypeptide or a functional fragment thereof, and human β2-microglobulin polypeptide or a functional fragment thereof. In another embodiment, each of the p * The MHCI monomer is HLA-A, HLA-B, or HLA-C. In another embodiment, each p * The MHCI monomer is HLA-A. In another embodiment, each p * MHCI monomers are soluble.

[0031] In another embodiment, each p * The MHCI monomer's MHCI heavy chain polypeptide or its functional fragment contains the MHCI α1 domain. In another embodiment, each p * The MHCI monomer's MHCI heavy chain polypeptide or its functional fragment contains an MHCI α1 / α2 heterodimer. In another embodiment, each p* The MHCI monomer's MHCI heavy chain polypeptide or its functional fragment contains the MHCI α1, α2, and α3 domains. In another embodiment, each p * The MHCI heavy chain polypeptide or functional fragment of the MHCI monomer contains an α-domain that is at least 80, 85, 90, 95, or 99% identical to any of the amino acid sequences shown in SEQ ID NOs. 28-159.

[0032] In another embodiment, each p * The MHCI monomer contains a β2-microglobulin domain. In one embodiment, each p * The β2-microglobulin polypeptide of the MHCI monomer is wild-type human β2-microglobulin. In another embodiment, the β2-microglobulin polypeptide comprises an amino acid sequence that is at least 80, 85, 90, 95, or 99% identical to the amino acid sequence of human β2-microglobulin (such as the amino acid sequence shown in SEQ ID NO: 2 or 160).

[0033] In another embodiment, each p * MHC monomers are fusion proteins. For example, in one embodiment, each p * The MHC monomer is a fusion protein comprising an MHC heavy chain or a functional fragment thereof, and β2-microglobulin or a functional fragment thereof. In another embodiment, p * MHC fusion proteins contain a peptide linker between the MHCI heavy chain or its functional fragment and the β2-microglobulin polypeptide or its functional fragment.

[0034] Any suitable placeholder peptide can be used in the methods described herein. In one embodiment, the placeholder peptide is a peptide or peptide-like compound that facilitates the folding of the MHCI polypeptide. In one embodiment, the placeholder peptide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids. In another embodiment, the placeholder peptide is 2 to 25 amino acids. In another embodiment, the placeholder peptide is 8 to 11 amino acids. In another embodiment, the placeholder peptide is 9 amino acids. In another embodiment, the placeholder peptide is 10 amino acids. In another embodiment, the placeholder peptide includes GILGFVFJL (SEQ ID NO: 7). In another embodiment, the placeholder peptide consists of GILGFVFJL (SEQ ID NO: 7). In yet another embodiment, the placeholder peptide has the sequence shown in any one of SEQ ID NOs: 8-27 or 271-279.

[0035] In another embodiment, the placeholder peptide has lower affinity to the MHCI peptide binding groove than the exchanged peptide epitope, and step (d) is performed in a competitive assay, p * This involves contacting an MHCI monomer with an excess peptide epitope. In another embodiment, the placeholder peptide has a KD about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times lower than the exchanged peptide epitope. In another embodiment, the placeholder peptide has a KD about 10 times lower than the exchanged peptide epitope. In another embodiment, the placeholder peptide has a higher affinity for the MHCI binding groove than the exchanged peptide epitope.

[0036] In another embodiment, the placeholder peptide is unstable at temperatures between approximately 30 and 37°C, and step (d) is performed in the presence of the peptide epitope at a temperature between approximately 30 and 37°C (e.g., 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C). * This includes exposure to MHCI monomers. In another embodiment, the placeholder peptide is unstable at acidic pH between approximately pH 2.0 and 5.5 (e.g., pH 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5). In another embodiment, p * MHCI monomers are exposed to a pH range of approximately 2.0 to 5.5 (e.g., pH 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5) in the presence of peptide epitopes. In another embodiment, the placeholder peptide is unstable at acidic pH levels between approximately pH 2.0 and 5.5 (e.g., pH 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5). Step (d) is to p in the presence of the peptide epitope to a pH between approximately pH 2.0 and 5.5 (e.g., pH 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5). * This includes exposure to MHCI monomers.

[0037] In another embodiment, the placeholder peptide is unstable at basic pH levels between approximately pH 9 and 11 (e.g., 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11). In another embodiment, the placeholder peptide is unstable at basic pH levels between approximately pH 9 and 11 (e.g., 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11), and p * MHCI monomers are exposed to pH levels between approximately pH 9 and 11 (e.g., 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11) in the presence of peptide epitopes. In another embodiment, the placeholder peptide is unstable at basic pH between approximately pH 9–11 (e.g., 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11), and step (d) is p in the presence of the peptide epitope at a pH between approximately pH 9–11 (e.g., 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11). * This includes exposure to MHCI monomers.

[0038] In some embodiments, the placeholder peptide comprises GILGFVFJL (SEQ ID NO: 7). In other embodiments, the placeholder peptide has the sequence shown in any one of SEQ ID NOs: 8-27 or 271-279.

[0039] In one embodiment, the placeholder peptide includes a cleavable portion. In one embodiment, the method is used to cleave the placeholder peptide under sufficient conditions. * The method includes the step of contacting an MHCI monomer with a peptide epitope. Any suitable cleavable moiety can be used. In one embodiment, the cleavable moiety is a photocleavable amino acid, and the method (e.g., step (d)) involves exposing the placeholder peptide to UV light under sufficient conditions to induce cleavage of the photocleavable moiety and binding of the peptide epitope to the MHCI monomer. * This includes exposure to MHCI monomers. In one embodiment, the photocleavable amino acid comprises a (2-nitro)phenyl side chain. In another embodiment, the photocleavable amino acid comprises 3-amino-3-(2-nitrophenyl)propionic acid. In yet another embodiment, the photocleavable amino acid is (2-nitro)phenylglycine.

[0040] In other embodiments, a photocleavable placeholder peptide and the corresponding MHC molecule to which it binds are A * 02:01, KILGFVFJV (sequence number 15) or GILGFVFJL (sequence number 7), A *01:01, STAPGJLEY (SEQ ID NO: 16); A*02:03, SVRDJLARL (SEQ ID NO: 271); A*02:06, LTAJFLIFL (SEQ ID NO: 272); A*02:07, LLDSDJERL (SEQ ID NO: 273); A*02:11, KMDIJVPLL (SEQ ID NO: 274); A*03:01, RIYRJGATR (SEQ ID NO: 17); A*11:01, RVFAJSFIK (SEQ ID NO: 18); A*24:02, VYGJVRACL (SEQ ID NO: 11); A*33:03, FYVJGAANR (SEQ ID NO: 275); B*07:02, AARGJTLAM (SEQ ID NO: 14); B*15:02, ILGPPGJVY (SEQ ID NO: 276); B* 35:01, KPIVVLJGY (sequence number 19); B*44:05, EEFGAAJSF (sequence number 277); B*46:01, KMKEIAJAY (sequence number 278); B*55:02, KPWDJIPMV (sequence number 279); C*03:04, FVYGJSKTSL (sequence number 20), B*08:01, FLRGRAJGL (sequence number 21); C*07:02, VRIJHLYIL (sequence number 22); C*04:01, QYDJAVYKL (sequence number 23); B*15:01, ILGPJGSVY (sequence number 24); B*40:01, TEADVQJWL (sequence number 25); B*58:01, ISARGQJLF (sequence number 26); and C * 08:01, selected from KAAJDLSHFL (SEQ ID NO: 27) (where J is 3-amino-3-(2-nitrophenyl)propionic acid).

[0041] In another embodiment, the cleavable portion is an amino acid containing a chemoselective moiety. In another embodiment, the method (e.g., step (d)) involves p under conditions sufficient to cleave the chemoselective moiety. * The method comprises contacting an MHCI monomer with a peptide epitope. In another embodiment, the chemoselective moiety is a sodium diothionite-sensitive azobenzene linker. In another embodiment, the method (e.g., step (d)) involves p in the presence of sodium diothionite. * This involves contacting an MHCI monomer with a peptide epitope.

[0042] In another embodiment, the cleavable portion is a periodate-sensitive amino acid. In another embodiment, the method (e.g., step (d)) is performed in the presence of periodate under conditions sufficient to cleave the placeholder peptide. * This involves contacting an MHCI monomer with a peptide epitope. In another embodiment, the periodate-sensitive amino acid comprises a vicinal diol moiety. In yet another embodiment, the periodate-sensitive amino acid comprises a vicinal amino alcohol. In yet another embodiment, the periodate-sensitive amino acid is α,γ-diamino-β-hydroxybutanoic acid (DAHB).

[0043] In another embodiment, the cleavable portion is a protease-recognizing portion. In one embodiment, the protease is an aminopeptidase. In another embodiment, the protease is a methionine aminopeptidase. In yet another embodiment, the protease is selected from FXa, thrombin, TEV, HRV3C, and furin.

[0044] In one embodiment, the placeholder peptide is a dipeptide. In another embodiment, the dipeptide binds to the F pocket of the MHCI binding groove. In another embodiment, the second amino acid of the dipeptide is hydrophobic. In yet another embodiment, the dipeptide is selected from the group consisting of glycylleucine (GL), glycylvaline (GV), glycylmethionine (methione) (GM), glycylcyclohexylalanine (GCha), glycylhomoleucine (GHle), and glycylphenylalanine (GF).

[0045] Any suitable multimerizing domain can be used. In one embodiment, each subunit of the multimerizing domain includes a conjugation moiety. In another embodiment, the multimerizing domain includes a peptide linker containing a conjugation moiety at the N-terminus of each subunit of the multimerizing domain. In another embodiment, the multimerizing domain includes a peptide linker containing a conjugation moiety at the C-terminus of each subunit of the multimerizing domain. In one embodiment, the multimerizing domain is a dimer, tetramer, hexamer, octamer, decamer, or dodecamer. In another embodiment, the multimerizing domain is a homomultimer. In another embodiment, the multimerizing domain is a heteromultimer. In another embodiment, the multimerizing domain includes streptavidin or a derivative thereof. In another embodiment, the multimerizing domain is a tetramer of streptavidin or a derivative thereof. In another embodiment, the multimerizing domain includes Strep-tag® or Strep-tactin®.

[0046] In one embodiment, the conjugation portion is each p * In another embodiment, the multimerizing domain is attached to the C-terminus of the MHCI heavy chain α1 domain of the MHCI monomer. In yet another embodiment, the conjugation moiety is attached to each p * In another embodiment, the multimerization domain is attached to the C-terminus of the MHCI heavy chain α2 domain. In yet another embodiment, the conjugation moiety is attached to each p * In another embodiment, the polymerizing domain is attached to the C-terminus of the MHCI heavy chain α3 domain. In yet another embodiment, the conjugation moiety is covalently conjugated to the C-terminus of the MHCI α3 domain. * Each p of the MHCI monomer * It is attached to the C-terminus of the β2-microglobulin of the MHC monomer. In another embodiment, the polymerizing domain is attached to each p *In another embodiment, each p is covalently conjugated to the C-terminus of the β2-microglobulin of the MHC monomer. * The covalent conjugation of MHCI monomers is mediated by cysteine ​​transpeptidases (e.g., saltase or its enzymatically active fragments).

[0047] In another embodiment, two or more p * The MHC monomer is chemically conjugated to a polymerizing domain. In another embodiment, the chemical conjugation is mediated by a cysteine ​​bioconjugation. In another embodiment, the chemical conjugation is mediated by a natural chemical conjugation. In yet another embodiment, the chemical conjugation is mediated by click chemistry.

[0048] In another embodiment, each p * The conjugation moiety of the MHCI monomer contains X, and the conjugation moiety of each subunit of the polymerizing domain contains Y. For example, in one embodiment, X is a terminal alkyne and Y is an azide. In another embodiment, X is an azide and Y is a terminal alkyne. In yet another embodiment, X is a strained alkyne and Y is an azide. In yet another embodiment, X is an azide and Y is a strained alkyne. In one particular embodiment, the azide is a copper chelated azide. In one embodiment, the copper chelated azide is a picolyl azide. In another embodiment, X is a diene and Y is a dienophile. In yet another embodiment, X is a dienophile and Y is a diene. In yet another embodiment, X is a thiol and Y is an alkene. In yet another embodiment, X is an alkene and Y is a thiol.

[0049] In another embodiment, each p *The conjugation portion of the MHC domain includes a peptide linker attached to the C-terminus, and the conjugation portion of each subunit of the polymerizing domain includes a peptide linker attached to the C-terminus of each subunit of the polymerizing domain. In another embodiment, each p * The C-terminal peptide linker of the MHC monomer contains (G)nX (where n is at least 2 and X is a suitable portion for chemical conjugation), and the C-terminal peptide linker of each subunit of the polymerization domain contains (G)nY (where n is at least 2 and Y is each p * It includes a portion that is suitable for chemical conjugation to the X portion of the MHC monomer.

[0050] In another embodiment, each p * The conjugation moiety of the MHCI monomer includes a C-terminal sortag, and the conjugation moiety of each subunit of the polymerization domain includes an N-terminal sortag. In another embodiment, each p * The conjugation moiety of the MHCI monomer includes an N-terminal sortag, and the conjugation moiety of each subunit of the polymerizing domain includes a C-terminal sortag. In another embodiment, the method (for example, step (c) of the method for preparing the MHC polymer described above) is p * The process involves adding saltase to a mixture of MHCI monomers and polymerizing domains, and each p * It catalyzes the formation of peptide bonds between MHC monomers and polymerizing domains, p * It produces MHC multimers.

[0051] In another embodiment, two or more p * The MHCI monomer (for example, in step (a) of the method for preparing the MHC polymer described above) contains a C-terminal sortag in the presence of a peptide linker containing a portion suitable for chemical conjugation. * It is produced by contacting the MHCI monomer with saltase or its enzymatically active fragment, and saltase or its enzymatically active fragment is p *The conjugation of the peptide linker to the MHC monomer is mediated, and the multimerizing domain (e.g., in step (b) of the method described above) is produced by contacting the multimerizing domain containing the N-terminal sortag with a saltase in the presence of a peptide linker containing a portion suitable for chemical conjugation, and the saltase or its enzymatically active fragment mediates the conjugation of the peptide linker to the N-terminus of each subunit of the multimerizing domain, and step (c) is p * Two or more p for producing MHC multimers * This involves a chemical conjugation between the C-terminal peptide linker of the MHC monomer and the N-terminal peptide linker of each subunit of the polymerizing domain.

[0052] In one embodiment, each p * The C-terminal peptide linker of the MHC monomer contains (G)nX (where n is at least 2 and X is a suitable portion for click chemistry conjugation), and the N-terminal peptide linker of each subunit of the polymerizing domain contains Y-(G)n (where n is at least 2 and Y is each p * It includes a portion that is suitable for click chemistry conjugation with the X portion of the MHC monomer. In another embodiment, X is a terminal alkyne and Y is an azide. In another embodiment, X is an azide and Y is a terminal alkyne. In another embodiment, X is a strained alkyne and Y is an azide. In another embodiment, X is an azide and Y is a strained alkyne. In a particular embodiment, the azide is a copper chelated azide. In one embodiment, the copper chelated azide is a picolyl azide. In another embodiment, X is a diene and Y is a dienophile. In another embodiment, X is a dienophile and Y is a diene. In another embodiment, X is a thiol and Y is an alkene. In another embodiment, X is an alkene and Y is a thiol.

[0053] In one embodiment, the saltase or its enzymatically active fragment is Ca2+-dependent. In another embodiment, the saltase or its enzymatically active fragment is Ca2+-independent. In another embodiment, the saltase or its enzymatically active fragment is a soluble fragment of wild-type saltase. In another embodiment, the saltase or its enzymatically active fragment is a variant or homolog of S. aureus saltase A. In another embodiment, the saltase or its enzymatically active fragment is modified saltase A. In another embodiment, the saltase or its enzymatically active fragment is a SrtAstaph mutant. In another embodiment, the SrtAstaph mutant is selected from the group consisting of F40, SrtAstaph quintuplet mutant, 2A-9, and 4S-9.

[0054] In one embodiment, each p into the polymerizing domain * The covalent conjugation of MHCI monomers is mediated by inteins. In one embodiment, the intein is selected from the group consisting of MxeGyrA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​Cfa (consensus DnaE split intein), gp41-1, gp41-8, and NrdJ-1. In another embodiment, the intein is a split intein pair. In yet another embodiment, each p * The MHCI monomer is conjugated to the multimerization domain by an intein peptide tag. In another embodiment, each p * The MHCI monomer contains an N-intane fragment at its C-terminus, and each subunit of the polymerizing domain contains an Npu-C-intane fragment at its N-terminus.

[0055] In one embodiment, the rescue peptide epitope includes an identifier. In one embodiment, the identifier is a nucleic acid identifier. In another embodiment, the nucleic acid identifier encodes a peptide. In another embodiment, the nucleic acid identifier is 25 to 500 nucleotides long. In another embodiment, the nucleic acid identifier is 80 to 120 nucleotides long.

[0056] In one embodiment, the disclosure provides a method for producing a library of diverse pMHCI polymers, and methods for their production, including high-throughput methods. In some embodiments, the pMHC polymers further include nucleic acid identifiers, which, for convenience, enable detection and quantification of binding, as described elsewhere in this specification.

[0057] In one embodiment, a method for producing a library containing diverse peptide epitope-probed MHCI (pMHCI) polymers, (a) a plurality of placeholder peptide loads MHCI (p * Steps to provide MHCI monomers; (b) A step of providing multiple polymerizing domains, wherein each subunit of the polymerizing domain includes a conjugation moiety; (c) Two or more p * For covalent conjugation between the MHCI monomer and the polymerizing domain, p under sufficient conditions * By combining the MHCI monomer and the polymerizing domain, p * A step of producing MHCI multimers; and (d) Multiple p * A step to produce multiple peptide-loaded MHCI (pMHCI) multimers by replacing placeholder peptides in an MHCI multimer with a peptide library containing multiple unique MHCI peptide epitopes. A method is provided that includes this.

[0058] In another embodiment, a method for producing a library containing diverse barcoded peptide-loaded major histocompatibility complex class I (pMHCI) multimers, (a) a plurality of placeholder peptide loads MHCI (p * Steps to provide MHCI monomers; (b) A step of providing a plurality of polymerizing domains, wherein each subunit of the polymerizing domain includes a conjugation moiety, and the polymerizing domain includes at least one non-covalent site; (c) Two or more p * Multiple p under sufficient conditions for covalent conjugation between MHCI monomers and polymerizing domains * By combining MHCI monomers and multiple polymerizing domains, multiple p * A step to produce MHCI multimers; (d)p * A step of producing multiple pMHCI multimers by replacing placeholder peptides bound in the peptide bond grooves of MHCI multimers with multiple unique rescue peptide epitopes; and (e) Step of binding an oligonucleotide barcode to a non-covalent binding site on a polymer domain. A method is provided that includes this.

[0059] In another embodiment, a method for producing a library containing diverse barcoded peptide-loaded major histocompatibility complex class I (pMHCI) multimers, (a) a plurality of placeholder peptide loads MHCI (p * Steps to provide MHCI monomers; (b) Providing a plurality of multimerization domains each comprising a peptide linker containing a conjugation moiety at the C-terminus of each subunit of the multimerization domain; (c) Two or more p * Under conditions sufficient for covalent conjugation between MHC I monomers, a plurality of p * Combining MHC I monomers and a plurality of multimerization domains to produce a plurality of p * Producing MHC I multimers; (d) p * Replacing placeholder peptides bound in the peptide binding groove of the MHC I multimer with a plurality of unique rescue peptide epitopes to produce a plurality of pMHC multimers; and (e) Binding an oligonucleotide barcode to a non-covalent binding site on the multimerization domain A method is provided that includes.

[0060] In another aspect, the present disclosure provides a library of peptide-loaded MHC class I (pMHC) multimers, wherein each pMHC multimer in the library comprises two or more pMHC monomers loaded with a unique peptide epitope, and each pMHC monomer is covalently linked to a subunit of a multimerization domain.

[0061] In one embodiment, the library of MHC I peptide epitopes is a highly diverse peptide library. In another embodiment, the peptide library comprises from about 10 3 ~1e1 20 In another embodiment, the peptide library comprises from about 10 3 About 10 4 About 10[[ID=#33]] 5 About 10 6 About 10 7 About 10 8 About 10 9 About 10 10 About 10 11 About 10 12 About 10 13, about 10 14 , about 10 15 , about 10 16 , about 10 17 , about 10 18 , about 10 19 , about 10 20 , or more, containing different MHCI peptide epitopes.

[0062] In one embodiment, the MHCI peptide epitope is derived from a single antigenic protein. In another embodiment, the MHCI peptide epitope comprises overlapping fragments of an antigenic protein. In yet another embodiment, multiple unique peptide epitopes are generated from the organism's genome, the organism's transcriptome, the organism's proteome, or the organism's peptides or proteins. In yet another embodiment, multiple unique peptide epitopes are generated from difference sequences between two genomes. In yet another embodiment, the MHC peptide epitope may be an altered peptide ligand (APL) of a specific peptide epitope of interest.

[0063] In another embodiment, each pMHC polymer contains a unique identifier portion. In one embodiment, the unique identifier portion is a nucleic acid.

[0064] In another embodiment, a polypeptide library is provided comprising a plurality of peptide load MHCI (pMHCI) polymers, wherein each peptide load pMHCI polymer comprises two or more pMHCI monomers conjugated to a polymerizing domain.

[0065] In another embodiment, a method for isolating lymphocytes bound to MHC multimers, (a) The step of contacting multiple lymphocytes with a library of pMHCI multimers; and (b) A step of generating multiple compartments, each compartment comprising lymphocytes bound to pMHCI multimers of a library and a capture support. A method is provided which includes the following: In one embodiment, the lymphocytes are T cells, B cells, or NK cells.

[0066] In another embodiment, a method for identifying lymphocytes bound to pMHC multimers, (a) A step of contacting multiple lymphocytes with a library of pMHCI multimers; (b) a step of compartmentalizing one lymphocyte from among multiple lymphocytes bound to a pMHCI multimer of a library into a single compartment, wherein the pMHCI multimer includes a unique identifier; and (c) A step of determining a unique identifier for pMHCI bound to compartmentalized lymphocytes. A method is provided that includes this.

[0067] For a more complete understanding of the nature and merits of this disclosure, the following detailed description should be considered in conjunction with the accompanying drawings. This disclosure is modifiable in various ways without departing from the disclosure. Therefore, the drawings and description of these embodiments are not restrictive. The present invention provides, for example, the following items: (Item 1) A method for producing major histocompatibility complex (MHC) multimers, (a) A step of providing two or more MHC monomers, each monomer comprising a conjugation moiety; (b) A step of providing a polymerizing domain, wherein each subunit of the polymerizing domain includes a conjugation moiety; (c) A step of producing an MHC polymer by combining the MHC monomer and the polymerizing domain under conditions sufficient for covalent conjugation between the MHC monomer and the polymerizing domain. Methods that include... (Item 2) The method according to item 1, wherein the MHC monomer is an MHC class I monomer. (Item 3) The method according to item 1, wherein the MHC monomer is an MHC class II monomer. (Item 4) The method according to any one of items 1 to 3, wherein the MHC monomer is loaded with a placeholder peptide before being combined with the polymerizing domain. (Item 5) The method according to any one of items 1 to 4, wherein the polymerizing domain includes a non-covalent binding site, and the MHC polymer is further labeled with an oligonucleotide barcode that binds to the non-covalent binding site of the polymerizing domain. (Item 6) The method according to any one of items 1 to 5, wherein the aforementioned polymerizing domain is streptavidin. (Item 7) The method according to item 5, wherein the polymerizing domain is streptavidin, and the oligonucleotide barcode binds to the biotin-binding site of streptavidin. (Item 8) Each MHC monomer's conjugation portion includes X, and each subunit of the polymerization domain's conjugation portion includes Y. (i) X is a terminal alkyne and Y is an azide; (ii) X is an azide and Y is a terminal alkyne; (iii) X is a distorted alkyne, and Y is an azid; (iv) X is Azid, and Y is a distorted Alkyne; (v) X is a diene and Y is a dienophil; (vi) X is a dienophile and Y is a diene; (vii) X is a thiol and Y is an alkene; or (viii) X is an alkene and Y is a thiol. The method described in any one of items 1 to 7. (Item 9) The method according to item 8, wherein the azide is a copper chelated azide. (Item 10) The method according to item 9, wherein the copper chelated azide is picolyl azide. (Item 11) The method according to any one of items 1 to 10, wherein the conjugation portion of each MHC monomer and the conjugation portion of each subunit of the polymerization domain include a sortag motif. (Item 12) Each subunit of the conjugation portion and the polymerization domain of each MHC monomer The method according to any one of items 1 to 10, wherein the conjugation portion of the knit includes an intein array. (Item 13) The method according to any one of items 1 to 12, wherein the polymer is a tetramer. (Item 14) The method according to item 4, further comprising the step of replacing the placeholder peptide with a rescue peptide epitope that binds to the MHC monomer. (Item 15) (a) Two or more MHC monomers; (b) A multimerizing domain comprising two or more subunits and having at least one non-covalent site; and (c) Oligolinear barcode A barcode-labeled MHC polymer containing, Each MHC monomer is linked to a subunit of the polymerizing domain through covalent linkage, The oligonucleotide barcode is bound to the polymerizing domain by non-covalent bonding to the non-covalent bonding site on the polymerizing domain. MHC multimer. (Item 16) The MHC polymer described in item 15, further comprising an MHC-binding peptide loaded onto each MHC monomer of the polymer. (Item 17) The MHC polymer according to item 15 or 16, wherein the MHC monomer is an MHC class I monomer. (Item 18) The MHC polymer described in item 15 or 16, wherein the MHC monomer is an MHC class II monomer. (Item 19) A tetramer, which is an MHC polymer described in any one of items 15-18. (Item 20) The MHC polymer described in item 19, wherein the aforementioned polymerizing domain is streptavidin. (Item 21) The MHC polymer described in item 20, wherein the oligonucleotide barcode is non-covalently bound to the biotin-binding site on streptavidin. (Item 22) Each MHC monomer contains a conjugation moiety X, and each subunit of the polymerization domain contains a conjugation moiety Y. (i) X is a terminal alkyne and Y is an azide; (ii) X is an azide and Y is a terminal alkyne; (iii) X is a distorted alkyne, and Y is an azid; (iv) X is Azid, and Y is a distorted Alkyne; (v) X is a diene and Y is a dienophil; (vi) X is a dienophile and Y is a diene; (vii) X is a thiol and Y is an alkene; or (viii) X is an alkene and Y is a thiol. An MHC polymer as described in any one of items 15-21. (Item 23) The MHC polymer described in item 22, wherein the azide is a copper chelated azide. (Item 24) The MHC polymer described in item 23, wherein the copper chelated azide is picolyl azide. (Item 25) An MHC polymer according to any one of items 15 to 24, wherein each MHC monomer and each subunit of the polymerizing domain includes a conjugation moiety, and each conjugation moiety includes a sortag motif. (Item 26) An MHC polymer according to any one of items 15 to 24, wherein each MHC monomer and each subunit of the polymerizing domain includes a conjugation moiety, and each conjugation moiety includes an intein sequence. (Item 27) A method for producing peptide load major histocompatibility complex class I (pMHCI) multimers, (a) Two or more placeholder peptide-loaded MHCIs (p * Steps to provide MHCI monomers; (b) A step of providing a polymerizing domain, wherein each subunit of the polymerizing domain includes a conjugation moiety; (c) Two or more p * Under conditions sufficient for covalent conjugation between the MHCI monomer and the polymerizing domain, the p * By combining the MHCI monomer and the aforementioned polymerizing domain, p * A step of producing MHCI multimers; and (d) the above p * The p in the MHCI polymer * A step to produce a pMHCI polymer by replacing the placeholder peptide bound in the peptide bond groove of each MHCI monomer with a rescue peptide epitope. Methods that include... (Item 28) A method for producing barcode peptide-loaded major histocompatibility complex class I (pMHCI) multimers, (a) Two or more placeholder peptide-loaded MHCIs (p * Steps to provide MHCI monomers; (b) A step of providing a multimerizing domain, wherein each subunit of the multimerizing domain includes a conjugation moiety, and the multimerizing domain includes at least one non-covalent site; (c) Two or more p * Under conditions sufficient for covalent conjugation between the MHCI monomer and the polymerizing domain, the p * By combining the MHCI monomer and the aforementioned polymerizing domain, p * A step to produce MHCI multimers; (d) the above p * The p in the MHCI polymer * A step of producing a pMHCI multimer by replacing the placeholder peptide bound in the peptide bond groove of each MHCI monomer with a rescue peptide epitope; and (e) The step of binding an oligonucleotide barcode to the non-covalent binding site on the polymerization domain. Methods that include... (Item 29) Peptide loaders produce major histocompatibility complex class I (pMHCI) multimers. It is a law, (a) Two or more placeholder peptide loads of MHCI (p * Steps to provide MHCI monomers; (b) Providing the multimerizing domain comprising a peptide linker having a conjugation moiety at the C-terminus of each subunit of the multimerizing domain; (c) Two or more p into the polymerizing domain * Under conditions sufficient for covalent conjugation between MHCI monomers, the p * By combining the MHCI monomer and the aforementioned polymerizing domain, p * A step of producing MHCI multimers; and (d) the above p * The p in the MHCI polymer * A step to produce a pMHCI polymer by replacing the placeholder peptide bound in the peptide bond groove of each MHCI monomer with a rescue peptide epitope. Methods that include... (Item 30) each p * The method described in any one of items 27-29, wherein the MHC monomers are of vertebrate origin. (Item 31) each p * The method according to any one of items 27 to 30, wherein the MHCI monomer comprises a human MHCI heavy chain polypeptide or a functional fragment thereof, and a human β2-microglobulin polypeptide or a functional fragment thereof. (Item 32) Each of those p * The method according to item 31, wherein the MHCI monomer is HLA-A, HLA-B, or HLA-C. (Item 33) each p * The method according to item 32, wherein the MHCI monomer is HLA-A. (Item 34) each p * The method according to any one of items 27 to 33, wherein the MHCI heavy chain polypeptide or functional fragment of the MHCI monomer comprises an MHCI α1 domain. (Item 35) each p * The method according to any one of items 27 to 34, wherein the MHCI heavy chain polypeptide or functional fragment of the MHCI monomer comprises an MHCI α1 / α2 heterodimer. (Item 36) each p * The method according to any one of items 27 to 35, wherein the MHCI heavy chain polypeptide of the MHCI monomer or its functional fragment comprises MHCI α1, α2, and α3 domains. (Item 37) each p * The method according to any one of items 27 to 36, wherein the MHCI heavy chain polypeptide or functional fragment of the MHCI monomer comprises an α-domain which is at least 80, 85, 90, 95, or 99% identical to any of the amino acid sequences shown in SEQ ID NOs. 28 to 159. (Item 38) each p * The method according to any one of items 27 to 37, wherein the MHCI monomer is soluble. (Item 39) each p * The method according to any one of items 27 to 38, wherein the β2-microglobulin polypeptide of the MHCI monomer is wild-type human β2-microglobulin. (Item 40) The method according to any one of items 27 to 39, wherein the β2-microglobulin polypeptide comprises an amino acid sequence that is at least 80, 85, 90, 95, or 99% identical to the amino acid sequence of human β2-microglobulin (SEQ ID NO: 2 or 160). (Item 41) each p * The method according to any one of items 27-40, wherein the MHC monomer is a fusion protein comprising an MHC heavy chain or a functional fragment thereof, and β2-microglobulin or a functional fragment thereof. (Item 42) The aforementioned p * The method according to item 41, wherein the MHC fusion protein comprises a peptide linker between the MHCI heavy chain or a functional fragment thereof and the β2-microglobulin polypeptide or a functional fragment thereof. (Item 43) The method according to any one of items 27 to 42, wherein the placeholder peptide is a peptide or peptide-like compound that promotes the folding of the MHCI polypeptide. (Item 44) The method according to any one of items 27 to 43, wherein the placeholder peptide consists of 2 to 25 amino acids. (Item 45) The method according to any one of items 27 to 44, wherein the placeholder peptide consists of 8 to 11 amino acids. (Item 46) The method according to any one of items 27 to 45, wherein the placeholder peptide consists of 9 to 10 amino acids. (Item 47) The placeholder peptide has a lower affinity to the MHCI peptide binding groove than the replaced peptide epitope, and step (d) is performed in the competitive assay, the p * The method according to any one of items 27 to 46, comprising contacting an MHCI monomer with an excess peptide epitope. (Item 48) The method according to item 47, wherein the placeholder peptide has a KD about 10 times lower than the exchanged peptide epitope. (Item 49) The placeholder peptide is unstable at temperatures between approximately 30 and 37°C, and step (d) is performed in the presence of the peptide epitope at temperatures between approximately 30 and 37°C. * A method according to any of items 27-46, including exposure to MHCI monomers. (Item 50) The placeholder peptide is unstable at an acidic pH between approximately pH 2.5 and 5.5, and step (d) is performed in the presence of the peptide epitope at a pH between approximately pH 2.5 and 5.5. * A method according to any of items 27-46, including exposure to MHCI monomers. (Item 51) The placeholder peptide is unstable at a basic pH between approximately pH 9 and 11, and step (d) is performed in the presence of the peptide epitope at a pH between approximately pH 9 and 11. * A method according to any of items 27-46, including exposure to MHCI monomers. (Item 52) The method according to any one of items 27 to 51, wherein the placeholder peptide comprises GILGFVFJL (SEQ ID NO: 7). (Item 53) The placeholder peptide consists of GILGFVFJL (SEQ ID NO: 7), item The method described in any of the following two methods (27-51). (Item 54) The placeholder peptide includes a cleavable portion, and step (d) is performed under conditions sufficient to cleave the placeholder peptide. * A method according to any one of items 27 to 46, comprising contacting an MHCI monomer with a peptide epitope. (Item 55) The cleavable portion is a photocleavable amino acid, and step (d) is exposed to UV light under conditions sufficient to induce cleavage of the photocleavable portion in the placeholder peptide and binding of the peptide epitope to the MHCI monomer. * The method described in item 54, which includes exposure to MHCI monomers. (Item 56) The method according to item 55, wherein the photocleavable amino acid includes a 2-nitrophenyl side chain. (Item 57) The method according to item 55, wherein the photocleavable amino acid comprises 3-amino-3-(2-nitrophenyl)propionic acid. (Item 58) The method according to item 55, wherein the photocleavable amino acid is (2-nitro)phenylglycine. (Item 59) The aforementioned photocleavable placeholder peptide is A * 02:01, KILGFVFJV (sequence number 15) or GILGFVFJL (sequence number 7), A * 01:01, STAPGJLEY (SEQ ID NO: 16); A*02:03, SVRDJLARL (SEQ ID NO: 271); A*02:06, LTAJFLIFL (SEQ ID NO: 272); A*02:07, LLDSDJERL (SEQ ID NO: 273); A*02:11, KMDIJVPLL (SEQ ID NO: 274); A*3:01, RIYRJGATR (SEQ ID NO: 17); A*11:01, RVFAJSFIK (SEQ ID NO: 18); A*24:02, VYGJVRACL (SEQ ID NO: 11); A*33:03, FYVJGAANR (SEQ ID NO: 275); B*07:02, AARGJTLAM (SEQ ID NO: 14); B*15:02, ILGPPGJVY (SEQ ID NO: 276); B* 35:01, KPIVVLJGY (sequence number 19); B*44:05, EEFGAAJSF (sequence number 277); B*46:01, KMKEIAJAY (sequence number 278); B*55:02, KPWDJIPMV (sequence number 279); C*3:04, FVYGJSKTSL (sequence number 20), B*08:01, FLRGRAJGL (sequence number 21); C*07:02, VRIJHLYIL (sequence number 22); C*04:01, QYDJAVYKL (sequence number 23); B*15:01, ILGPJGSVY (sequence number 24); B*40:01, TEADVQJWL (sequence number 25); B*58:01, ISARGQJLF (sequence number 26); and C * The method described in item 55, selected from the group consisting of 08:01, KAAJDLSHFL (SEQ ID NO: 27) (where J is 3-amino-3-(2-nitrophenyl)propionic acid). (Item 60) The cleavable portion is an amino acid containing a chemoselective portion, and step (d) is performed under conditions sufficient to cleave the chemoselective portion, * The method according to item 54, comprising contacting an MHCI monomer with a peptide epitope. (Item 61) The chemoselective portion is a sodium dithionite-sensitive azobenzene linker, and step (d) is the p in the presence of sodium dithionite.* The method according to item 60, comprising contacting an MHCI monomer with a peptide epitope. (Item 62) The cleavable portion is a periodate-sensitive amino acid, and step (d) is performed in the presence of periodate under conditions sufficient to cleave the placeholder peptide, and the p * The method according to item 54, comprising contacting an MHCI monomer with a peptide epitope. (Item 63) The method according to item 62, wherein the periodate-sensitive amino acid comprises a vicinaldiol moiety or a vicinalamino alcohol. (Item 64) The method according to item 63, wherein the periodate-sensitive amino acid is α,γ-diamino-β-hydroxybutanoic acid (DAHB). (Item 65) The method according to item 54, wherein the cleavable portion is a protease-recognizing portion. (Item 66) The method according to item 65, wherein the protease is an aminopeptidase. (Item 67) The method according to item 66, wherein the protease is methionine aminopeptidase. (Item 68) The method according to any one of items 27 to 46, wherein the placeholder peptide is a dipeptide. (Item 69) The method according to item 68, wherein the dipeptide binds to the F pocket of the MHCI binding groove. (Item 70) The method according to item 68 or 69, wherein the second amino acid of the dipeptide is hydrophobic. (Item 71) The method according to any one of items 68 to 70, wherein the dipeptide is selected from the group consisting of glycyl-leucine (GL), glycyl-valine (GV), glycyl-methionine (GM), glycyl-cyclohexylalanine (GCha), glycyl-homoleucine (GHle), and glycyl-phenylalanine (GF). (Item 72) The method according to any one of items 27 to 71, wherein the polymerizing domain is a dimer, tetramer, hexamer, octamer, decamer, or dodecamer. (Item 73) The method according to any one of items 27 to 72, wherein the aforementioned polymerizing domain is a tetramer. (Item 74) The method according to any one of items 27 to 73, wherein the polymerizing domain is a homopolymer. (Item 75) The method according to any one of items 27 to 73, wherein the polymerizing domain is a heteropolymer. (Item 76) The method according to any one of items 27 to 75, wherein the polymerizing domain comprises streptavidin or a derivative thereof. (Item 77) The method according to item 76, wherein the polymerizing domain is a tetramer of streptavidin or a derivative thereof. (Item 78) The method according to item 76, wherein the aforementioned polymerizing domain includes Strep-tag® or Strep-tactin®. (Item 79) The aforementioned conjugation portion, each p* The method according to any one of items 27 to 78, wherein step (c) comprises covalent conjugation of the polymerizing domain to the C-terminus of the MHCI heavy chain α1 domain of the MHCI monomer. (Item 80) The aforementioned conjugation portion, each p * The method according to any one of items 27 to 78, wherein step (c) comprises covalent conjugation of the polymerizing domain to the C-terminus of the MHCI heavy chain α2 domain. (Item 81) The aforementioned conjugation portion, each p * The method according to any one of items 27 to 78, wherein step (c) comprises covalent conjugation of the polymerizing domain to the C-terminus of the MHCI heavy chain α3 domain. (Item 82) The aforementioned conjugation portion, each p * Each p of the MHCI monomer * It is attached to the C-terminus of the β2-microglobulin of the MHC monomer, and step (c) is performed at each p * The method according to any one of items 27 to 78, comprising covalent conjugation of the polymerizing domain to the C-terminus of the β2-microglobulin of the MHC monomer. (Item 83) Step (c) involves the two or more p to the polymerizing domain. * The method described in any one of items 27 to 82, including the chemical conjugation of MHC monomers. (Item 84) The method according to item 83, wherein the chemical conjugation is mediated by cysteine ​​bioconjugation. (Item 85) The method according to item 83, wherein the chemical conjugation is mediated by a natural chemical conjugation. (Item 86) The method according to item 83, wherein the chemical conjugation is mediated by click chemistry. (Item 87) each p * The conjugation portion of the MHCI monomer includes X, and the conjugation portion of each subunit of the polymerization domain includes Y. (i) X is a terminal alkyne and Y is an azide; (ii) X is an azide and Y is a terminal alkyne; (iii) X is a distorted alkyne, and Y is an azid; (iv) X is Azid, and Y is a distorted Alkyne; (v) X is a diene and Y is a dienophil; (vi) X is a dienophile and Y is a diene; (vii) X is a thiol and Y is an alkene; or (viii) X is an alkene and Y is a thiol. The method described in item 86. (Item 88) The method according to item 87, wherein the azide is a copper chelated azide. (Item 89) The method according to item 88, wherein the copper chelated azide is picolyl azide. (Item 90) each p * The method according to any one of items 27 to 89, wherein the conjugation portion of the MHC domain includes a peptide linker attached to its N-terminus, and the conjugation portion of each subunit of the polymerizing domain includes a peptide linker attached to the C-terminus of each subunit of the polymerizing domain. (Item 91) each p * The peptide linker at the C-terminus of the MHC monomer is (G) n -X (where n is at least 2, and X is a portion suitable for chemical conjugation), wherein the peptide linker at the C-terminus of each subunit of the polymerizing domain is (G) n -Y(where n is at least 2 and Y is each p * The method according to item 90, comprising a portion that is suitable for chemical conjugation of the X portion of the MHC monomer. (Item 92) Each p into the aforementioned polymerizing domain * The method according to any one of items 27 to 78, wherein the covalent conjugation of MHCI monomers is mediated by a cysteine ​​transpeptidase. (Item 93) The method according to item 92, wherein the cysteine ​​transpeptidase is a saltase or an enzymatically active fragment thereof. (Item 94) each p * The conjugation portion of the MHCI monomer includes a C-terminal sortag, and the conjugation portion of each subunit of the polymerization domain includes an N-terminal sortag, and step (c) is p * The process involves adding the saltase to a mixture of MHCI monomers and polymerizing domains, each p * Catalyst for the formation of a peptide bond between the MHC monomer and the polymerizing domain, p * The method described in item 93, which produces MHC multimers. (Item 95) The two or more p in step (a) * The MHCI monomer contains a C-terminal sortag in the presence of a peptide linker containing a moiety suitable for chemical conjugation. * The MHCI monomer is produced by contacting the saltase or an enzymatically active fragment thereof, and the saltase or an enzymatically active fragment thereof is the p * It mediates the conjugation of the peptide linker to the MHC monomer, The multimerization domain in step (b) is produced by contacting the multimerization domain, including the C-terminal sortag, with the saltase in the presence of a peptide linker containing a portion suitable for chemical conjugation, wherein the saltase or an enzymatically active fragment thereof mediates the conjugation of the peptide linker at the N-terminus of each subunit of the multimerization domain. Step (c) is the p * Two or more p for producing MHC multimers * The method according to item 92, comprising a chemical conjugation between the peptide linker at the C-terminus of an MHC monomer and the peptide linker at the C-terminus of each subunit of the polymerizing domain. (Item 96) each p * The peptide linker at the C-terminus of the MHC monomer is (G) n -X (where n is at least 2, and X is a portion suitable for click chemistry conjugation), wherein the peptide linker at the N-terminus of each subunit of the polymerizing domain is (G) n -Y(where n is at least 2 and Y is each p * The method according to item 95, comprising a portion that is suitable for click chemistry conjugation with the aforementioned X portion of the MHC monomer. (Item 97) (i) X is a terminal alkyne and Y is an azide; (ii) X is an azide and Y is a terminal alkyne; (iii) X is a distorted alkyne, and Y is an azid; (iv) X is Azid, and Y is a distorted Alkyne; (v) X is a diene and Y is a dienophil; (vi) X is a dienophile and Y is a diene; (vii) X is a thiol and Y is an alkene; or (viii) X is an alkene and Y is a thiol. The method described in item 96. (Item 98) The method according to item 97, wherein the azide is a copper chelated azide. (Item 99) The method according to item 98, wherein the copper chelated azide is picolyl azide. (Item 100) The method according to any one of items 93 to 99, wherein the saltase or its enzymatically active fragment is Ca2+-dependent. (Item 101) The method according to any one of items 93 to 99, wherein the saltase or an enzymatically active fragment thereof is Ca2+ independent. (Item 102) The method according to any one of items 93 to 99, wherein the saltase is a soluble fragment of wild-type saltase. (Item 103) The method according to any one of items 93 to 99, wherein the saltase is a variant or homolog of S. aureus saltase A. (Item 104) The method according to any one of items 93 to 99, wherein the saltase is a soluble fragment of modified saltase A. (Item 105) The method according to any one of items 93 to 99, wherein the saltase is a SrtAstaph mutant. (Item 106) The method according to item 105, wherein the SrtAstaph mutant is selected from the group consisting of F40, SrtAstaph quintuple mutant, 2A-9, and 4S-9. (Item 107) Each p into the aforementioned polymerizing domain * The method according to any one of items 27 to 78, wherein the covalent conjugation of the MHCI monomer is mediated by an intein. (Item 108) The method according to item 107, wherein the intein is selected from the group consisting of MxeGyrA, SspDnaE, ​​NpuDnaE, ​​AvaDnaE, ​​Cfa (consensus DnaE split intein), gp41-1, gp41-8, and NrdJ-1. (Item 109) The method according to item 107, wherein the intein is a split intein pair. (Item 110) each p * The method according to any one of items 27 to 78, wherein an MHCI monomer is conjugated to the polymerizing domain by an intein peptide tag. (Item 111) each p * The method according to any one of items 27 to 78, wherein the MHCI monomer contains an N-intane fragment at its C-terminus, and each subunit of the polymerizing domain contains an Npu-C-intane fragment at its N-terminus. (Item 112) The aforementioned polymerizing domain includes an identifier as described in any one of items 27 to 111. method. (Item 113) The method according to item 112, wherein the identifier is a nucleic acid identifier. (Item 114) The method according to item 113, wherein the nucleic acid identifier encodes the peptide. (Item 115) The method according to item 113 or 114, wherein the nucleic acid identifier is 25 nucleotides to 500 nucleotides in length. (Item 116) The method according to item 113 or 114, wherein the nucleic acid identifier is 40 nucleotides to 120 nucleotides in length. (Item 117) A method for producing a library containing diverse peptide epitope-probed MHCI (pMHCI) polymers, (a) a plurality of placeholder peptide loads MHCI (p * Steps to provide MHCI monomers; (b) A step of providing a plurality of polymerizing domains, wherein each subunit of the polymerizing domain includes a conjugation moiety; (c) Two or more p * Under conditions sufficient for covalent conjugation between the MHCI monomer and the polymerizing domain, the p * By bringing the MHCI monomer and the polymerizing domain into contact, p * A step of producing MHCI multimers; and (d) The plurality of p * A step of producing multiple peptide-loaded MHCI (pMHCI) polymers by replacing the placeholder peptide in the MHCI polymer with a peptide library containing multiple unique MHCI peptide epitopes. Methods that include... (Item 118) A method for producing a library containing diverse barcoded peptide-loaded major histocompatibility complex class I (pMHCI) multimers, (a) a plurality of placeholder peptide loads MHCI (p * Steps to provide MHCI monomers; (b) A step of providing a plurality of polymerizing domains, wherein each subunit of the polymerizing domain includes a conjugation moiety, and the polymerizing domain includes at least one non-covalent site; (c) Two or more p * For covalent conjugation between the MHCI monomer and the polymerizing domain, the plurality of p * By contacting the MHCI monomer with the plurality of polymerizing domains, a plurality of p * A step to produce MHCI multimers; (d) the above p * A step of producing multiple pMHCI multimers by replacing the placeholder peptide bound in the peptide bond groove of the MHCI multimer with a plurality of unique rescue peptide epitopes; and (e) The step of binding an oligonucleotide barcode to the non-covalent binding site of the polymerization domain. Methods that include... (Item 119) A method for producing a library containing diverse barcoded peptide-loaded major histocompatibility complex class I (pMHCI) multimers, (a) a plurality of placeholder peptide loads MHCI (p * Steps to provide MHCI monomers; (b) Providing a plurality of the multimerizing domains, each of which contains a peptide linker with a conjugation moiety at the C-terminus of each subunit of the multimerizing domain; (c) Two or more p into the multimerization domain * Under conditions sufficient for covalent conjugation between MHCI monomers, the plurality of p * By contacting the MHCI monomer with the plurality of polymerizing domains, a plurality of p * A step of producing MHCI multimers; and (d) the above p * A step of producing multiple pMHCI multimers by replacing the placeholder peptide bound in the peptide bond groove of the MHCI multimer with multiple unique rescue peptide epitopes. Methods that include... (Item 120) The method according to any one of items 117 to 119, wherein the library of MHCI peptide epitopes is a high-diversity peptide library. (Item 121) The aforementioned peptide library is approximately 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 10 14 , about 10 15 , about 10 16 , about 10 17 , about 10 18 , about 10 19 , about 10 20 The method according to item 120, comprising, or more different MHCI peptide epitopes. (Item 122) The method according to any one of items 117 to 120, wherein the MHCI peptide epitope is derived from a single antigenic protein. (Item 123) The method according to item 122, wherein the MHCI peptide epitope comprises a duplicated fragment of an antigenic protein. (Item 124) The method according to any one of items 117 to 121, wherein the plurality of unique peptide epitopes are generated from the genome of an organism, the transcriptome of an organism, the proteome of an organism, or peptides or proteins of an organism. (Item 125) The method according to any one of items 117-121, wherein the plurality of unique peptide epitopes are generated from different sequences between two genomes. (Item 126) The method according to any one of items 117 to 125, wherein each of the pMHC polymers includes a unique identifier portion. (Item 127) The method according to item 126, wherein the unique identifier portion is a nucleic acid. (Item 128) A polypeptide library comprising multiple peptide load MHCI (pMHCI) polymers, wherein each of the peptide load pMHCI polymers comprises two or more pMHCI monomers conjugated to a polymerizing domain. (Item 129) A method for isolating lymphocytes bound to MHC multimers, (a) The step of contacting multiple lymphocytes with a library of pMHCI multimers; and (b) A step of generating a plurality of compartments, each compartment comprising lymphocytes bound to pMHCI multimers of the library and a capture support. Methods that include... (Item 130) The method according to item 129, wherein the lymphocytes are T cells, B cells, or NK cells. (Item 131) A method for identifying lymphocytes bound to pMHC multimers, (a) A step of contacting multiple lymphocytes with a library of pMHCI multimers; (b) a step of compartmentalizing one lymphocyte from among the plurality of lymphocytes bound to a pMHCI multimer of the library into a single compartment, wherein the pMHCI multimer includes a unique identifier; and (c) A step of determining a unique identifier for the pMHCI bound to the compartmentalized lymphocyte. Methods that include...

[0068] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0069] [Figure 1] Figure 1 illustrates various click chemistry handles and reactions. [Figure 2] Figure 2 illustrates various peptide exchange methods. [Figure 3-1]Figures 3A-3E show SDS-PAGE or Western blot analysis of the conjugation reaction. The schematic images represent SAv tetramers linked to one, two, three, or four HLA molecules. Arrows indicate unwanted byproducts. Figure 3A: Anti-His Western blot analysis of the SAv conjugation reaction. A description of each lane is shown in the table. The degree of reaction is approximately 94-97% based on comparison with the reference SA protein. Figure 3B: SDS-PAGE image of HLA-A2-DBCO-SAv-Az. Lane 1: SeeBlue and protein standard, Lane 2: SA-Az (unboiled), Lane 3: SA-Az (boiled), Lane 4: HLA-A2-DBCO-SAv-Az (unboiled, unreduced), Lane 5: HLA-A2-DBCO-SAv-Az (boiled, reduced). Figure 3C: SDS-PAGE image of HLA-A2-Az-SAv-DBCO. Lane 1: SeeBlue and protein standard, Lane 2: HLA-A2-Az (unboiled), Lane 3: HLA-A2-Az-SAv-DBCO (unboiled), Lanes 4-7: HLA-A2-Az-SAv-DBCO reaction (unboiled). Figure 3D: SDS-PAGE image of HLA-A2-Alk-SAv-Az. Lane 1: SeeBlue and protein standard, Lane 3: HLA-A2-Alk-SAv-Az (unboiled, unreduced), Lane 5: HLA-A2-alkyne-SAv-Az (boiled, reduced). Figure 3E: SDS-PAGE images of HLA-A*01:01, HLA-A*03:01, and HLA-A*24:02 in a conjugated tetramer format. The samples were either boiled / not reduced (NB / NR) or boiled / reduced (boiled / R). [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4] SDS-PAGE analysis of the intent splicing reaction between the HLA-A2-N-inteiin / β2m / peptide complex and SAv-C-inteiin. [Figure 5]Figures 5A and 5B show UV exchange monitored by differential scanning fluorescence (SFluorescence) quantification. Figure 5A shows the differential scanning fluorescence (DSF) quantification of the HLA-A*02:01-Alk-SAv-Az conjugate tetramer after UV exchange, produced in the same manner as in Example 1 containing the placeholder GILGFVFJL peptide (SEQ ID NO: 7), or in the presence of excess NLVPMVATV peptide (SEQ ID NO: 8), showing a 20°C increase in stability indicating exchange to a higher affinity peptide. Figure 5B shows the DSF of the HLA-A*02 biotin-mediated tetramer produced by UV exchange in the monomer and subsequent tetramerization, or by UV exchange in the tetramer itself, confirming that the polymeric state does not affect the efficiency of UV exchange and that the polymer of the present invention has the same stability as industry-standard pMHCs. [Figure 6] Figures 6A–6F show flow cytometry after peptide exchange in biotinylated HLA-A*02 monomers and tetramers. Expanded donor PBMCs with NLVPMVGTV peptide (SEQ ID NO: 9) were stained as follows: anti-CD8-BV785 and anti-Flag-APC secondary only (Figure 6A), 50 nM HLA-A*02 biotin-mediated tetramers loaded with placeholder peptide GILGFVFJL (SEQ ID NO: 7) (Figure 6B), 50 nM HLA-A*02 biotin-mediated tetramers refolded with NLVPMVATV peptide (SEQ ID NO: 8) (Figure 6C), and UV exchange in monomeric form followed by streptavidin Figure 6D shows a 50 nM HLA-A*02 biotin-mediated tetramer loaded with NLVPMVATV peptide (SEQ ID NO: 8) via tetramerization; Figure 6E shows a 50 nM HLA-A*02 biotin-mediated tetramer loaded with NLVPMVATV peptide (SEQ ID NO: 8) via UV exchange within the tetramer itself; and Figure 6F shows a 50 nM HLA-A*02 biotin-mediated tetramer loaded with NLVPMVATV peptide (SEQ ID NO: 8) via dipeptide exchange within the tetramer itself. [Figure 7]Figures 7A-7B show flow cytometry after UV exchange in HLA-A*02:01-Alk-SAv-Az conjugate tetramers. Expanded donor PBMCs with NLVPMVATV peptide (SEQ ID NO: 8) were stained as follows: anti-streptavidin PE and anti-Flag-APC secondary only (Figure 7A), or 1 nM HLA-A*02:01-Alk-SAv-Az conjugate tetramers loaded with NLVPMVATV peptide (SEQ ID NO: 8) via direct UV exchange in the tetrameric form (Figure 7B). [Figure 8] Figures 8A–8C show a comparison of ELISA and DSF as stability tests for UV-exchanged HLA-A*02 tetramers. Specifically, Figure 8A shows the ELISA analysis of HLA-A*02:01-Alk-SAv-Az conjugate tetramers UV-exchanged to a 192-member peptide panel representing the modified peptide ligand (APL) of the NLVPMVATV peptide (SEQ ID NO: 8). The ELISA OD is plotted against the net MHC predicted IC50 for each peptide. Different peptides extend the range of the ELISA signal. Figure 8B shows the DSF curve for a subset of NLVPMVATV (SEQ ID NO: 8) APL peptides UV-exchanged to biotin-mediated tetramers, demonstrating the range of stability. Figure 8C shows the DSF / ELISA correlation for a subset of NLVPMVATV (SEQ ID NO: 8) APL peptides UV-exchanged to biotin-mediated tetramers. [Figure 9]Figures 9A-9D represent the quality control analysis of the HLA-A*01:01-Alk-SAv-Az conjugate tetramer. Specifically, Figure 9A shows the analytical SEC chromatogram of a low-aggregation HLA-A*01:01 tetramer. Figure 9B shows the SDS-PAGE of the HLA-A*01:01-Alk-SAv-Az conjugate tetramer without boiling / no reduction (NB / NR) or boiling / reducing (boil / R). Figure 9C shows the DSF of the HLA-A*01:01-Alk-SAv-Az conjugate tetramer after UV exchange under the load of placeholder peptide STAPGJLEY (SEQ ID NO: 16) (no UV), or in the absence of rescue peptide (no UV peptide) or in the presence of rescue peptide (UV+VTEHDTLLY (SEQ ID NO: 10)). Figure 9D is enlarged with VTEHDTLLY peptide (SEQ ID NO: 10) and shows flow cytometry data for PBMCs stained with 20 nM HLA-A*01:01 biotin-mediated tetramers loaded with VTEHDTLLY peptide (SEQ ID NO: 10) by refolding (refolded VTE), HLA-A*01:01-Alk-SAv-Az conjugate tetramers loaded with STAPGJLEY (SEQ ID NO: 16) (without UV), or HLA-A*01:01-Alk-SAv-Az conjugate tetramers after UV exchange in the presence of rescue peptide VTEHDTLLY (SEQ ID NO: 10) (UV+VTE). Both the percentage of tetramer-positive cells (tetramer+%) and mean fluorescence intensity (MFI) are shown. [Figure 10]Figures 10A-10D represent the quality control analysis of the HLA-A*24:02-Alk-SAv-Az conjugate tetramer. Specifically, Figure 10A shows the analytical SEC chromatogram of a low-aggregation HLA-A*24:02 tetramer. Figure 10B shows the SDS-PAGE of the HLA-A*24:02-Alk-SAv-Az conjugate tetramer without boiling / no reduction (NB / NR) or boiling / reducing (boil / R). Figure 10C shows the DSF of the HLA-A*24:02-Alk-SAv-Az conjugate tetramer after UV exchange under the load of placeholder peptide VYGJVRACL (SEQ ID NO: 11) (no UV), or in the absence of rescue peptide (no UV peptide) or in the presence of rescue peptide (UV + QYDPVAALF (SEQ ID NO: 12)). Figure 10D is enlarged with the QYDPVAALF peptide (SEQ ID NO: 12) and shows flow cytometry data for PBMCs stained with: 20 nM HLA-A*24:02-Alk-SAv-Az conjugate tetramer loaded with QYDPVAALF peptide (SEQ ID NO: 12) by secondary refolding only (refolded QYD); 20 nM HLA-A*24:02-Alk-SAv-Az conjugate tetramer loaded with VYGJVRACL (SEQ ID NO: 11) (no UV); or 20 nM HLA-A*24:02-Alk-SAv-Az conjugate tetramer after UV exchange in the presence of rescue peptide QYDPVAALF (SEQ ID NO: 12) (UV+QYD). Both the percentage of tetramer-positive cells (tetramer+%) and mean fluorescence intensity (MFI) are shown. [Figure 11]Figures 11A-11C represent the quality control analysis of the HLA-B*07:02-Alk-SAv-Az conjugate tetramer. Specifically, Figure 11A shows the analytical SEC chromatogram of the HLA-B*07:02 tetramer without aggregation. Figure 11B shows the SDS-PAGE of the HLA-B*07:02-Alk-SAv-Az conjugate tetramer without boiling / reduction (NB / NR). Figure 11C is enlarged with the RPHERNGFTVL peptide (SEQ ID NO: 13) and shows flow cytometry data for PBMCs stained with 20 nM HLA-B*07:02-Alk-SAv-Az conjugate tetramer loaded with RPHERNGFTVL peptide (SEQ ID NO: 13) by secondary refolding only (refolded RPH), 20 nM HLA-B*07:02-Alk-SAv-Az conjugate tetramer loaded with AARGJTLAM (SEQ ID NO: 14) (no UV), or 20 nM HLA-B*07:02-Alk-SAv-Az conjugate tetramer after UV exchange in the presence of rescue peptide RPHERNGFTVL (SEQ ID NO: 13) (UV+RPH). Both the percentage of tetramer-positive cells (tetramer+%) and mean fluorescence intensity (MFI) are shown. [Figure 12] Figure 12 shows the labeling of the HLA-A*02:01-Alk-SAv-Az conjugate tetramer by a discriminative oligonucleotide tag. The HLA-A*02:01-Alk-SAv-Az conjugate tetramer produced as described in Example 1 was incubated with 5' biotinylated oligonucleotides and separated by Western blotting with an anti-Flag antibody. The band that shifted upon oligonucleotide addition indicated the labeling of the tetramer. [Figure 13]Figure 13 shows single-cell sequencing of the barcoded HLA-A*02:01-Alk-SAv-Az APL library. Heatmap of pMHC binding to individual T cells identified by single-cell sequencing. Columns representing 2008 individual cells are clustered by TCR clone type, and each column represents one of the 192 APL variants of NLVPMATV (SEQ ID NO: 8). Warm colors indicate readings of strong pMHC-TCR interactions by identifying oligonucleotide tags. [Figure 14] Figure 14 shows the PCR amplification of a peptide coding template on a hydrogel under single-template conditions. PCR was performed on hydrogel beads either in bulk or after droplet encapsulation under single-template conditions. The supernatant released upon droplet disruption after PCR was electrophoresed alongside products released from the beads by XbaI or mock digestion. [Figure 15] Figure 15 shows the verification of single-template amplification in droplets. Hydrogels after PCR amplification of the template in bulk or droplets under single-template conditions were stained with streptavidin-PE. Fluorescence in the hydrogels was quantified for all hydrogels to confirm the single-template conditions. [Figure 16] Figures 16A-16B show the loading of HLA-A*02:01-Alk-SAv-Az conjugate tetramers on PCR-amplified hydrogels. The signal-to-noise ratio (Figure 16A) is for hydrogels stained with anti-Flag-APC or anti-β2M-Alexa488 after loading by the conjugate tetramer or subsequent release by benzonase or SmaI. The concentration of HLA-A*02:01-Alk-SAv-Az conjugate tetramers determined by ELISA in the supernatant after the hydrogel loading step or released from the hydrogel loaded with benzonase or SmaI (Figure 16B). [Figure 17]Figures 17A–17B illustrate IVTT peptide production for generating a functional UV-exchange tetramer. Western blotting of the IVTT reaction (+ / - Ulp1 protease) driven by a PCR amplicon template encoding the SUMO-NLVPMVATV (SEQ ID NO: 8) peptide fusion, searched for with an anti-SUMO domain antibody, was performed in lanes 10–11 (Figure 17A). Lanes 2–9 contained a dilution series of SUMO domain-containing standards, which were used to quantify the SUMO domain yield down to approximately 1 μM (Figure 17A). Flow analysis of the tetramer produced by UV exchange from the IVTT-produced peptide is shown (Figure 17B). The tetramers were UV-exchanged in the presence of equimolar synthetic NLVPMVATV (SEQ ID NO: 8) peptide (UV ex 1:1 NLV-synthetic) or IVTT reaction (+Ulp1) (UV ex NLV-IVTT) driven by a SUMO-NLVPMVATV (SEQ ID NO: 8) peptide template, and stained with 1 nM in NLVPMVATV (SEQ ID NO: 8)-enlarged PBMCs (Figure 17B). Positive and negative control tetramers refolded with NLVPMVATV (SEQ ID NO: 8) or GILGFVFJL (SEQ ID NO: 7) peptides were also stained with 1 nM as shown (Figure 17B). [Figure 18] Figure 18 shows flow cytometry results for pMHC tetramers produced and released from hydrogels used in the droplet method, stained in antigen-specific CD8+ T cells. [Figure 19] Figure 19 is a schematic diagram illustrating high-throughput barcoded antigen library production using interchangeable barcodeable tetramers. [Figure 20] Figure 20 is a schematic diagram illustrating the use of sortag and click chemistry for the conjugation of p*MHCII to SAv, cleavage of the peptide linker within the placeholder peptide, replacement of the placeholder peptide by the rescue peptide, and binding to the TCR. [Figure 21]Figures 21A-21E illustrate the formation of p*MHCII multimers. Figure 21A: Anti-Myc Western blot analysis of GGG-alkyne conjugation of monomeric p*MHCII to the α-chain. Figure 21B: SDS-PAGE analysis after click reactions of p*MHCII-Alk and SAv-Az. Figure 21C: HiLoad 26 / 600 Superdex 200 SEC elution chromatogram of click reaction samples. Figure 21D: Anti-FLAG Western blot analysis of main peaks obtained from SEC. Lane 1: Chameleon Duo Pre-stained protein ladder (Licor), Lane 2: Click reaction before loading the sample into the SEC column, Lanes 3 and 4: SEC sample from peak I, Lanes 5 and 6: SEC sample from peak II, Lane 7: No SAv. Lane numbers correspond to unboiled samples, except lane numbers labeled with an asterisk which correspond to boiled samples. Figure 21E: Anti-His Western blot analysis of the main peak obtained after SEC. The lane numbers are the same as those shown in Figure 21D. [Figure 22] Figures 22A–22C show the digestion, exchange, and TCR binding of pMHCII. Figure 22A: SDS-PAGE analysis of boiled and unboiled samples before and after factor Xa cleavage. Figure 22B: ELISA assay to detect the ability of biotinylated exchanged peptides to bind to p↓MHCII multimers. Figure 22C: BLI assay to measure the interaction between HA-specific TCRs and p↓MHCII multimers exchanged to present cognitive HA peptides. The black, light gray, and dark gray curves correspond to signals obtained from moving a TCR-loaded biosensor into wells containing either exchanged p↓MHCII, unexchanged p*MHCII, and BLI buffer, respectively. The dashed line defines the movement of the biosensor to a well lacking the analyte (dissociation). [Figure 23]Figures 23A-B show the staining of a pMHCII tetramer library in antigen-specific T cells. Figure 23A: Donor CD4+ PBMCs expressing the DRB1*01:01 allele and expanded with the influenza hemagglutinin epitope PKYVKQNTLKLAT (SEQ ID NO: 281) were stained with a 10-member DRB1*01:01 library by anti-streptavidin-PE and anti-CD4-BV510 secondary. Cells in the tetramer-positive gate were sorted and mixed with MART1 antigen-specific CD8+ T cells stained with a 6-member library of the ELAGIGILTV (SEQ ID NO: 282) variant loaded onto the A*02:01 tetramer. The resulting pool was subjected to single-cell sequencing, and the resulting heatmap is shown in Figure 23B. Most of the dominant TCR clone types distributed along the y-axis specifically bind to the HA peptide-loaded DRB1*01:01 tetramer. [Modes for carrying out the invention]

[0070] Detailed explanation definition All technical and scientific terms used herein are intended to have the same meaning as those ordinarily understood by those skilled in the art, unless otherwise defined below. References to techniques used herein are intended to refer to techniques as ordinarily understood in the art, including variations or substitutions of equivalent techniques that would be recognized by those skilled in the art. The following terms are expected to be well understood by those skilled in the art, but their definitions are provided below to facilitate the explanation of the subject matter disclosed herein.

[0071] Where used herein, “about” is to be understood by those skilled in the art and to some extent varies depending on the context in which it is used. Where there is a use of this term that is not clear to those skilled in the art considering the context in which the term is used, “about” means up to 10 percent plus or minus a particular value.

[0072] As used herein, “modified peptide ligand” or “APL” refers to a modified or mutant version of a peptide ligand, such as an MHC-binding peptide. A modified or mutant version of a peptide ligand contains at least one structural alteration (e.g., amino acid substitution) compared to the peptide ligand from which it is derived. For example, a panel of APLs can be prepared by systematic or random mutation of known MHC-binding peptides to create a pool of APLs that can be used as a library of MHC-binding peptides for loading onto MHC-conjugated polymers as described herein.

[0073] As used herein, the term "and / or," when used in the context of a list of entities, refers to entities that exist individually or in any possible combination or subcombination.

[0074] The terms “antigen determinant” or “epitope” refer to a site on an antigen to which a variable domain of a T cell receptor, MHC molecule, or antibody specifically binds. Epitopes can be formed from both continuous amino acids or discontinuous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in their unique spatial conformation. Methods for determining which epitopes are bound by a given TCR or antibody (i.e., epitope mapping) are well known in the art, including, for example, immunoblotting and immunoprecipitation assays, where duplicate or continuous peptides derived from the antigen are tested for reactivity with a given TCR or immunoglobulin. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallographic nuclear magnetic resonance, cryo-electron microscopy (cryo-EM), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and site-directed mutagenesis (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0075] As used herein, the term "avidity" refers to the binding strength as a function of the cooperative interactivity of multiple binding sites between a polyvalent molecule (e.g., soluble multimer pMHC-immunoglobulin protein) and its target molecule. Several techniques exist to characterize the avidity of molecular interactions, including switchSENSE and surface plasmon resonance (Gjelstrup et al., J. Immunol. 188:1292-1306, 2012; Vorup-Jensen, Adv. Drug. Deliv. Rev. 64:1759-1781, 2012).

[0076] As used herein, “barcode” is also referred to as an oligonucleotide barcode and is a short nucleotide sequence (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length) that identifies the molecule to which it is conjugated. Barcodes can be used, for example, to identify molecules in a reaction mixture. Barcodes uniquely identify the molecule to which they are conjugated by performing reverse transcription using primers each containing, for example, a “unique molecular identifier” barcode. In other embodiments, primers containing “molecular barcodes” unique to each molecule can be utilized. The process of labeling molecules with barcodes is referred to herein as “barcoding.” A “DNA barcode” is a DNA sequence used to identify a target molecule during DNA sequencing. In some embodiments, a library of DNA barcodes is generated randomly, for example, by assembling oligos in a pool. In other embodiments, a library of DNA barcodes is rationally designed and then manufactured in silico.

[0077] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., TCR, pMHC) and its binding partner. Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., TCR and antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (kd). For example, kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or stronger, up to a maximum of 1 μM. Affinity can be measured by common methods known in the art, including the methods described herein. Low-affinity TCRs generally tend to bind slowly to antigens and dissociate quickly, while high-affinity TCRs generally tend to bind more quickly to antigens and remain bound for a longer period. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of this disclosure.

[0078] The term "biochorthogonal chemistry" refers to any chemical reaction that can occur within a biological system without interfering with natural biochemical processes. This term includes chemical reactions that occur in vitro at physiological pH or in the presence of water. In biochorthogonal chemistry, the reaction is selective, and side reactions with other functional groups found in the starting compounds are avoided. Furthermore, the covalent bonds formed between reaction partners must be strong and chemically inert to the biological reaction, and should not affect the biological activity of the desired molecule.

[0079] As used herein, the terms “carrier” and “pharmaceutically acceptable carrier” include any physiologically compatible solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent and absorption retarder, and the like.

[0080] As used herein, the term "chelator ligand" refers to a bifunctional conjugate moiety that covalently links a radiolabeled prosthetic group to a biologically active target molecule (e.g., a peptide or protein). The bifunctional conjugate moiety utilizes a functional group, for example, a carboxylic acid or activated ester for amide coupling, an isothiocyanate for thiourea coupling, and a maleimide for thiol coupling.

[0081] As used herein, the term “cleavable portion” refers to a motif or sequence that can be cleaved. In some embodiments, the cleavable portion includes a protein, for example, an enzymatic cleavage site. In some embodiments, the cleavable portion includes a chemical cleavage site, for example, by exposure to oxidation / reduction conditions, light / ultrasound, temperature, pH, pressure, etc.

[0082] The term "click chemistry" refers to a set of reliable, selective, bioorthogonal reactions for the rapid synthesis of new compounds and combinatorial libraries. Characteristics of click reactions include modularity, broad range, high yield, stereoselectivity, and simple product isolation (separation from inert by-products by non-chromatographic methods) for producing compounds stable under physiological conditions. In radiochemistry and radiopharmacy, click chemistry is a general term for a set of labeling reactions that use selective, modular building blocks to enable chemoselective ligation for radiolabeling biologically relevant compounds in the absence of catalysts. "Click reactions" can be carried out using copper, or they may be copper-free click reactions. A non-limiting example of a click chemistry handle and reaction is shown in Figure 1.

[0083] As used herein, the term “sufficient conditions for covalent conjugation” refers to reaction conditions, including but not limited to, suitable temperature, pH, and concentrations of reaction components, such that a desired covalent conjugation chemical reaction occurs.

[0084] As used herein, the term “conjugated polymer” is also referred to as a pMHC conjugated polymer and refers to a reaction product resulting from the reaction of a pMHC monomer containing a conjugation moiety with a polymerizing domain containing a conjugation moiety, where two conjugation moieties react with each other to form a covalent link between the pMHC monomer and the polymerizing domain, thereby forming a conjugated polymer. In one embodiment, the conjugated polymer is a conjugated tetramer, where four pMHC monomers react with a polymerizing domain through their conjugation moieties to form a tetramer. In one embodiment, the conjugated polymer is a pMHCI conjugated polymer (e.g., a tetramer), where a pMHC class I monomer is polymerized. In one embodiment, the conjugated polymer is a pMHCII conjugated polymer (e.g., a tetramer), in which a pMHC class II monomer is polymerized.

[0085] As used herein, the term “crosslinking unit” may refer to a molecule linked to another (same or different) molecule. In some embodiments, the crosslinking unit is a monomer. In some embodiments, the crosslinking is a chemical bond. In some embodiments, the crosslinking is a covalent bond. In some embodiments, the crosslinking is an ionic bond. In some embodiments, the crosslinking alters at least one physical property of the linked molecules, for example, the physical properties of a polymer.

[0086] As used herein, the term "endoprotease" refers to a protease that cleaves peptide bonds of non-terminal amino acids.

[0087] As used herein, the term “epitope” (as in “peptide epitope”) refers to a portion of an antigen (e.g., an antigenic protein) that binds to (interacts with or is recognized by) an immune receptor. Thus, a T cell receptor recognizes and binds to an MHC molecule that is complexed with (loaded with) a peptide epitope.

[0088] The terms “exchangeable pMHC polypeptide,” “exchangeable pMHC polymer,” and “placeholder peptide-loaded MHC polypeptide” are used interchangeably herein and refer to an MHC monomer or MHC polymer containing a placeholder peptide in the binding groove of the MHC polypeptide. * MHC is also called monomer or polymer. "Interchangeable" means that the placeholder peptide can be exchanged with the antigen peptide. * MHC monomer or p * This refers to the properties of MHC polymers. In one embodiment, replaceable pMHC or p * The MHC polypeptide comprises an MHC class I molecule having an MHC class I-binding peptide in the binding groove of the MHC class I molecule. In another embodiment, interchangeable pMHC or p * MHC polypeptides contain MHC class II molecules that have MHC class II-binding peptides in the binding groove of the MHC class II molecule.

[0089] As used interchangeably in this specification, “fusion protein” or “fusion polypeptide” refers to a recombinant protein prepared by linking or fusing two polypeptides into a single protein molecule.

[0090] The term “isolated” as applied to MHC monomers herein refers to MHC glycoproteins that are other than their native state and, for example, do not associate with the cell membrane of cells that normally express MHC. This term encompasses the full-length subunit chain and the functional fragment of the MHC monomer. The functional fragment contains the antigen-binding site and the sequence necessary for recognition by the appropriate T cell receptor. It typically contains at least about 60–80%, typically 90–95%, of the sequence of the full-length chain. “Isolated” MHC subunit components can be recombinantly produced or solubilized from a suitable cellular origin. In one embodiment, the “isolated” MHC monomer is a soluble form of an MHC class I monomer, e.g., an MHC class I heavy chain (α chain) associated with β2-microglobulin. In another embodiment, the “isolated” MHC monomer is a soluble form of an MHC class II monomer, e.g., an MHC class II α / β chain.

[0091] As used herein, the term “identifier” refers to a readable representation of data that provides information such as identity corresponding to an identifier.

[0092] As used herein, the terms “linked,” “conjugated,” “fused,” or “fused” are interchangeable when referring to joining two or more elements or components or domains together by any means, including recombinant or chemical means.

[0093] The term “Major Histocompatibility Complex” or “MHC” refers to a genomic locus containing a group of genes encoding diverse cell membrane-bound glycoproteins known as MHC classical class I and class II molecules, respectively, which modulate the immune response by presenting fragmentation protein peptides to circulating cytotoxic and helper T lymphocytes. In humans, this group of genes is also called the “Human Leukocyte Antigen” or “HLA” system. Human MHC class I genes encode, for example, HLA-A, HLA-B, and HLA-C molecules. HLA-A is one of the three main types of human MHC class I cell surface receptors. The others are HLA-B and HLA-C. The HLA-A protein is a heterodimer, consisting of a heavy α chain and a smaller β chain. The α chain is encoded by the variant HLA-A gene, and the β chain is the invariant β2-microglobulin (β2m) polypeptide. The β2-microglobulin polypeptide is encoded by separate regions of the human gene. * 02(A * 02) is a human leukocyte antigen serotype within the HLA-A serotype group. Serotype is determined by antibody recognition of the α2 domain of the HLA-A α chain. * Regarding 02, the α chain is HLA-A * Encoded by the O2 gene, the β chain is encoded by the B2M locus. Human MHC class II genes encode, for example, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, and HLA-DRB1. The complete nucleotide sequences and gene maps of the human major histocompatibility complex are publicly available (e.g., The MHC sequencing consortium, Nature 401:921-923, 1999).

[0094] As used herein, the terms “MHC molecule” and “MHC protein” are used herein to refer to the diverse glycoproteins encoded by MHC class I and MHC class II genes, which are involved in the presentation of peptide epitopes to T cells. The terms “MHC class I” or “MHC I” are used interchangeably to refer to protein molecules comprising an α chain consisting of three domains (α1, α2, and α3) and a second invariant β2-microglobulin. The α3 domain is transmembrane and anchors the MHC class I molecule to the cell membrane. The antigen-derived peptide epitope is located in the peptide bond groove in the central region of the α1 / α2 heterodimer. MHC class I molecules, such as HLA-A, are part of the process of presenting short polypeptides to the immune system. These polypeptides are typically 9–11 amino acid long and originate from proteins expressed by cells. MHC class I molecules present antigens to CD8+ cytotoxic T cells. The terms "MHC class II" or "MHC II" are used interchangeably to refer to protein molecules containing an α chain with two domains (α1 and α2) and a β chain with two domains (β1 and β2). The peptide bond groove is formed by an α1 / β1 heterodimer. MHC class II molecules present antigens to specific CD4+ T cells. Antigens delivered endogenously to APCs are primarily processed for association with MHC class I. Antigens delivered exogenously to APCs are primarily processed for association with MHC class II.

[0095] As used herein, MHC proteins (MHC class I or class II proteins) also include MHC variants, which involve amino acid substitutions, deletions, or insertions and still bind to MHC peptide epitopes (MHC class I or MHC class II peptide epitopes). The term also includes all fragments of these proteins, such as the extracellular domain that holds the peptide bond.

[0096] The term "MHC protein" also includes MHC proteins from non-human vertebrate species. MHC proteins from non-human vertebrate species play a role in the experimentation and treatment of diseases in these vertebrate species, for example in veterinary medicine, and in animal studies where human diseases are investigated in animal models, such as in experimental autoimmune encephalomyelitis (EAE) in mice (mus musculus), an animal model of the human disease multiple sclerosis. Non-human vertebrate species include, more specifically, mice (mus musculus), rats (rattus norvegicus), cattle (bos taurus), horses (equus equus), and green monkeys (macaca mulatta). Mouse MHC proteins are, for example, referred to as H-2 proteins, where MHC class I proteins are encoded by loci H2K, H2L, and H2D, and MHC class II proteins are encoded by locus H2I.

[0097] When used herein, "peptide-free MHC polypeptide" or "peptide-free MHC polymer" refers to an MHC monomer or MHC polymer that does not contain peptides in the binding grooves of the MHC polypeptide. Peptide-free MHC monomers and polymers are also referred to as "empty." In one embodiment, the peptide-free MHC polypeptide or polymer is an MHC class I polypeptide or polymer. In another embodiment, the peptide-free MHC polypeptide or polymer is an MHC class II polypeptide or polymer.

[0098] As used herein, the term “multimer” refers to multiple units. In some embodiments, a multimer comprises one or more different units. In some embodiments, the units in the multimer are the same. In some embodiments, the units in the multimer are different. In some embodiments, the multimer comprises a mixture of the same and different units.

[0099] The terms “peptide epitope,” “MHC peptide epitope,” “MHC peptide antigen,” and “MHC ligand” are used interchangeably herein and refer to MHC ligands that can be bound in the peptide bond groove of an MHC molecule. Peptide epitopes can typically be presented by an MHC molecule. Peptide epitopes typically have 8 to 25 amino acids linked via peptide bonds. Peptides may contain modifications, such as, but are not limited to, side chains of amino acid residues, the presence of labels or tags, the presence of synthetic amino acids, or functional equivalents of amino acids. Typical modifications include those produced by cellular mechanisms, such as glycan addition and phosphorylation. However, other types of modifications are also within the scope of this disclosure.

[0100] As used herein, the term “peptide exchange” refers to a competitive assay in which a placeholder peptide is removed and replaced by a “rescue peptide” (or “rescue peptide epitope”) or a “competitive peptide” (or “competitive peptide epitope”), also referred herein as the “exchanged peptide” (or “exchanged peptide epitope”). Typically, peptide exchange occurs under conditions in which the placeholder peptide is released by peptide cleavage, or under suitable conditions that allow the rescue peptide to compete for binding to an MHC monomer or polymer binding pocket. For example, peptide exchange can be achieved by UV-induced exchange, dipeptide-induced exchange, temperature-induced exchange, or other exchange methods known in the art and disclosed herein. An exemplary method of peptide exchange is shown in Figure 2.

[0101] As used herein, the term “peptide library” refers to a group of peptides. In some embodiments, the library comprises one or more peptides having unique sequences. In some embodiments, each peptide in the library has a different sequence. In some embodiments, the library comprises a mixture of peptides having the same and different sequences.

[0102] As used herein, the term “high diversity peptide library” refers to a peptide library having a high degree of peptide diversity. For example, a high diversity peptide library may have approximately 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 , about 10 14 , about 10 15 , about 10 16 , about 10 17 , about 10 18 , about 10 19 , about 10 20 It contains, or more than, different peptides.

[0103] As used herein, the term “library peptide” refers to a single peptide in a library.

[0104] As used herein, the terms “placeholder peptide” or “exchangeable peptide” are used interchangeably to refer to a peptide or peptide-like compound that binds with sufficient affinity to an MHC protein (e.g., MHCI or MHCII protein) and causes or promotes the proper folding of the MHC protein from an unfolded state or the stabilization of a folded MHC protein. The placeholder peptide may then be exchanged for a peptide of a different purpose (referred to as an exchange peptide or rescue peptide). This exchange can be achieved by UV-induced exchange, dipeptide-induced exchange, temperature-induced exchange, or other exchange methods known in the art.

[0105] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to polymers of amino acids, where one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as polymers of naturally occurring amino acids and polymers of naturally occurring amino acids. The terms “isolated protein” and “isolated polypeptide” are used interchangeably to refer to proteins (e.g., soluble polymer proteins) that have been isolated or purified from other components (e.g., proteins, cellular materials) and / or chemicals. Typically, a polypeptide is purified if it constitutes at least 60% by weight (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) of the total protein in a sample.

[0106] As used herein, the term "protein folding" refers to the spatial organization of a peptide. In some embodiments, the amino acid sequence influences the spatial organization or folding of a peptide. In some embodiments, a peptide may be folded into a functional conformation. In some embodiments, the folded peptide has one or more biological functions. In some embodiments, the folded peptide acquires a three-dimensional structure.

[0107] As used herein, the term “N-terminal amino acid residue” refers to one or more amino acids at the N-terminus of a polypeptide.

[0108] As used herein, the terms “small ubiquitin-like modifying factor moiety,” “SUMO domain,” or “SUMO moiety” are used interchangeably and refer to a specific protease-recognizing portion.

[0109] As used herein, the term “tag” refers to an oligonucleotide component, generally DNA, which provides a means of dealing with a target molecule (e.g., a conjugated multimer) to which it is conjugated. For example, in some embodiments, the tag includes a nucleotide sequence that enables identification, recognition, and / or molecular or biochemical manipulation of the molecule to which the tag is attached (e.g., by providing a unique sequence and / or oligonucleotide, such as a primer for extension by DNA polymerase, or a site for annealing of oligonucleotide for capture or ligation reactions). The process of conjugating the tag to a target molecule may be referred to herein as “tagging,” and a target molecule that undergoes tagging or contains a tag is referred to as “tagged” (e.g., “tagged conjugated multimer”). The tag may be a barcode, an adapter sequence, a primer hybridization site, or a combination thereof.

[0110] The term "T cell" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of T cell receptors on its cell surface. There are several subsets of T cells, and are not limited to helper T cells (also known as T cells). H Cell or CD4 + T cells) and subtypes, for example, T H 1. T H 2, T H 3, T H 17, T H 9, and T FH cells, cytotoxic T cells (also known as T C cells, CD8 + T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, for example, central memory T cells (T CM Cells), effector memory T cells (T EM and T EMRA T cells, and resident memory T cells (T RM cells), regulatory T cells (also known as T reg Cells or suppressor T cells) and subtypes, e.g., CD4 +FOXP3 + T reg cells, CD4 + FOXP3 - T reg Cells, Tr1 cells, Th3 cells, and T reg Examples include 17 cells, natural killer T cells (also known as NKT cells), mucosa-associated invariant T cells (MAITs), and gamma delta T cells (γδT cells), such as Vγ9 / Vδ2 T cells. The term "T-cytotoxic" includes any immune response mediated by CD8+ T cell activation.

[0111] As used herein, the terms “T cell receptor” and “TCR” refer to a surface protein of a T cell that typically binds to one or more major histocompatibility complex (MHC) molecules, enabling the T cell to recognize an antigen and / or its epitope. The TCR functions to recognize antigenic determinants and initiate an immune response. Typically, the TCR is a heterodimer containing two distinct protein chains. In most T cells, the TCR contains an alpha (α) chain and a beta (β) chain. Each chain contains two extracellular domains: a variable (V) region and a constant (C) region, the latter being close to the membrane. The variable domains of the α and β chains are composed of three hypervariable regions, also called complementarity-determining regions (CDRs). The CDRs, particularly CDR3, are the primary cause of contact with the antigen and thus define the specificity of the TCR, although CDR1 of the α chain can interact with the N-terminal portion of the antigen, and CDR1 of the β chain interacts with the C-terminal portion of the antigen. Approximately 5% of T cells possess a TCR composed of gamma and delta (γ / δ) chains. All numbering and naming of the amino acid sequences of the TCR protein loops and sheets follows the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; http: / / imgt.cines.fr; Lefranc et al., (2003) Dev Comp Immunol 27:55 77.; Lefranc et al. (2005) Dev Comp Immunol 29:185-203).

[0112] As used herein, the terms “soluble T cell receptor” and “sTCR” refer to a heterodimerized shortened variant of a TCR, which includes the extracellular portions of the α and β chains of the TCR (e.g., linked by disulfide bonds) but lacks the transmembrane and cytosolic domains of the full-length protein. The sequences (amino acids or nucleic acids) of the soluble TCR α and β chains may be identical to the corresponding sequences in the native TCR, or they may include variant soluble TCR α and β chain sequences compared to the corresponding native TCR sequences. As used herein, the term “soluble T cell receptor” encompasses soluble TCRs having variant or non-variant soluble TCR α and β chain sequences. Variants may be, but are not limited to, variable or constant regions of the soluble TCR α and β chain sequences, including amino acid deletions, insertions, substitutions, and changes to nucleic acid sequences that do not alter the amino acid sequence. Variants retain the binding functionality of their parent molecules.

[0113] As used herein, “TCR / pMHC complex” refers to a protein complex formed by the binding of a T cell receptor (TCR) or its soluble portion to a peptide-loaded MHC molecule. Thus, “components of the TCR / pMHC complex” refers to one or more subunits of the TCR (e.g., Vα, Vβ, Cα, Cβ), or one or more subunits of an MHC or pMHC class I or II molecule.

[0114] As used herein, the term “unbiased” means lacking one or more selection criteria. overview This disclosure provides a method for high-throughput production of libraries containing peptide-loaded MHC (pMHC) polymers, which contain multiple unique peptides in the MHC binding groove and have oligonucleotide barcode labeling to facilitate identification of library members. The method provided herein performs all the challenging and potentially inefficient chemical steps for pMHC polymer production in a single bulk reaction, including chromatographic cleansing and purification, followed by highly efficient peptide exchange and oligonucleotide barcoding. In particular, pMHC monomers are ligated to the polymerizing domain by the use of a conjugation moiety on the monomer and polymerizing domain, which react to form a stable chemical linkage (i.e., covalent bond) between the monomer and the polymerizing domain, thereby forming a pMHC conjugated polymer, such as a pMHC conjugated tetramer. Various conjugation moieties and reactions are suitable for use in the formation of conjugated polymers described herein, including the use of bioorthogonal chemistry, such as click chemistry, which enables easy and efficient reactions. Furthermore, if the multimerizing domain is streptavidin, the biotin-binding site is not used to attach to the pMHC monomer; therefore, this biotin-binding site is available for the convenient attachment of biotinylated oligonucleotide barcodes, thereby allowing for easy and efficient labeling of the multimer.

[0115] The library of pMHC multimers provided herein is useful in a range of therapeutic, diagnostic, and research applications, particularly in any situation where pMHC multimers are useful. For example, the pMHC multimers described herein can be used in various methods, such as methods for identifying and isolating specific T cells in a wide range of applications. In one embodiment, the pMHC multimer is a pMHC class I multimer, which is useful for determining the antigen specificity of CD8+ T cells (e.g., cytotoxic T cells). In another embodiment, the pMHC multimer is a pMHC class II multimer, which is useful for determining the antigen specificity of CD4+ T cells (e.g., helper T cells). I.MHC polypeptide A.MHC class I polypeptide The Class I histocompatibility triple complex consists of three parts associated by non-covalent bonds. The MHCI heavy chain is a diverse transmembrane glycoprotein of approximately 45 kDa composed of three extracellular domains, each containing approximately 90 amino acids (α1, α2, and α3 at the N-terminus), a transmembrane domain of approximately 40 amino acids, and a cytoplasmic tail of approximately 30 amino acids. The α1 and α2 domains of the MHCI heavy chain contain two segments of an alpha-helix that form a peptide binding groove or gap. Short peptides of approximately 8-10 amino acids bind non-covalently to this groove between the two alpha-helices ("fit"). The α3 domain of the MHCI heavy chain is close to the cell membrane. The MHCI heavy chain non-covalently binds to the β2-microglobulin (β2m) polypeptide to form a triple complex. In MHCI, the binding groove is closed at both ends by conserved tyrosine residues, resulting in a size limitation of the bound peptide to typically 8-10 residues by its C-terminal docking into the F-pocket.

[0116] This disclosure provides a multimeric protein comprising two or more MHCI or MHCI-like polypeptides. The MHCI molecules may preferably be vertebrate MHC molecules such as human, mouse, rat, pig, bovine, or avian MHC molecules.

[0117] In some embodiments of the multimeric MHCI polymers described herein, the MHC molecule is a human MHC class I protein: HLA-A, HLA-B, or HLA-C. In some embodiments, the polymer includes, but is not limited to, MHCI class I-like molecules (including non-classical MHC class I molecules), such as CD1d, HLA E, HLA G, HLA F, HLA H, MIC A, MIC B, ULBP-1, ULBP-2, and ULBP-3. The amino acid sequences of MHCI heavy chains, β2m polypeptides, and MHC class I-like molecules from various vertebrate species are known and publicly available in the art.

[0118] In some embodiments, the MHCI heavy chain alpha domain is human, for example, HLA-A * 01:01, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 24:02, HLA-B * 07:02, HLA-C * 04:01, HLA-C * 07:02, HLA-B * 08:01, HLA-B * 35:01, HLA-B * 57:01, HLA-B * 57:03, HLA-E, HLA-C * 16:01, HLA-C * 08:02, HLA-C * 07:01, HLA-C * 05:01, HLA-B * 44:02, HLA-A * 29:02, HLA-B * 44:03, HLA-C * 03:04, HLA-B * 40:01, HLA-C * 06:02, HLA-B * 15:01, HLA-C * 03:03, HLA-A * 30:01, HLA-B * 13:02, HLA-C * 12:03, HLA-A* 26:01、HLA-B * 38:01、HLA-B * 14:02、HLA-A * 33:01、HLA-A * 23:01、HLA-A * 25:01、HLA-B * 18:01、HLA-B * 37:01、HLA-B * 51:01、HLA-C * 14:02, HLA-C * 15:02, HLA-C * 02:02、HLA-B * 27:05、HLA-A * 31:01、HLA-A * 30:02、HLA-B * 42:01、HLA-C * 17:01、HLA-B * 35:02、HLA-B * 39:06、HLA-C * 03:02、HLA-B * 58:01、HLA-A * 33:03、HLA-A * 68:02、HLA-C * 01:02、HLA-C * 07:04、HLA-A * 68:01、HLA-A * 32:01、HLA-B * 49:01、HLA-B * 53:01、HLA-B * 50:01、HLA-A * 02:05、HLA-B * 55:01、HLA-B * 45:01、HLA-B * 52:01、HLA-C * 12:02, HLA-B * 35:03、HLA-B * 40:02、HLA-B * 15:03, and / or HLA-A *The molecules contain MHCI heavy chain alpha domains derived from human MHC class I molecules selected from the group consisting of 74:01. The full-length amino acid sequences (including signal sequences and transmembrane domains) of these MHCI molecules are shown in SEQ ID NOs. 28-93, respectively. The soluble amino acid sequences (lacking signal sequences and transmembrane domains) of these MHCI molecules are shown in SEQ ID NOs. 94-159, respectively.

[0119] In some embodiments, the pMHCI polymers described herein include the α1 and α2 domains of the MHCI heavy chain. In some embodiments, the compounds described herein include the α1, α2, and α3 domains of the MHCI heavy chain.

[0120] In some embodiments, two or more pMHCI or pMHCI-like polypeptides in the polymer include β2-microglobulin polypeptide, e.g., human β2-microglobulin. In some embodiments, the β2-microglobulin is wild-type human β2-microglobulin. In some embodiments, the β2-microglobulin includes an amino acid sequence that is at least 80, 85, 90, 95, or 99% identical to the amino acid sequence of human β2-microglobulin, the full-length sequence of which is shown in SEQ ID NO: 160 (UniProt Id. P61769). Alternatively, the human β2-microglobulin polypeptide used in the pMHCI polymer may include, or consist of, the amino acid sequence shown in SEQ ID NO: 2.

[0121] In some embodiments, the multimer protein comprises a soluble MHCI polypeptide. In some embodiments, the MHC multimer protein comprises a soluble MHCI α domain and a β2-microglobulin polypeptide. In some embodiments, the soluble MHCI protein comprises an MHCI heavy chain α1 domain and an MHCI heavy chain α2 domain.

[0122] Alternatively, in some embodiments, the MHCI monomer is a fusion protein comprising a β2m polypeptide or a functional fragment covalently linked to an MHCI heavy chain or a functional fragment thereof. In some embodiments, the carboxy(-COOH) terminus of the β2m is covalently linked to the amino(-NH2) terminus of the MHCI heavy chain.

[0123] In some embodiments, the MHC monomer includes one or more linkers between the individual components of the MHCI monomer. In some embodiments, the MHCI monomer includes a heavy chain fused to β2m through a linker. In some embodiments, the linker between the heavy chain and β2m is a flexible linker made of, for example, glycine and serine. In some embodiments, the flexible linker between the heavy chain and β2m is between 5 and 20 residues in length. In other embodiments, the linker between the heavy chain and β2m is rigid with a defined structure and is made of, for example, amino acids such as glutamic acid, alanine, lysine, and leucine. In one embodiment, the linker is a (G4S)4 linker (SEQ ID NO: 181, where n=4).

[0124] The amino acid sequences of many MHC class I proteins are publicly known, their genes have been cloned, and therefore, heavy chain monomers can be expressed using recombination. Methods for the expression and purification of MHCI molecules are widely described (e.g., Altman et al., Curr. Protoc. Enz. 17.3.1-17.2-44, 2016). For example, MHCI heavy chains and β2-microglobulin can be expressed in separate cells, isolated by purification, and then refolded in vitro. For example, MHC polypeptide chains can be expressed in E. coli, where they accumulate as insoluble inclusion bodies in bacterial cells. In vitro refolding occurs in a refolding buffer to which the polypeptides are added, for example, by dialysis or dilution. The refolding buffer can be any buffer that allows the MHC polypeptide chains and peptides to reconstitute their native trimer folds. The buffer may contain oxidizing and / or reducing agents to create a redox buffer system that helps MHC proteins establish the correct fold. Suitable refolding buffers include, but are not limited to, Tris buffer, CAPS buffer, TAPs buffer, PBS buffer, other phosphate buffers, carbonate buffers, and Ches buffer. Chaperone molecules or other molecules that improve correct protein folding may also be added, as may agents that increase solubility and agents that prevent aggregate formation. Examples of such molecules include, but are not limited to, arginine, GroE, HSP70, HSP90, small organic compounds, DnaK, CIpB, proline, glycine betaine, glycerol, tween, salts, and PLURONIC®.

[0125] Once expressed, the MHCI complex can be directly purified from MHCI-expressing cells as whole MHCI or MHCI peptide monomers. MHCI monomers may be expressed on the surface of cells and then isolated by disrupting the cell membrane, for example, using a surfactant, followed by purification of MHCI. In some embodiments, MHC monomers are expressed in the periplasm, and the expressing cells are lysed to release purified MHCI monomers. Alternatively, MHC monomers may be purified from the supernatant of cells that secrete the expressed protein into the culture supernatant. Methods for purifying MHCI monomers are well known in the art and include, for example, the use of other techniques including affinity chromatography with affinity tags, ant-tag coated beads, and / or immobilization of MHCI proteins on an affinity matrix, size exclusion chromatography using gel filtration, ion exchange, or other methods that can separate MHC molecules from cells and / or cell lysates.

[0126] In some embodiments, recombinant expression of MHC class I polypeptides allows for the modification of many MHC monomers. For example, recombinant techniques provide a method for carboxyl terminator shortening, which involves the deletion of a hydrophobic transmembrane domain. The carboxyl terminator can also be optionally selected to facilitate ligand or label conjugation by introducing, for example, cysteine ​​and / or lysine residues into the molecule. The synthetic gene typically contains restriction sites to facilitate insertion into an expression vector and manipulation of the gene sequence. The gene encoding the appropriate monomer is then inserted into an expression vector and expressed in a suitable host, e.g., E. coli, yeast, insect, or other suitable cells, to obtain the recombinant protein. For example, the production of an MHC class I polypeptide involves bacterial expression and folding of the MHC class I light chain, β2-microglobulin (β2m), as well as the formation of a complex consisting of the MHC class I heavy chain, β2m, and placeholder peptides.

[0127] In some embodiments, MHC monomers are biotinylated in either their heavy chain or β2m. In some embodiments, MHC monomers are biotinylated before peptide loading, either by refolding or peptide exchange. Biotinylation of MHC monomers can be achieved, as is known in the art, by attaching biotin to a specific attachment site, which is a recognition site of a biotinylating enzyme. In some embodiments, the biotinylating enzyme is BirA. In some embodiments, biotinylation is performed in vivo on the desired protein chain as a post-translational modification during protein expression. B.MHC Class II Polypeptide MHC class II molecules are heterodimers composed of α and β chains, both encoded by MHC. The α chain consists of α1 and α2 domains. The β chain consists of β1 and β2 domains. The α1 and β1 domains of the chains interact non-covalently to form peptide-binding domains distal to the membrane, while the α2 and β2 domains form immunoglobulin-like domains proximal to the membrane. The antigen-binding groove to which the peptide epitope binds is constructed from two α-helices and β-sheets. Since the antigen-binding groove of MHC class II molecules is open at both ends, the groove can accommodate longer peptide epitopes than those of MHC class I molecules. Peptide epitopes presented by MHC class II molecules are typically about 15–24 amino acid residues long.

[0128] This disclosure provides a multimeric protein comprising two or more MHCII or MHCII-like polypeptides. The MHCII molecules may preferably be vertebrate MHCII molecules such as human, mouse, rat, pig, cattle, or avian MHCII molecules.

[0129] In some embodiments of the multimeric MHCII polymers described herein, the MHC molecules are human MHC class II proteins: HLA-DR, HLA-DQ, HLA-DX, HLA-DO, HLA-DZ, and HLA-DP. The amino acid sequences of MHCII α and β chains from various vertebrate species, including humans, are known and publicly available in the art.

[0130] In some embodiments, the human MHCII molecule is DRB1 * 0101 (see, for example, Cameron et al. (2002) J. Immunol. Methods, 268:51-69; Cunliffe et al. (2002) Eur. J. Immunol., 32:3366-3375; Danke et al. (2003) J. Immunol., 171:3163-3169), DRB1 * 1501 (see, for example, Day et al. (2003) J. Clin. Invest, 112:831-842), DRB5 * 0101 (for example, Day et al., see the book), DRB1 * 0301 (see, for example, Bronke et al. (2005) Hum. Immunol., 66:950-961), DRB1 * 0401 (see, for example, Meyer et al. (2000) PNAS, 97:11433-11438; Novak et al. (1999) J. Clin. Invest, 104:R63-R67; Kotzin et al. (2000) PNAS, 97:291-296), DRB1 * 0402 (see, for example, Veldman et al. (2007) Clin. Immunol., 122:330-337), DRB1 * 0404 (see, for example, Gebe et al. (2001) J. Immunol. 167:3250-3256), DRB1 *1101 (see, for example, Cunliffe, ibid.; Moro et al. (2005) BMC Immunol., 6:24), DRB1 * 1302 (see, for example, Laughlin et al. (2007) Infect. Immunol. 75:1852-1860), DRB1 * 0701 (for example, Danke, see the book), DQA1 * 0102 (see, for example, Kwok et al. (2000) J. Immunol., 164:4244-4249), DQB1 * 0602 (for example, Kwok, see the book), DQA1 * 0501 (see, for example, Quarsten et al. (2001) J. Immunol., 167:4861-4868), DQB1 * 0201 (for example, Quarsten, see the book), DPA1 * 0103 (see, for example, Zhang et al. (2005) Eur. J. Immunol, 35:1066-1075; Yang et al. (2005) J. Clin. Immunol., 25:428-436), and DPB1 * It is an allotype selected from the group consisting of 0401 (see, for example, Zhang, ibid.; Yang, ibid.).

[0131] In some embodiments, the MHCII molecule is human, for example, HLA-DRA * 01:01, HLA-DRB1 * 01:01, HLA-DRB1 * 01:02, HLA-DRB1 * 03:01, HLA-DRB1 * 04:01, HLA-DRB1 * 04:04, HLA-DRB1 * 07:01, HLA-DRB1 * 08:01, HLA-DRB1 * 10:01, HLA-DRB1 * 11:01, HLA-DRB1* 11:04, HLA-DRB1 * 13:01, HLA-DRB1 * 13:02, HLA-DRB1 * 14:01, HLA-DRB1 * 15:01, HLA-DRB1 * 15:03, HLA-DQA1 * 01:01, HLA-DQB1 * 05:01, HLA-DQA1 * 01:02, HLA-DQB1 * 06:02, HLA-DQA1 * 03:01, HLA-DQB1 * 03:02, HLA-DQA1 * 05:01, HLA-DQB1 * 02:01, HLA-DQB1 * 03:01, HLA-DQB1 * 03:03, HLA-DQB1 * 04:02, HLA-DQB1 * 05:03, HLA-DQB1 * 06:03, and HLA-DQB1 * The MHCII alpha and beta chains are selected from the group consisting of 06:04. The full-length amino acid sequences (including signal sequences and transmembrane domains) of these MHCII chains are shown in SEQ ID NOs. 194-223, respectively. The soluble amino acid sequences (lacking signal sequences and transmembrane domains) of these MHCII chains are shown in SEQ ID NOs. 224-253, respectively.

[0132] In certain embodiments, additional amino acid sequences may be added to the C-terminal sequence of the alpha or beta chain of the MHCII molecule for the purpose of labeling and / or attaching a moiety that mediates attachment (e.g., conjugation) to a polymerization domain, for example. For example, avitag (mediating binding through the biotin-binding site of Sav), such as avitage (SEQ ID NO: 254) with Myc and His tags, or avitag (SEQ ID NO: 255) with a Myc tag, may be added. In another embodiment, sortag (which can mediate the conjugation of the click chemistry moiety through saltase, as described herein), such as sortag shown in SEQ ID NO: 257, or sortag with a His tag shown in SEQ ID NO: 256, may be added. In yet another embodiment, a V5 tag (SEQ ID NO: 258) may be added to the C-terminus.

[0133] In certain embodiments, heterodimerization pairs can be added to the C-terminal sequences of the alpha and / or beta chains of an MHCII molecule. Non-limiting examples of such heterodimerization pair sequences include Fos and Jun (e.g., having the amino acid sequences shown in SEQ ID NOs. 259 and 260, respectively), acidic and basic leucine zippers (e.g., having the amino acid sequences shown in SEQ ID NOs. 261 and 262, respectively), knob and hole sequences for knob-into-hole technology (e.g., having the amino acid sequences shown in SEQ ID NOs. 263 and 264, respectively), or spytab and spycatcher sequences (e.g., having the amino acid sequences shown in SEQ ID NOs. 265 and 266, respectively).

[0134] In certain embodiments, the MHCII-binding placeholder peptide is included in the expression construct for one of the MHCII chains, preferably the beta chain, and as a result, the placeholder peptide and digestible linker are encoded in a construct that is operable with a coding sequence for the MHCII chain upstream (N-terminus). For example, the expression construct may encode (from N-terminus to C-terminus): the placeholder peptide, the digestible linker, the MHCII chain (e.g., the beta chain), and the C-terminal tag (e.g., encoding the amino acid sequence shown in SEQ ID NO: 192). In certain embodiments, the N-terminal tag is also added upstream of the placeholder peptide, which allows for the removal of non-exchangeable peptide species after peptide exchange. Non-limiting examples of such N-terminal tags include the FLAG tag (e.g., having the amino acid sequence shown in SEQ ID NO: 267), the Strep tag (e.g., having the amino acid sequence shown in SEQ ID NO: 268), and the Protein C tag (e.g., having the amino acid sequence shown in SEQ ID NO: 269).

[0135] In some embodiments, the pMHCII multimer described herein comprises the α1 and α2 domains of the MHCII alpha chain, and the β1 and β2 domains of the MHCII beta chain. In some embodiments, the multimer described herein comprises only the α1 and β1 domains of the MHCII heavy chain. In other embodiments, the pMHCII multimer comprises the alpha and beta chains combined with a peptide. Other embodiments include an MHCII molecule consisting only of the alpha and beta chains (so-called "empty" MHC II without a peptide loaded), a shortened alpha chain (e.g., α1 domain) combined with an empty or full-length beta chain loaded with a peptide, a shortened beta chain (e.g., β1 domain) combined with an empty or full-length alpha chain loaded with a peptide, or a shortened alpha chain (e.g., α1 and β1 domains) combined with an empty or shortened beta chain loaded with a peptide.

[0136] In some embodiments, the multimer protein comprises a soluble MHCII polypeptide. In some embodiments, the MHC multimer protein comprises a soluble MHCII lacking transmembrane and intracellular domains.

[0137] The amino acid sequences of numerous MHC class II proteins, including human MHCII, are publicly known in the art, and their genes have been cloned. Therefore, alpha and beta chain monomers can be expressed using recombinant methods. Methods for the expression and purification of MHCII molecules are widely described (e.g., Crawford et al. (1998) Immunity, 8:675-682; Novak et al. (1999) J. Clin. Invest., 104:R63-R67; Nepom et al. (2002) Arthrit. Rheum., 46:5-12; Day et al. (2003) J. Clin. Invest., 112:831-842; Vollers and Stern (2008) Immunol., 123:305-313; Cecconi et al. (2008) Cytometry, 73A:1010-1018, the full contents of each of these are incorporated herein by reference).

[0138] For MHC II molecules, the alpha and beta chains may be expressed as individual polypeptides in separate cells or as a fusion protein in the same cell. The peptides of the MHC II-peptide complex may be produced separately and added after purification of the entire MHC complex, or added during in vitro refolding, or expressed together with the alpha and / or beta chains linked to either chain via a linker. The genetic material may encode all or only fragments of the MHC class II alpha and beta chains. The genetic material may be fused with genes encoding other proteins, including proteins useful for purifying expressed polypeptide chains (e.g., purification tags), proteins useful for increasing / decreasing polypeptide solubility, proteins useful for detecting polypeptides, and proteins involved in coupling the MHC complex to the multimerization domain and / or to the labeled MHC complex and / or MHC multimers.

[0139] In contrast to the MHC I complex, the MHC II complex does not readily refold after denaturation in vitro. Only some MHC II alleles are expressed in E. coli and can be refolded in vitro. Therefore, preferred expression systems for the production of MHC II molecules are eukaryotic cell lines that do not require refolding after protein expression. Preferred expression systems include mammalian expression systems, such as CHO cells, HEK cells, or other mammalian cell lines suitable for human protein expression. Other expression systems include soluble Drosophila cell transfectants, baculovirus-infected insect cells, or other mammalian cell lines suitable for protein expression.

[0140] The stabilization of soluble MHC II complexes is even more critical than that of MHC I molecules, as both the alpha and beta chains are involved in the formation of peptide bond grooves and tend to dissociate if not embedded in the cell membrane. Therefore, in one embodiment, MHC II monomers are prepared by covalently linking a peptide to an MHC II molecule. For example, one technique is the covalent synthesis of single-stranded MHC class II chain-peptide complexes, directed by manipulating a peptide-specific cDNA sequence adjacent to the beta chain complementary DNA (cDNA) (as described in Crawford et al. (1999) Immunity, 8:675-682). In this strategy, the resulting polypeptide is refolded by a peptide sequence extended from the amino terminus of the class II molecule. The anchoring of the linker sequence in the peptide allows for sufficient flexibility for the peptide to occupy the peptide bond groove in the mature class II molecule. Cleavable linkers can be used to enable the cleavage of covalent links between peptides and MHCII molecules (as described, for example, Day et al. (2003) J. Clin. Invest., 112:831-842), thereby enabling peptide exchange with other peptides (e.g., libraries of different peptides) and loading of MHCII molecules.

[0141] Once expressed, the MHCII complex can be directly purified from MHCII-expressing cells as whole MHCII or MHCII peptide monomers. MHCII monomers may be expressed on the surface of cells and then isolated by disrupting the cell membrane, for example, using a surfactant, followed by purification of MHCII. In some embodiments, MHC monomers are expressed in the periplasm, and the expressing cells are lysed to release the purified MHCII monomers. Alternatively, MHC monomers may be purified from the supernatant of cells that secrete the expressed protein into the culture supernatant. Methods for purifying MHCII monomers are well known in the art and include, for example, the use of other techniques including affinity tags, ant-tag coated beads, and / or immobilization of MHCII proteins on an affinity matrix in conjunction with affinity chromatography, size exclusion chromatography using gel filtration, ion exchange, or other methods that can separate MHC molecules from cells and / or cell lysates.

[0142] In some embodiments, recombinant expression of MHCII polypeptides allows for the modification of many MHC monomers. For example, recombinant techniques provide a method for carboxyl terminator shortening, which involves the deletion of a hydrophobic transmembrane domain. The carboxyl terminator can also be optionally selected to facilitate ligand or label conjugation by introducing, for example, cysteine ​​and / or lysine residues into the molecule. The synthetic gene typically contains restriction sites to facilitate insertion into an expression vector and manipulation of the gene sequence. The gene encoding the appropriate monomer is then inserted into an expression vector and expressed in a suitable host, e.g., E. coli, yeast, insects, or other suitable cells, to obtain the recombinant protein.

[0143] In some embodiments, MHCII monomers are biotinylated in either their alpha or beta chain. In some embodiments, MHCII monomers are biotinylated before peptide loading, either by refolding or peptide exchange. Biotinylation of MHC monomers can be achieved, as is known in the art, by attaching biotin to a specific attachment site, which is a recognition site of a biotinylating enzyme. In some embodiments, the biotinylating enzyme is BirA. In some embodiments, biotinylation is performed in vivo on the desired protein chain as a post-translational modification during protein expression. II. Placeholder Peptides A. MHC Class I Placeholder Peptide In the method provided herein, the MHCI monomer is loaded with a placeholder peptide to facilitate proper folding of the MHCI monomer, and the placeholder peptide loaded MHCI(p) is used before polymerization. * MHCI produces placeholder peptides. Examples of placeholder peptides and methods for inducing the folding of MHCI heavy chains and β2-microglobulin in vitro in the presence of placeholder peptides have been described in the art (e.g., Bakker et al., PNAS 105:3825-3830, 2008; Rodenko et al., Nat. Prot. 1: 1120-1132, 2006).

[0144] In some embodiments, the placeholder peptide is an HLA-A, HLA-B, or HLA-C peptide. In some embodiments, the placeholder peptide is an HLA-A1 peptide (e.g., A * 1:01 binding peptide). In some embodiments, the placeholder peptide is an HLA-A2 peptide (e.g., A * 02:01 Binding peptide, A * 02:02 Binding peptide, A * 02:06 binding peptide). In other embodiments, the placeholder peptide is an HLA-A3 peptide (e.g., A* 3:01 binding peptide), HLA-A11 peptide (e.g., A * 11:01 binding peptide), HLA-A23 peptide (e.g., A * 23:01 binding peptide), HLA-A24 peptide (for example, A * 24:02 binding peptide), HLA-A26 peptide (e.g., A * 26:01 binding peptide), HLA-A29 peptide (e.g., A * 29:02 binding peptide), HLA-A30 peptide (e.g., A * 30:01 Binding peptide; A * 30:02 binding peptide), HLA-A31 peptide (e.g., A * 31:01 binding peptide), HLA-A32 peptide (e.g., A * 32:01 binding peptide), HLA-A33 peptide (e.g., A * 33:01 Binding peptide; A * 33:03 binding peptide), HLA-A68 peptide (e.g., A * 68:02 binding peptide), HLA-B7 peptide (e.g., B * 07:02 binding peptide), HLA-B8 peptide (e.g., B * 08:01 binding peptide), HLA-B15 peptide (e.g., B * 15:01 Binding peptide; B * 15:03 binding peptide), HLA-B18 peptide (e.g., B * 18:01 binding peptide), HLA-B35 peptide (e.g., B * 35:01 binding peptide), HLA-B38 peptide (e.g., B * 38:01 binding peptide), HLA-B40 peptide (e.g., B * 40:01 Binding peptide; B * 40:02 binding peptide), HLA-B45 peptide (e.g., B * 45:01 binding peptide), HLA-B51 peptide (e.g., B * 51:01 binding peptide), HLA-B53 peptide (e.g., B *53:01 binding peptide), HLA-B58 peptide (e.g., B * 58:01 binding peptide), HLA-C3 peptide (e.g., C * 03:03 Binding peptide; C * 03:04 binding peptide), HLA-C4 peptide (e.g., C * 04:01 binding peptide), HLA-C7 peptide (e.g., C * 07:01 Binding peptide; C * 07:02 binding peptide), or HLA-C8 peptide (e.g., C * 08:01 is a binding peptide. In some embodiments, the placeholder peptide is a synthetic peptide.

[0145] In some embodiments, the affinity of the placeholder peptide to the MHCI binding groove is lower than that of the rescue peptide. In some embodiments, the affinity of the placeholder peptide to the MHCI binding groove is about 10 times lower than that of the rescue peptide. In some embodiments, the affinity of the placeholder peptide to the MHCI binding groove is higher than that of the rescue peptide; however, the placeholder peptide can still be replaced by the rescue peptide by using an excess concentration of the rescue peptide.

[0146] In some embodiments, the placeholder peptide is thermally unstable. In some embodiments, the placeholder peptide is thermally unstable at temperatures between approximately 30 and 37°C. In some embodiments, the placeholder peptide is unstable at temperatures of 30°C or above, 32°C or above, 34°C or above, 35°C or above, 36°C or above, or approximately 37°C. Thermally unstable placeholder peptides and methods for identifying and producing them are described (e.g., WO93 / 10220; WO2005 / 047902; US2008 / 0206789; Luimstra et al., Curr. Protoc. Immunol. 126(1):e85, 2019; Luimstra et al., J. Exp. Med. 215(5):1493-1504, 2018).

[0147] In some embodiments, the placeholder peptide is unstable at acidic pH. In some embodiments, the placeholder peptide is unstable at pH approximately 2.5 to 6.5. In some embodiments, the placeholder peptide is unstable at pH approximately 2.5 to 6.0, 3.0 to 6.0, 3.0 to 6.5, 3.5 to 6.0, 3.5 to 6.5, 4.0 to 6.0, 4.0 to 6.5, 4.5 to 6.0, 4.5 to 6.5, 5.0 to 6.0, 5.0 to 6.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, the placeholder peptide is unstable at basic pH. In some embodiments, the placeholder peptide is unstable between pH approximately 9 and 11. In some embodiments, the placeholder peptide is unstable at pH 9 or above, pH 9.5 or above, pH 10 or about pH 10, pH 10.5 or about pH 10.5, or pH 11 or about pH 11. Methods for generating and using pH-sensitive placeholder peptides are publicly available, for example, as described in WO93 / 10220; US2008 / 0206789; and Cameron et al., J. Immunol. Meth. 268:51-59.

[0148] In some embodiments, the placeholder peptide includes a cleavable portion. Various types of cleavable portions are known in the art, including, for example, portions that can be cleaved by light irradiation, enzymes, nucleophiles or electrophiles, reducing agents and oxidizing agents (see, for example, Leriche et al., Biorg. Med. Chem. 20(2):571-582, 2012).

[0149] In some embodiments, the cleavable placeholder peptide comprises one or more photocleavable non-native β-amino acids. In some embodiments, the placeholder peptide comprises 3-amino-3-(2-nitrophenyl)-propionic acid. In some embodiments, the placeholder peptide comprises (2-nitro)phenylglycine. In some embodiments, the placeholder peptide comprises an azobenzene group. In some embodiments, the HLA-A2 placeholder peptide comprises A * 02:01, KILGFVFJV (SEQ ID NO: 15) or GILGFVFJL (SEQ ID NO: 7) (where J is 3-amino-3-(2-nitro)phenyl-propionic acid). In some embodiments, the placeholder peptides are A*01:01, STAPGJLEY (SEQ ID NO: 16); A*03:01, RIYRJGATR (SEQ ID NO: 17); A*11:01, RVFAJSFIK (SEQ ID NO: 18); A*24:02, VYGJVRACL (SEQ ID NO: 11); B*07:02, AARGJTLAM (SEQ ID NO: 14); B*35:01, KPIVVLJGY (SEQ ID NO: 19); C*03 :04, FVYGJSKTSL (sequence number 20), B*08:01, FLRGRAJGL (sequence number 21); C*07:02, VRIJHLYIL (sequence number 22); C*04:01, QYDJAVYKL (sequence number 23); B*15:01, ILGPJGSVY (sequence number 24); B*40:01, TEADVQJWL (sequence number 25); B*58:01, ISARGQJLF (sequence number 26); and C *08:01, selected from the group consisting of KAAJDLSHFL (SEQ ID NO: 27) (where J is 3-amino-3-(2-nitro)phenyl-propionic acid). In another embodiment, the placeholder peptide comprises the sequence shown in any one of SEQ ID NOs: 7-27 or 271-279. In another embodiment, the placeholder peptide consists of the sequence shown in any one of SEQ ID NOs: 7-27 or 271-279.

[0150] Methods for generating placeholder peptides containing photocleavable amino acids are known and previously described in the art (e.g., Toebes et al., Curr. Protoc. Immunol. 87:18.16.1-18.16.20, 2009; Bakker et al., see above; Rodenko et al., see above). In various embodiments, the photocleavable placeholder peptide is cleaved upon exposure to UV light using previously described methods (e.g., Toebes et al., Nat Med. 2006 Feb; 12(2):246-51; Bakker et al., Proc Natl Acad Sci US A. 2008 Mar 11; 105(10):3825-30; Rodenko et al., Nat Protoc. 2006; 1(3):1120-32; Frosig et al., Cytometry A. 2015 Oct; 87(10):967-75).

[0151] In some embodiments, the placeholder peptide comprises a chemoselective moiety. In some embodiments, the chemoselective moiety comprises a sodium dithionite-sensitive azobenzene linker, where azobenzene comprises at least one aromatic group containing an electron-donating group and is located between two amino acid residues. Methods for azobenzene linker and chemoselective peptide exchange are known in the art and are described, for example, in U.S. Patent No. 10,400,024.

[0152] In some embodiments, the placeholder peptide includes a cleavable portion that is cleaved upon exposure to an aminopeptidase. In some embodiments, cleavage of the amino acid residue occurs via the use of methionine aminopeptidase. Methionine aminopeptidase can cleave methionine from the peptide when the amino acid residue at position 2 is, for example, glycine, alanine, serine, cysteine, or proline. In some embodiments, the cleavable portion includes a thrombin cleavage domain.

[0153] In some embodiments, the placeholder peptide includes a cleavable moiety that is sensitive to a chemical trigger. In some embodiments, the placeholder peptide includes a periodate-sensitive amino acid. In some embodiments, the periodate-sensitive amino acid includes a vicinal diol moiety. In some embodiments, the periodate-sensitive amino acid includes a vicinal amino alcohol. In some embodiments, the periodate-sensitive amino acid is a 1,2-amino alcohol-containing amino acid. In some embodiments, the periodate-sensitive amino acid is α,γ-diamino-β-hydroxybutanoic acid (DAHB). Methods for producing and using peptides containing periodate-sensitive amino acids are published and described, for example, in Rodenko et al. (J. Am. Chem. Soc. 131:12605-12313, 2009) and Amore et al. (ChemBioChem 14:123-131, 2013).

[0154] In some embodiments, the placeholder peptide is a dipeptide. In some embodiments, the dipeptide binds to the F pocket of the MHCI binding groove. In some embodiments, the second amino acid of the dipeptide is hydrophobic. In some embodiments, the dipeptide is selected from the group consisting of glycyl-leucine (GL), glycyl-valine (GV), glycyl-methionine (GM), glycyl-cyclohexylalanine (GCha), glycyl-homoleucine (GHle), and glycyl-phenylalanine (GF). Methods for producing and using dipeptides as placeholder peptides are published and are described, for example, in Saini et al. (PNAS 112:202-207, 2015).

[0155] In some embodiments, the placeholder peptide comprises GILGFVFJL (SEQ ID NO: 7). In other embodiments, the placeholder peptide comprises the sequence shown in any one of SEQ ID NOs: 8-27 or 271-279. In other embodiments, the placeholder peptide comprises the sequence shown in any one of SEQ ID NOs: 8-27 or 271-279.

[0156] In some embodiments, the placeholder peptide further includes a fluorescent label. In some embodiments, the fluorescent label is attached to a cysteine ​​residue in the placeholder peptide.

[0157] In some embodiments, p * The MHCI molecule is purified and stored to serve as a source of stock molecules that can be exchanged for the target peptide epitope upon exposure to the peptide exchange conditions described herein. B.MHC Class II Placeholder Peptide In the method provided herein, the MHCII monomer is loaded with a placeholder peptide to facilitate proper folding of the MHCII monomer, and the placeholder peptide loaded MHCII(p) is used before polymerization. *MHCII produces HLA-DR, HLA-DQ, HLA-DX, HLA-DO, HLA-DZ, or HLA-DP. In various embodiments, the placeholder peptide is a peptide that binds to HLA-DR, HLA-DQ, HLA-DX, HLA-DO, HLA-DZ, or HLA-DP. In some embodiments, the placeholder peptide is a synthetic peptide.

[0158] In some embodiments, the affinity of the placeholder peptide to the MHCII binding groove is lower than that of the rescue peptide. In some embodiments, the affinity of the placeholder peptide to the MHCII binding groove is about 10 times lower than that of the rescue peptide.

[0159] In some embodiments, the placeholder peptide is thermally unstable. In some embodiments, the placeholder peptide is thermally unstable at temperatures between approximately 30 and 37°C. In some embodiments, the placeholder peptide is unstable at temperatures of 30°C or above, 32°C or above, 34°C or above, 35°C or above, 36°C or above, or approximately 37°C. Thermally unstable placeholder peptides and methods for identifying and producing them are described (e.g., WO93 / 10220; WO2005 / 047902; US2008 / 0206789; Luimstra et al., Curr. Protoc. Immunol. 126(1):e85, 2019; Luimstra et al., J. Exp. Med. 215(5):1493-1504, 2018).

[0160] In some embodiments, the placeholder peptide is unstable at acidic pH. In some embodiments, the placeholder peptide is unstable at pH approximately 2.5 to 6.5. In some embodiments, the placeholder peptide is unstable at pH approximately 2.5 to 6.0, 3.0 to 6.0, 3.0 to 6.5, 3.5 to 6.0, 3.5 to 6.5, 4.0 to 6.0, 4.0 to 6.5, 4.5 to 6.0, 4.5 to 6.5, 5.0 to 6.0, 5.0 to 6.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, the placeholder peptide is unstable at basic pH. In some embodiments, the placeholder peptide is unstable between pH approximately 9 and 11. In some embodiments, the placeholder peptide is unstable at pH 9 or above, pH 9.5 or above, pH 10 or about pH 10, pH 10.5 or about pH 10.5, or pH 11 or about pH 11. Methods for generating and using pH-sensitive placeholder peptides are publicly available, for example, as described in WO93 / 10220; US2008 / 0206789; and Cameron et al., J. Immunol. Meth. 268:51-59.

[0161] In some embodiments, the placeholder peptide includes a cleavable portion. Various types of cleavable portions are known in the art, including, for example, portions that can be cleaved by light irradiation, enzymes, nucleophiles or electrophiles, reducing agents and oxidizing agents (see, for example, Leriche et al., Biorg. Med. Chem. 20(2):571-582, 2012).

[0162] In one embodiment, a placeholder peptide is fused to a degradation tag, and peptide exchange is facilitated by proteolysis in the presence of a corresponding protease (which digests the degradation tag) along with the presence of a rescue peptide.

[0163] In some embodiments, the cleavable placeholder peptide is a photocleavable peptide, which is cleaved, for example, upon exposure to UV light. For example, the placeholder peptide may contain one or more photocleavable non-natural amino acids. For example, an MHCII-binding photocleavable peptide incorporating the UV-sensitive amino acid analog 3-amino-3-(2-nitrophenyl)-propionate has been described (see, for example, Negroni and Stern (2018) PLos One, 13(7):e0199704).

[0164] In one embodiment, the MHCII placeholder peptide is a CLIP peptide, for example, having the amino acid sequence KPVSKMRMATPLLMQA (SEQ ID NO: 189) or ATPLLMQALPMGA (SEQ ID NO: 280). In one embodiment, the CLIP peptide is cleavable. In one embodiment, the MHCII monomer is synthesized using a cleavable CLIP peptide, which is covalently attached, for example, by the synthesis of a single-stranded MHC class II chain-peptide complex and directed by manipulating the peptide-specific complementary DNA (cDNA) sequence proximal to the beta chain cDNA (see, for example, Day et al. (2003) J. Clin. Invest., 112:831-842). Cleavage of the covalent linkage between the CLIP peptide (as a placeholder peptide) and MHCII thus enables peptide exchange with other MHCII-binding peptides.

[0165] Other MHCII-binding peptides that can be used as placeholder peptides, based on the appropriate pairing of MHCII molecules and known MHCII-binding peptides, are described in the Art. Non-limiting examples of known MHCII molecule / MHCII-binding peptide pairs include DRA1 * 0101 / DRB1 * 0401 and HA of hemagglutinins 307~319The immunodominant peptide (see Novak et al. (1999) J. Clin. Invest., 104:R63-R67), as well as HLA-DR * Tetanus toxoid (TT)-derived p2 peptide (TT) having amino acid sequence 1101 and QIYKANSKFIGITEL (SEQ ID NO: 190) 830~844 (See Cecconi et al. (2008) Cytometry, 73A:1010-1018.) III.p * Production of MHC multimers The multimerization domains provided herein for use in the production of pMHC multimers are two or more pMHCs or p * The multimer comprises a protein, polypeptide, or other multimerized portion, which is suitable for covalent conjugates of MHC monomers and does not interfere with the binding of pMHC polypeptides to cells. In some embodiments, the multimerizing domain comprises a protein subunit. In some embodiments, the multimerizing domain is a homomultimer of protein subunits. In some embodiments, the multimerizing domain is a heteromultimer of protein subunits. In one embodiment, the multimer is a dimer, trimer, tetramer, pentamer, hexamer, octamer, decamer, or dodecamer. In one preferred embodiment, the pMHC multimer is a tetramer.

[0166] Examples of suitable binding entities include streptavidin (SA) and avidin and their derivatives, biotin, immunoglobulins, antibodies (monoclonal, polyclonal, and recombinant), their antibody fragments and derivatives, the leucine zipper domain of AP-1 (jun and fos), hexa-his (metal chelate moiety), and hexa-hat. GST (Glutathione S-Transferase) glutathione affinity, calmodulin-binding peptide (CBP), Strep-tag (registered trademark), cellulose-binding domain, maltose-binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, VSV epitope, lecithin mediating binding to a variety of compounds including carbohydrates, lipids, and proteins, such as Con A (Canavaliaensi formis) or WGA (Wheat Germ Agglutinin), as well as tetranectin, or protein A or G (antibody affinity), or coiled-coil polypeptide, such as leucine zipper. Combinations of such binding entities are also included.

[0167] In some embodiments, the multimerizing domain is a tetramer of streptavidin (SA or SAv) or a derivative thereof. In some embodiments, the multimerizing domain is tetrameric streptavidin. In some embodiments, the tetramer comprises Strep-tag® or Strep-tactin®. Strep-tag® and Strep-tactin® are described in U.S. Patent No. 5,506,121 and U.S. Patent No. 6,103,493, respectively, and are commercially available from several sources. To non-covalently attach the MHC monomer to streptavidin via the biotin-binding site of SAv, avitag (e.g., having the amino acid sequence shown in SEQ ID NO: 161, which includes the 6×His tag and FLAG tag) can be incorporated into the MHC monomer, for example, at the C-terminus (see, for example, Example 3).

[0168] In the method provided herein, the pMHC multimer is formed by adding each p to the N or C terminus of each subunit of the multimerization domain. * The reaction product is produced by the covalent conjugation of MHC monomers and is referred to herein as a conjugated polymer. In one embodiment, the conjugated polymer is a pMHC class I (pMHCI) conjugated polymer. In another embodiment, the conjugated polymer is a pMHC class II (pMHCII) conjugated polymer.

[0169] In some embodiments, pMHCI multimers are produced by covalent conjugation of a multimerizing domain to the C-terminus of the MHCI α1 domain. In some embodiments, pMHCI multimers are produced by covalent conjugation of a multimerizing domain to the C-terminus of the MHCI α2 domain. In some embodiments, pMHCI multimers are produced by covalent conjugation of a multimerizing domain to the C-terminus of the MHCI α3 domain. In some embodiments, pMHCI multimers are produced by each p *It is produced by the covalent conjugation of a multimerization domain to the C-terminus of β2-microglobulin, a MHC monomer.

[0170] In a preferred embodiment, the pMHCII multimer is produced by covalent conjugation of the multimerizing domain to the MHCII α chain. In another embodiment, the pMHCII multimer is produced by covalent conjugation of the multimerizing domain to the MHCII β chain. In a particular embodiment, the pMHCII multimer is produced by covalent conjugation of the multimerizing domain to the C-terminus of the MHCII α1 domain. In a particular embodiment, the pMHCII multimer is produced by covalent conjugation of the multimerizing domain to the C-terminus of the MHCII α2 domain. In a particular embodiment, the pMHCII multimer is produced by covalent conjugation of the multimerizing domain to the C-terminus of the MHCII β1 domain. In a particular embodiment, the pMHCII multimer is produced by covalent conjugation of the multimerizing domain to the C-terminus of the MHCII β2 domain.

[0171] Several preferred methods for forming covalent bonds between each MHC monomer and a polymerizing domain are provided herein. A. Chemical bioconjugation In some embodiments, p * MHC multimers are produced by chemical conjugation. In some embodiments, chemical conjugation involves p to the multimerization domain. *The bioconjugation of MHC polypeptides is mediated by cysteine ​​bioconjugation. In some embodiments, cysteine ​​bioconjugation is mediated by alkylation of cysteine. In some embodiments, cysteine ​​bioconjugation is mediated by oxidation of cysteine. In other embodiments, cysteine ​​bioconjugation is mediated by a desulfurization reaction. In some embodiments, cysteine ​​bioconjugation is mediated by iodoacetamide. In some embodiments, cysteine ​​bioconjugation is mediated by maleimide. Methods for utilizing cysteine-mediated linkage of two parts that can be used to produce the pMHC polymers disclosed herein are described; see, for example, Chalker et al., Chem Asian J.4(5):630-40, 2009; Spicer et al., Nat Commun. 5:4740, 2015.

[0172] In some embodiments, MHC polymers are produced by chemical modifications of amino acids other than cysteine, including, but not limited to, lysine, tyrosine, arginine, glutamic acid, aspartic acid, serine, threonine, methionine, histidine, and tryptophan side chains, as well as N-terminal amines or C-terminal carboxyls, as previously described (Basle et al., M Chem Biol. 17(3):213-27, 2010; Hu et al., Chem Soc Rev. 45(6):1691-719, 2016; Lin et al., Science 355(6325):597-602, 2017). B. Natural chemical ligation In some embodiments, pMHC polymers are produced by natural chemical ligation (NCL), where each p * MHC polypeptides contain a C-terminal thioester, and each subunit of the polymerizing domain contains an N-terminal cysteine ​​residue or a functional equivalent thereof, and the reaction between the cysteine ​​side chain and the thioester irreversibly forms a native peptide bond, thus p* MHC monomers are ligated into the polymerizing domain. Methods for NCLs have been described (Hejjaoui et al., M Protein Sci. 24(7):1087-99. 2015; Mandal et al., Proc Natl Acad Sci USA 109(37):14779-84, 2012; Torbeev et al., Proc Natl Acad Sci USA 110(50):20051-6, 2013).

[0173] In some embodiments, β- and / or γ-thioamino acids are p * It is incorporated into the MHC monomer. In some embodiments, β- and / or γ-thioamino acids replace cysteine-like residues at the N-terminal position of each subunit of the polymerizing domain to provide, for example, a reactive thiol for trans-thioesterification. A desulfurization protocol can then be used to produce the desired native side chain. In some embodiments, NCL is performed at an alanine residue. In other embodiments, NCL is phenylalanine (Crich & Banerjee, 2007), valine (Chen et al. 2008; Haase et al. 2008), leucine (Harpaz et al. 2010; Tan et al. 2010), threonine (Chen et al. 2010b), lysine (El Oualid et al. 2010; Kumar et al. 2009; Yang et al. 2009), proline (Shang et al. 2011), glutamine (Siman et al. 2012), arginine (Malins et al. 2013), tryptophan (Malins et al. 2014), aspartic acid (Thompson et al. 2013), glutamic acid (Cergol et al. This is done with asparagine (2014) and asparagine (Sayers et al. 2015). Ligation / desulfurization methods that eliminate the purification step and increase the yield of the ligated product have been described (Moyal et al. 2013; Thompson et al. 2014). C. Crick chemistry-mediated bioorthogonal conjugation In some embodiments, p * MHC multimers contain each p * This is produced by bioorthogonal conjugation between the C-terminal conjugation moiety of the MHC monomer and the N-terminal conjugation moiety of each subunit of the polymerizing domain. In some embodiments, bioorthogonal conjugation is mediated by "click chemistry" (see, e.g., Kolb, Finn and Sharpless, Angewandte Chemie International Edition (2001) 40: 2004-2021). Suitable conjugation moieties, reaction conditions, and related methods for click chemistry are available in the art (e.g., Kolb et al., Angewandte Chemie International Edition 40:2004-2021, 2001; Evans, Australian Journal of Chemistry 60: 384-395, 2007; Lahann, Click Chemistry for Biotechnology and Materials Science, John Wiley & Sons Ltd, ISBN 978-0-470-69970-6, 2009). In some embodiments, the click chemistry moiety may include, or consist of, terminal alkynes, azides, strained alkynes, dienes, dienophiles, alkoxyamines, carbonyls, phosphines, hydrazides, thiols, or alkene moieties. In certain embodiments, the azide is a copper chelated azide. In one embodiment, the copper chelated azide is a picolyl azide, for example, Gly-Gly-Gly-(PEG)4-picolyl azide. Reagents for use in click chemistry reactions are commercially available, for example, from Click Chemistry Tools (Scottsdale, AZ) or GenScript (Piscataway, NJ).

[0174] Each p of the polymerizing domain to a subunit via click chemistry * For the conjugation of MHC monomers, the click chemistry moiety of the protein is, for example, each p * The click chemistry moieties of the MHC monomers must be reactive with each other in such a way that one reactive group on the click chemistry moiety reacts with a reactive group on a second click chemistry moiety on a subunit of the polymerizing domain to form a covalent bond. Such reactive pairs of click chemistry handles are well known to those skilled in the art, but are not limited to those shown in Figure 1.

[0175] In some embodiments, each p * The MHC conjugation moiety can be covalently conjugated to the conjugation moiety of each subunit of the polymerization domain under click chemistry reaction conditions. In some embodiments, each p * Saltase-mediated conjugation is used to install the reaction of the second click chemistry moiety into each subunit of the C-terminal first click chemistry moiety and the polymerization domain of the MHC monomer. In the method provided herein, two or more p containing the first click chemistry moiety are used. * MHC monomers are conjugated under click chemistry conditions to a second click chemistry moiety at the C-terminus of each subunit of the polymerizing domain. A method for attaching the click chemistry moiety using saltase is described, for example, in WO2013 / 00355, the full contents of which are incorporated herein by reference. Non-limiting examples of pMHC polymers prepared using alkyne-azido click chemistry in combination with saltase-mediated conjugation are described in detail in Examples 1, 5, 6, and 7.

[0176] In some embodiments, each p *Intein-mediated conjugation is used to install the reaction of the second click chemistry moiety into the first click chemistry moiety at the C-terminus of the MHC monomer and into each subunit of the polymerization domain. Methods utilizing intein-mediated conjugation are further described herein.

[0177] In some embodiments, p to the polymerizing domain provided herein * Methods for click chemistry-mediated covalent conjugation of MHC monomers include the natural chemical ligation of a C-terminal thioester with a β-aminothiol (Xiao J, Tolbert TJ Org Lett. 2009 Sep 17; 11(18):4144-7).

[0178] In some embodiments, p * Click chemistry used to produce MHC polymers includes 1,3-dipolar cycloaddition (e.g., stepwise variants of copper(I) catalysts, often simply referred to as "click reactions"; see, e.g., Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057-3064). Copper and ruthenium are commonly used catalysts in the reaction. The use of copper as a catalyst results in the formation of 1,4-positional isomers, while ruthenium results in the formation of 1,5-positional isomers.

[0179] In some embodiments, MHC monomers are ligated to alkyned peptides by expressed protein ligation (EPL) and then conjugated to azide-labeled polymerization domains by copper(I)-catalyzed azide-alkyne cyclization (CuAAC).

[0180] In some embodiments, click chemistry conjugation involves cycloaddition reactions such as the Diels-Alder reaction. In some embodiments, the MHCI and polymerizing domains are conjugated by azide-alkyne 1,3-dipolar cycloaddition ("click chemistry"). In some embodiments, the cycloaddition is facilitated by the presence of copper(I) catalyzed cycloaddition (CuAAC).

[0181] In some embodiments, click chemistry conjugation involves nucleophilic addition to small, distorted rings such as epoxides and aziridines. In some embodiments, cyclization is facilitated by a distorted cyclooctin system, as described, for example, Agard NJ, Prescher JA, Bertozzi CR J Am Chem Soc. 2004 Nov 24; 126(46):15046-7.

[0182] In some embodiments, click chemistry conjugation involves nucleophilic addition to an activated carbonyl group.

[0183] In some embodiments, the conjugation of the pMHC monomer and the polymerizing domain occurs by a bioorthogonal reaction. In some embodiments, the MHC and polymerizing domain are conjugated by a reverse electron-demanding Diels-Alder reaction between a strained dienophile and a tetrazindiene, as described, for example, Blackman ML, Royzen M, Fox JM J Am Chem Soc. 2008 Oct 15; 130(41):13518-9; and Devaraj NK, Weissleder R, Hilderbrand SA Bioconjug Chem. 2008 Dec; 19(12):2297-9. In some embodiments, the dienophile is trans-cyclooctene. In some embodiments, the dienophile is norbornene. D. saltase-mediated conjugation In some embodiments, p *Conjugation between MHC monomers and polymerizing domains is mediated by cysteine ​​transpeptidases. In some embodiments, the cysteine ​​transpeptidase is a saltase or an enzymatically active fragment thereof. Various saltase enzymes have been described and are commercially available (e.g., Antos et al., Curr. Opin. Struct. Biol. 38:111-118, 2016). Saltase recognizes and cleaves an amino acid motif referred to as "sortag" to create a peptide bond between acyl donor and acceptor sites on two polypeptides, resulting in ligation of different polypeptides containing N- or C-terminal sortag. Non-limiting examples of pMHC polymers prepared using saltase-mediated conjugation (in combination with alkyne-azidocric chemistry) are described in detail in Examples 1, 5, 6, and 7.

[0184] Therefore, in some embodiments, each p * The MHC monomer contains a C-terminal sortag, and each subunit of the polymerizing domain contains an N-terminal sortag. In other embodiments, each p * The MHC monomer contains an N-terminal sortag, and each subunit of the polymerizing domain contains a C-terminal sortag. In some embodiments, the saltase catalyzes the formation of peptide bonds between the MHC polypeptide and each of the subunits of the polymerizing domain.

[0185] In some embodiments, the recognition motif is attached to the C-terminus of each pMHC monomer, and the oligo-glycine motif is attached to the N-terminus of each subunit of the polymerizing domain. Upon addition of saltase to the mixture of MHC monomers and polymerizing domains, the polypeptides are covalently linked through innate peptide bonds to produce pMHC polymers.

[0186] In some embodiments, MHC monomers and / or multimerizing domains are expressed in frame along with sortag. In some embodiments, additional tags may include, for example, a 6×-His tag (Sinisi et al. Bioconjug. Chem 23:1119-1126, 2012), a nucleophilic fluorescent dye (Nair et al. Immun. Inflamm. Dis. 1:3-13, 2013), and / or a FLAG tag (Greineder et al. Bioconjug. Chem. 29:56-66, 2018).

[0187] In some embodiments, sortag contains modified amino acids suitable for chemical conjugation between MHC monomers and polymerizing domains. In some embodiments, sortag contains a C-terminal azidrisine residue capable of directing the click-click chemistry conjugation described herein.

[0188] In some embodiments, the MHC polypeptide and / or polymerizing domain includes a linker between the polypeptide and sortag. In some embodiments, each subunit of each MHC polypeptide and polymerizing domain includes sortag together with the linker. Preferred linkers are described, for example, in Greineder et al., Bioconjug. Chem. 29:56-66, 2018. In some embodiments, the linker is a semi-rigid linker. In some embodiments, the linker includes (SSSSG)2SAA (SEQ ID NO: 182). In some embodiments, the linker includes (G)5 (SEQ ID NO: 183).

[0189] In some embodiments, sortag contains fluorophore-modified lysine residues to facilitate the measurement of reaction progress and efficiency.

[0190] In some embodiments, the saltase is Ca2+-dependent. In some embodiments, the saltase is Ca2+-independent.

[0191] In some embodiments, the sortag-labeled MHC molecule is a soluble HLA-A2 (HLA-A) molecule having a C-terminal sortag and a 6×His tag, for example, having the amino acid sequence shown in SEQ ID NO: 1. * 02:01) In some embodiments, the sortag-labeled polymerizing domain is a streptavidin molecule having a C-terminal sortag and a 6×His tag, for example, having the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the sortag label with the 6×His tag has the amino acid sequence shown in SEQ ID NO: 162. Various other sortag sequences are known in the art and are suitable for use in the preparation of the conjugated polymers of this disclosure, and non-limiting examples thereof are further described below.

[0192] In some embodiments, sortag comprises the amino acid sequence LPXTG (SEQ ID NO: 163) (where X is any amino acid), and the sortase cleaves between the threonine and glycine skeleton within the motif.

[0193] In some embodiments, the sortase recognizes sortag containing an amino acid sequence selected from IPKTG (SEQ ID NO: 164), MPXTG (SEQ ID NO: 165), LAETG (SEQ ID NO: 166), LPXAG (SEQ ID NO: 167), LPESG (SEQ ID NO: 168), LPELG (SEQ ID NO: 169), or LPEVG (SEQ ID NO: 170).

[0194] In some embodiments, the saltase is a SrtAstaph mutant. In some embodiments, the SrtAstaph mutant is F40, and the recognition motif is XPKTG (SEQ ID NO: 171) (Piotukh et al., J. Am. Chem. Soc. 2011 133:17536-17539). In some embodiments, the SrtAstaph mutant is F40, and the recognition motif is APKTG (SEQ ID NO: 172), DPKTG (SEQ ID NO: 173), or SPKTG (SEQ ID NO: 174).

[0195] In some embodiments, the SrtAstaph mutant is a SrtAstaph quintuple mutant, and the recognition motif is LPXTG (SEQ ID NO: 163) (where X is any amino acid), LPEXG (SEQ ID NO: 175) (where X is any amino acid), or LAETG (SEQ ID NO: 166). In some embodiments, the mutant is a SrtAstaph quintuple mutant, and the recognition motif is LPEAG (SEQ ID NO: 176), LPECG (SEQ ID NO: 177), or LPESG (SEQ ID NO: 168). In some embodiments, the SrtAstaph mutant is 2A-9, and the recognition motif is LAETG (SEQ ID NO: 166). In some embodiments, the SrtAstaph mutant is 4S-9, and the recognition motif is LPEXG (SEQ ID NO: 178) (where X = A, C, or S).

[0196] In some embodiments, the saltase is a soluble fragment of wild-type saltase. In some embodiments, the saltase is a soluble fragment of modified saltase A (Mao H, Hart SA, Schink A, Pollok BA, J Am Chem Soc. 2004 Mar 10; 126(9):2670-1 A).

[0197] In some embodiments, the saltase is a variant or homolog of S. aureus saltase A (Antos JM, Truttmann MC, Ploegh HL Curr Opin Struct Biol. 2016 Jun; 38:111-8; Dorr BM, Ham HO, An C, Chaikof EL, Liu DR Proc Natl Acad Sci US A. 2014 Sep 16; 111(37):13343-8; Glasgow JE, Salit ML, Cochran JR J Am Chem Soc. 2016 Jun 22; 138(24):7496-9).

[0198] Methods for conjugating sortag to proteins are also described (Matsumoto T, Furuta K, Tanaka T, Kondo A ACS Synth Biol. 2016 Nov 18; 5(11):1284-1289; Williams FP, Milbradt AG, Embrey KJ, Bobby R PLoS One. 2016; 11(4):e0154607; and Witte MD, Cragnolini JJ, Dougan SK, Yoder NC, Popp MW, Ploegh HL Proc Natl Acad Sci US A. 2012 Jul 24; 109(30):11993-8; Mao H, Hart SA, Schink A, Pollok BA J Am Chem Soc. 2004 Mar 10; 126(9):2670-1; Guimaraes CP, Witte MD, Theile CS, Bozkurt G, Kundrat L, Blom AE, Ploegh HL Nat Protoc. 2013 Sep; 8(9):1787-99 and Theile CS, Witte MD, Blom AE, Kundrat L, Ploegh HL, Guimaraes CP Nat Protoc. 2013 Sep; 8(9):1800-7).

[0199] In some embodiments, the aminoglycine peptide fragments generated by the saltase reaction are removed, for example, by dialysis or centrifugation while the reaction is progressing (Freiburger L, Sonntag M, Hennig J, Li J, Zou P, Sattler MJ Biomol NMR. 2015 Sep; 63(1):1-8). In some embodiments, affinity immobilization strategies or flow systems platforms are used for the selective removal of reaction components (Policarpo RL, Kang H, Liao X, Rabideau AE, Simon MD, Pentelute BL Angew Chem Int Ed Engl. 2014 Aug 25; 53(35):9203-8).

[0200] In some embodiments, the reaction equilibrium can be controlled by inactivation of the ligation product or byproduct. For example, in some embodiments, the reaction is controlled by ligation of the WTWTW (SEQ ID NO: 179) motif added to the donor and acceptor, as described in Yamamura Y, Hirakawa H, Yamaguchi S, Nagamune T Chem Commun (Camb). 2011 Apr 28; 47(16):4742-4. In other embodiments, the byproduct is inactivated by chemical modification of the glycine of the acyl donor, as described, for example, in Liu F, Luo EY, Flora DB, Mezo AR J Org Chem. 2014 Jan 17; 79(2):487-92; and Williamson DJ, Webb ME, Turnbull WB Nat Protoc. 2014 Feb; 9(2):253-62. E. Intein-mediated conjugation Inteins are naturally occurring subdomains of self-splicing proteins that can excise their own protein subdomains from a larger protein structure while simultaneously conjugating two original adjacent peptide regions ("extines") together to form a mature host protein. Intein-based methods for protein modification and ligation have been developed. Inteins are endogenous protein sequences that can catalyze protein splicing reactions, cleaving an intein sequence from a precursor protein and conjugating adjacent sequences (N or C extensions) via peptide bonds. Non-limiting examples of pMHC multimers prepared using intein-mediated conjugation are described in detail in Example 2.

[0201] As used herein, the term “split intein” refers to any intein in which one or more peptide bonds present between the N-terminal and C-terminal intein segments are broken so that the N-terminal and C-terminal intein segments become separate molecules that can be non-covalently reassembled or reconstructed into an intein that is functional for splicing or cleavage reactions. A split intein for use in the systems and methods disclosed herein can be derived using any catalytically active intein or its fragments. For example, in one embodiment, a split intein may be derived from a eukaryotic intein. In another embodiment, a split intein may be derived from a bacterial intein. In yet another embodiment, a split intein may be derived from an archaeal intein. Preferably, the split intein thus derived has only the amino acid sequence essential for catalyzing the splicing reaction.

[0202] As used herein, “N-terminal intein segment” refers to any intein sequence comprising an N-terminal amino acid sequence that, when combined with the corresponding C-terminal intein segment, is functional for splicing and / or cleavage reactions. Thus, an N-terminal intein segment also includes the sequence to be spliced ​​if splicing occurs. An N-terminal intein segment may include a sequence that is a modification of the N-terminal portion of a naturally occurring (native) intein sequence. For example, an N-terminal intein segment may include additional amino acid residues and / or mutated residues, provided that the inclusion of such additional and / or mutated residues does not render the intein non-functional for splicing or cleavage. Preferably, the inclusion of additional and / or mutated residues improves or enhances the splicing activity and / or regulatory properties of the intein. Non-intene residues may also be genetically fused to an intein segment to provide additional functionality, such as the ability to be affinity-purified or covalently immobilized.

[0203] As used herein, “C-terminal intein segment” refers to any intein sequence comprising a C-terminal amino acid sequence that, when combined with a corresponding N-terminal intein segment, is functional for splicing or cleavage reactions. In one embodiment, the C-terminal intein segment comprises a sequence to be spliced ​​if splicing occurs. In another embodiment, the C-terminal intein segment is cleaved from a peptide sequence fused to its C-terminus. The C-terminal intein segment may include a sequence that is a modification of the C-terminal portion of a naturally occurring (native) intein sequence. For example, the C-terminal intein segment may include additional amino acid residues and / or mutated residues, provided that the inclusion of such additional and / or mutated residues does not render the C-terminal intein segment non-functional for splicing or cleavage.

[0204] Expressed protein ligation (EPL) refers to the innate chemical ligation between a recombinant protein having a C-terminal thioester and a second drug having an N-terminal cysteine. The C-terminal thioester can be readily introduced into any recombinant protein (i.e., target ligand) by the use of autoprocessing, also known as intein (intervening protein)-mediated protein splicing. The intein is a protein that can cleave itself from a larger precursor polypeptide chain by utilizing a process that results in the formation of innate peptide bonds between adjacent extein (external protein) fragments. When the autoprocessing protein is cloned downstream of the target ligand, a thiol (e.g., 2-mercaptoethanesulfonic acid, MESNA) can be used to induce site-directed cleavage of the autoprocessing protein, resulting in the formation of a reactive thioester. The thioester then reacts with any drug having an N-terminal cysteine. EPL operates in a site-specific manner, and the reaction is known to be highly efficient when both functional groups are present at high concentrations (reviewed in Elias et al. Small 6:2460-2468).

[0205] Therefore, in some embodiments, MHC monomers are ligated to alkyned peptides by expressed protein ligation (EPL) and then conjugated to azide-labeled polymerizing domains by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).

[0206] In some embodiments, the MHC monomer is conjugated to a polymerizing domain by an intein peptide tag. In some embodiments, the MHC polypeptide contains a C-terminal thioester, and the polymerizing domain contains an N-extine fused to a modified intein lacking the ability to trans-esterify, with trans-esterification occurring by the addition of an exogenous thiol.

[0207] Many inteins have been described, and this list is not limited to them, but includes: MxeGyrA (Frutos et al. (2010); Southworth et al. (1999)); SspDnaE (Shah et al. (2012); Wu et al. (1998)); NpuDnaE (Shah et al. (2012); Vila-Perello et al. (2013)); AvaDnaE (David et al. (2015); Shah et al. (2012)); Cfa (Consensus DnaE Split Intein) (Stevens et al. (2016)); gp41-1 and gp41-8 (Carvajal-Vallejos et al. (2012)); NrdJ-1 (Carvajal-Vallejos et al. (2012)); IMPDH-1 (Carvajal-Vallejos et Examples include (al) and AceL-TerL (Thiel et al. (2014)). The properties and uses of these inteins are summarized in Table 1.

[0208] [Table 1]

[0209] In some embodiments, the intein is a 198-residue gyrase A intein (Mxe GyrA) derived from Mycobacterium xenopi (Southworth MW, Amaya K, Evans TC, Xu MQ, Perler FB Biotechniques. 1999 Jul; 27(1):110-4, 116, 118-20). In some embodiments, the intein is derived from the cyanobacterium Synechocystis sp. strain PCC6803(Ssp).

[0210] In some embodiments, the intein is a split intein pair. In some embodiments, the split intein pair is an orthogonal split intein pair (Carvajal-Vallejos P, Pallisse R, Mootz HD, Schmidt SR J Biol Chem. 2012 Aug 17; 287(34):28686-96; Shah NH, Vila-Perello M, Muir TW Angew Chem Int Ed Engl. 2011 Jul 11; 50(29):6511-5).

[0211] In some embodiments, the split intein pair is an artificial split intein pair that is short, approximately 6 or 11 residues (Appleby JH, Zhou K, Volkmann G, Liu XQ J Biol Chem. 2009 Mar 6; 284(10):6194-9; Ludwig C, Pfeiff M, Linne U, Mootz HD Angew Chem Int Ed Engl. 2006 Aug 4; 45(31):5218-21).

[0212] In some embodiments, the intein is DnaE intein. In some embodiments, the DnaE intein is derived from Nostoc punctiforme (Npu). In some embodiments, the intein is gp41-1 intein. In some embodiments, the intein is gp41-8 intein. In some embodiments, the intein is IMPDH-1 intein. In some embodiments, the intein is NrdJ intein.

[0213] In some embodiments, the split-intane pair is AceL-TerL (Thiel IV, Volkmann G, Pietrokovski S, Mootz HD Angew Chem Int Ed Engl. 2014 Jan 27; 53(5):1306-10).

[0214] In some embodiments, the intein includes a consensus split intein sequence (Cfa) (Stevens AJ, Brown ZZ, Shah NH, Sekar G, Cowburn D, Muir TW. Design of a split intein with exceptional protein splicing activity. Journal of the American Chemical Society. 2016;138(7):2162-2165).

[0215] Several protocols for intein-mediated conjugation are available, and exemplary methods are provided in Example 2 herein. Suitable intein sequences and protocols for use in protein conjugation are, for example, Stevens, et al. J. Am. Chem. Soc., 138, 2162-2165, 2016; Shah et al. J. Am. Chem. Soc., 134, 11338-11341, 2012; and Vila-Perello et al., J. Am. Chem. Soc., 135, 286-292, 2013; Batjargal S, Walters CR, Petersson EJ J Am Chem Soc. 2015 Feb 11; 137(5):1734-7; and Guan D, Ramirez M, Chen Z Biotechnol Bioeng. 2013 Sep; This is described in 110(9):2471-81, and the entire contents of each of these are incorporated herein by reference.

[0216] In some embodiments, the intein-labeled MHC molecule is a soluble HLA-A2 (HLA-A) molecule having an N-intine tag, for example, having the amino acid sequence shown in SEQ ID NO: 4. * 02:01) In some embodiments, the intein-labeled polymerizing domain is a streptavidin molecule having a C-intane tag and a FLAG tag, having, for example, the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the N-intane tag including the FLAG tag has the amino acid sequence shown in SEQ ID NO: 180. Various other N-intane and C-intane sequences are known in the art and are suitable for use in the preparation of the conjugated polymers of this disclosure, and non-limiting examples thereof are described in the references cited above. F. Additional Bioconjugation Methods In some embodiments, the conjugation of MHCs and polymerizing domains is enzymatically mediated. In some embodiments, the enzyme is a formylglycine-producing enzyme (FGE) (Wu P, Shui W, Carlson BL, Hu N, Rabuka D, Lee J, Bertozzi CR Proc Natl Acad Sci US A. 2009 Mar 3; 106(9):3000-5) that recognizes the CXPXR amino acid sequence motif and converts cysteine ​​residues to formylglycine, thus introducing an aldehyde functional group. This enzyme is then subjected to bioorthogonal transformations such as oximation and the hydrazino-Picte-Spengler reaction (Agarwal P, Kudirka R, Albers AE, Barfield RM, de Hart GW, Drake PM, Jones LC, Rabuka D Bioconjug Chem. 2013 Jun 19; 24(6):846-51; Dirksen A, Dawson PE Bioconjug Chem. 2008 Dec; 19(12):2543-8).

[0217] Site-specific bioconjugation strategies offer numerous possibilities for target protein modification. Among various enzyme-based conjugation protocols, formylglycinases allow for the post-translational introduction of the amino acid Cα-formylglycine (FGly) into recombinant proteins, starting from cysteine ​​or serine residues within a distinct consensus motif. Aldehyde-retaining FGly residues exhibit orthogonal reactivity to all other native amino acids and can therefore be used for site-specific labeling reactions in protein scaffolds (reviewed in Kruger et al., Biol Chem. 2019 Feb 25;400(3):289-297. doi: 10.1515 / hsz-2018-0358).

[0218] Formylglycine-producing enzyme (FGE) recognizes the pentapeptide consensus sequence CxPxR, which specifically oxidizes the cysteine ​​in this sequence to abnormal aldehyde-containing formylglycine. The FGE recognition sequence or aldehyde tag can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine ​​to formylglycine can be achieved by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can selectively react with α-nucleophiles to generate site-selectively modified bioconjugates (Rabuka et al. Nat Protoc. 2012 May 10; 7(6): 1052-1067).

[0219] In some embodiments, the enzyme is a lipoic acid ligase that modifies the lysine side chain within a 13-residue target sequence (Uttamapinant C, White KA, Baruah H, Thompson S, Fernandez-Suarez M, Puthenveetil S, Ting AY Proc Natl Acad Sci US A. 2010 Jun 15; 107(24):10914-9) to introduce bioorthogonal groups containing azides, aryl aldehydes and hydrazines, p-iodophenyl derivatives, norbornene, and trans-cyclooctene (reviewed in Debelouchina et al. Q. Rev Biophys. 2017; 50 e7. doi:10.1017 / S0033583517000021).

[0220] In other embodiments, the enzyme is biotin ligase, farnesyltransferase, transglutaminase, or N-myristoyltransferase (reviewed in Rashidian M, Dozier JK, Distefano MD Bioconjug Chem. 2013 Aug 21; 24(8):1277-94). G. peptide linker In another embodiment, p *MHC polymers contain peptide linkers. The term "peptide linker" refers to a linear amino acid chain of natural and / or synthetic origin. Linkers have the function of ensuring that conjugated polypeptides can perform their biological activities by enabling polypeptides to be properly folded and presented. Peptide linkers may contain repeating amino acid sequences or sequences of naturally occurring polypeptides. In some embodiments, peptide linkers have a length of 2 to 50 amino acids. In some embodiments, peptide linkers are 3 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, or 10 to 20 amino acids.

[0221] In some embodiments, the peptide linker is rich in glycine, glutamine, and / or serine residues. These residues are arranged in small repeating units of, for example, up to five amino acids. This small repeating unit may be repeated 1 to 5 times. Up to six additional any naturally occurring amino acids may be added to the amino and / or carboxyl termini of the multimeric unit. Other synthetic peptide linkers consist of a single amino acid, which is repeated 10 to 20 times and may contain up to six additional any naturally occurring amino acids at the amino and / or carboxyl termini. All peptide linkers may be encoded by nucleic acid molecules and therefore may be recombinantly expressed. Since the linker is a peptide itself, the polypeptide linked by the linker is connected to the linker via a peptide bond formed between two amino acids.

[0222] Suitable peptide linkers are well known in the art and are disclosed, for example, in US2010 / 0210511, US2010 / 0179094, and US2012 / 0094909, which are incorporated herein by reference in their entirety. Other linkers are provided, for example, in U.S. Patent No. 5,525,491; Alfthan et al., Protein Eng., 1995, 8:725-731; Shan et al., J. Immunol., 1999, 162:6589-6595; Newton et al., Biochemistry, 1996, 35:545-553; Megeed et al.; Biomacromolecules, 2006, 7:999-1004; and Perisic et al., Structure, 1994, 12:1217-1226, each of which is incorporated herein by reference in its entirety.

[0223] In some embodiments, the polypeptide linker is synthetic. As used herein, the term “synthetic” with respect to a polypeptide linker includes a peptide (or polypeptide) comprising an amino acid sequence (which may or may not be naturally present) in which a linear sequence of amino acids is linked to a sequence (which may or may not be naturally present) that is not actually linked in nature. For example, a polypeptide linker may be a modified form of a naturally present polypeptide (including mutations such as addition, substitution, or deletion, for example) or may comprise a naturally non-natural polypeptide comprising the initial amino acid sequence (which may or may not be naturally present). The polypeptide linker may be used, for example, to ensure that the binding moiety (TCR or MHC), the multimerization domain, and the Igg framework of each multimer fusion polypeptide are juxtaposed to ensure proper folding and formation of the functional multimer protein complex. Preferably, the polypeptide linker is relatively non-immunogenic among the monomeric subunits of the binding protein and does not inhibit any non-covalent association.

[0224] In some embodiments, the linker is a Gly-Ser polypeptide linker, i.e., a peptide composed of glycine and serine residues. One exemplary Gly-Ser polypeptide linker contains the amino acid sequence (Gly4Ser)n (where n=1 to 6) (SEQ ID NO: 181). In certain embodiments, n=1. In certain embodiments, n=2. In certain embodiments, n=3. In certain embodiments, n=4. In certain embodiments, n=5. In certain embodiments, n=6. Another exemplary Gly-Ser polypeptide linker contains the amino acid sequence Ser(Gly4Ser)n (where n=1 to 10) (SEQ ID NO: 184). In certain embodiments, n=1. In certain embodiments, n=2. In certain embodiments, n=3, i.e., Ser(Gly4Ser)3. In certain embodiments, n=4, i.e., Ser(Gly4Ser)4. In certain embodiments, n=5. In a particular embodiment, n=6. In a particular embodiment, n=7. In a particular embodiment, n=8. In a particular embodiment, n=9. In a particular embodiment, n=10.

[0225] Other exemplary linkers include the GS linker (i.e., (GS)n), the GGSG linker (i.e., (GGSG)n) (SEQ ID NO: 185), the GSAT linker (SEQ ID NO: 186), the SEG linker, and the GGS linker (i.e., (GGSGGS)n) (SEQ ID NO: 187) (where n is a positive integer (e.g., 1, 2, 3, 4, or 5)). Other suitable linkers for use in multimeric fusion proteins can be found using publicly available databases such as the Linker Database (ibi.vu.nl / programs / linkerdbwww). The Linker Database is a database of interdomain linkers in multifunctional enzymes that may act as linkers in novel multimeric fusion proteins (see, for example, George et al., Protein Engineering 2002;15:871-9).

[0226] Polypeptide linkers can be introduced into polypeptide sequences using techniques known in the art. Modifications can be confirmed by DNA sequencing analysis. Host cells can be transformed using plasmid DNA for stable production of the polypeptide. H. Additional peptide linkers and tags Suitable additional tags for use in the methods and compositions provided herein include, but are not limited to, affinity tags, which include small polypeptides that bind with high specificity to enzymes, protein domains, or a range of substrates such as carbohydrates, small biomolecules, metal chelates, and antibodies, enabling rapid and efficient purification of proteins. Solubility tags enhance the proper folding and solubility of proteins and are frequently used in tandem with affinity tags.

[0227] Small tags, including but not limited to 6×His, FLAG, Strep II, and calmodulin-binding peptide (CBP) tags, have the advantage of minimizing their effect on the structure, activity, and characteristics of MHC polypeptides (Zhao et al. J. Anal. Chem. 2013 581093).

[0228] In some embodiments, the tag is a FLAG tag. The FLAG tag is a hydrophilic octapeptide epitope tag that binds to several specific anti-FLAG monoclonal antibodies, such as M1, M2, and M5, which have different recognition and binding characteristics (Einhauer et al. J. Biochem. Biophys. 49:455-465, 2001; Hopp et al. Mol. Immunol. 33:601-608, 1996). The FLAG fusion protein can be recognized by the monoclonal antibody in a calcium-dependent (e.g., M2) or calcium-independent manner. In particular, the tag appended to the N-terminus of the fusion protein is required for immunoaffinity purification by the M1 monoclonal antibody, while M2 is site-insensitive. IV. MHC peptide epitopes A. Peptide epitope selection Various processes have been developed to identify novel MHC-binding peptides that may be T cell epitopes. Many experimental methods begin by constructing a duplicate library of peptide fragments derived from a given protein sequence by synthesizing n-mer amino acid sequences of a fixed length that cancel each other out along a fixed number of amino acids. The MHC binding properties and potential of each sequence for T cell activation can then be evaluated in several assays.

[0229] Existing MHC-binding peptides identified by the methods outlined above and other methods, such as crystallographic analysis of conformations and the distribution of charges in MHC binding grooves, yield binding motifs defined for the most common MHC alleles, establishing rules for what kinds of putative MHC-binding peptides can actually bind sufficiently to the MHC molecules of a given allele. These motifs have been translated into predictive computer algorithms for predicting peptide binding to MHC molecules, such as the SYFPEITHI algorithm (Rammensee H.-G., et al. (1995), Immunogenetics 41:178-228).

[0230] Protein sequences for desired antigens are analyzed for potential HLA-specific antigens using artificial neural network (ANN) and stabilized matrix (SMM) algorithms from SYFPEITHY (Rammensee et al. Immungenetics 50:213-219, 1999) and IEDB (Peters et al. PLoS Biol. 3:e91, 2005). Peptides are selected based on a predicted binding value of either >21 for SYFPEITHY, <6000 for ANN, or <600 for SMM. The selected peptides are synthesized.

[0231] Binding assays can be performed using previously described fluorescence polarization (FP) assays (e.g., Buchi et al. Biochemistry 43:14852-14863, 2004; Sette et al., Mol. Immunol. 31:813-822). To determine the peptide binding capacity, the inhibition percentage relative to the control can be determined in an FP competition assay using a placeholder peptide.

[0232] In some embodiments, the peptide bound to the pMHC polymer is derived from a peptide-unbiased library. In some embodiments, the peptide is a 9-mer. In some embodiments, the peptide bound to the pMHCI polymer is a 9-mer containing an HLA-A2 binding motif having important amino acids at positions 2 and 9, which may include isoleucine (I), valine (V), or leucine (L).

[0233] In some embodiments, the library includes all k-mer peptides produced by the transcription and translation of any polynucleotide sequence of interest, for example, by in silico production of transcription and translation products of both the forward and reverse strands of the genome or metagenomics in all six reading frames.

[0234] In some embodiments, the library of this disclosure includes all k-mer peptides that may be derived from the in silico translation of the exome of interest.

[0235] In some embodiments, the library of this disclosure includes all k-mer peptides that may be derived from the in silico translation of the transcriptome of interest.

[0236] In some embodiments, the library of this disclosure includes all k-mer peptides that may be derived from the proteome of interest.

[0237] In some embodiments, the library of this disclosure includes all k-mer peptides that may be derived from the in silico translation of the ORFeome of interest.

[0238] In some embodiments, an algorithm can be used to select peptides from a peptide library. For example, an algorithm can be used to predict which peptides are most likely to fold or dock in MHC / HLA-binding pockets, and peptides that exceed a certain threshold can be selected for inclusion in the library.

[0239] In some embodiments, the library of this disclosure comprises all peptides that may be derived from in silico transcription and translation or translation of a group of genomes, proteomes, transcriptomes, ORFeomes, or any combination thereof.

[0240] In some embodiments, the peptides are derived from in silico transcription and translation or translation of polynucleotide sequences from a group of samples, such as clinical samples from a patient population or a group of pathogen genomes.

[0241] In some embodiments, the peptides originate from different genomes, proteomes, transcriptomes, ORFeomes, or any combination thereof, where two or more genomes, proteomes, transcriptomes, ORFeomes, or combinations thereof are compared to identify differential sequences (e.g., sequences that are different between them). In some embodiments, the peptide sequences are identified by comparison with the tissue of interest. In some embodiments, the peptide sequences are identified by comparison with the cells of interest. In some embodiments, the peptide sequences are identified by comparison with diseased cells or tissues to healthy cells or tissues. In some embodiments, the diseased cells or tissues are cancer cells or tissues. In some embodiments, the diseased cells originate from an individual with an autoimmune disorder.

[0242] In some embodiments, the peptides are derived from homologous sequences of a genome, proteome, transcriptome, ORFeome, or any combination thereof, where two or more genomes, proteomes, transcriptomes, ORFeomes, or combinations thereof are compared to identify homologous sequences.

[0243] In some embodiments, the peptides are derived from mutations in all 9-mer peptides that can be generated from a single nucleotide mutation in a polynucleotide sequence encoding a sequence of interest, such as an antigen or epitope.

[0244] In some embodiments, the peptides in the duplicated peptide library include duplicated peptides from a template sequence (e.g., an in silico-translated genome), where duplicated peptides of a set length are offset by a defined number of residues.

[0245] In some embodiments, peptide selection includes prioritizing peptides based on their predicted binding affinity to a particular HLA type.

[0246] In some embodiments, the selection of peptides for the library of this disclosure is prioritized by HLA type or allele based on the prevalence in a population, for example, a human population.

[0247] In some embodiments, the library includes all k-mer peptides produced by in silico production of transcription and translation of any polynucleotide sequence of interest, e.g., both forward and reverse strand transcription and translation products of a genome or metagenome in all six reading frames. In some embodiments, the library of the disclosure includes all k-mer peptides that may be derived from in silico transcription and translation of mammalian genomes, e.g., mouse genome, human genome, patient genome, autoimmune patient genome, or cancer genome. In some embodiments, the library of the disclosure includes all k-mer peptides that may be derived from in silico transcription and translation of microbial genomes, e.g., bacterial genome, viral genome, protist genome, protist genome, yeast genome, archaeal genome, or bacteriophage genome. In some embodiments, the library of the disclosure includes all k-mer peptides that may be derived from in silico transcription and translation of pathogen genomes, e.g., bacterial pathogen genome, viral pathogen genome, fungal pathogen genome, opportunistic pathogen genome, conditional pathogen genome, or eukaryotic parasite genome. In some embodiments, the libraries of the Disclosure may be derived from a plant genome or a fungal genome. In some embodiments, the libraries of the Disclosure include k-mer peptides derived from in silico transcription and translation of a genome, where the genome is modified during in silico transcription and translation, for example, by in silico mutation to produce k-mer peptides containing mutations (e.g., substitutions, insertions, deletions).

[0248] In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from in silico translation of an exome of interest, e.g., mammalian exome, human exome, mouse exome, patient exome, autoimmune patient exome, cancer exome, viral exome, protist exome, protist exome, yeast exome, pathogen exome, eukaryotic parasite exome, plant exome, or fungal exome. In some embodiments, the library of the Disclosure comprises k-mer peptides that may be derived from in silico translation of an exome, where the exome is modified during in silico translation, e.g., mutated in silico to produce a k-mer peptide containing a mutation (e.g., substitution, insertion, deletion).

[0249] In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from in silico translation of a transcriptome of interest, such as a mammalian transcriptome, human transcriptome, mouse transcriptome, patient transcriptome, autoimmune patient transcriptome, cancer transcriptome, microbial transcriptome, bacterial transcriptome, viral transcriptome, protist transcriptome, protist transcriptome, yeast transcriptome, archaeal transcriptome, bacteriophage transcriptome, pathogen transcriptome, eukaryotic parasite transcriptome, plant transcriptome, fungal transcriptome, RNA sequencing-derived transcriptome, microbiome transcriptome, or metagenomic RNA sequencing-derived transcriptome. In some embodiments, the library of the Disclosure includes k-mer peptides derived from in silico translation of a transcriptome, where the transcriptome is modified during in silico translation, for example, mutated in silico to produce k-mer peptides containing mutations (e.g., substitutions, insertions, deletions).

[0250] In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from the proteome of interest, e.g., mammalian proteome, human proteome, mouse proteome, patient proteome, autoimmune patient proteome, cancer proteome, microbial proteome, bacterial proteome, viral proteome, protist proteome, protist proteome, yeast proteome, archaeal proteome, bacteriophage proteome, pathogen proteome, eukaryotic parasite proteome, plant proteome, or fungal proteome. In some embodiments, the library of the Disclosure comprises k-mer peptides derived from the proteome, where the k-mer peptide is a k-mer peptide that has been modified from the proteome sequence, e.g., through mutations (e.g., substitutions, insertions, deletions).

[0251] In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from in silico translation of an ORFeome of interest, such as a mammalian ORFeome, human ORFeome, mouse ORFeome, patient ORFeome, autoimmune patient ORFeome, cancer ORFeome, microbial ORFeome, bacterial ORFeome, viral ORFeome, protist ORFeome, protist ORFeome, yeast ORFeome, archaeal ORFeome, bacteriophage ORFeome, pathogen ORFeome, eukaryotic parasite ORFeome, plant ORFeome or fungal ORFeome, ORFeome derived from next-generation sequencing, microbiome ORFeome, or ORFeome derived from metagenomic sequencing. In some embodiments, the library of the Disclosure includes k-mer peptides derived from in silico translation of an ORFeome, where the ORFeome is modified during in silico translation, for example, mutated in silico to produce a k-mer peptide containing a mutation (e.g., substitution, insertion, deletion).

[0252] In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from in silico transcription and translation or translation of a group of genomes, proteomes, transcriptomes, ORFeomes, or any combination thereof. In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from in silico transcription and translation or translation of polynucleotide sequences from a group of samples, e.g., clinical samples from a patient population, or a group of pathogen genomes. In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from in silico transcription and translation of a group of viral genomes, e.g., human vilomes. In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from in silico transcription and translation of a group of genomes, proteomes, transcriptomes, ORFeomes, or any combination thereof, wherein the origin sequence is modified during in silico translation, e.g., mutated in silico to produce a k-mer peptide containing a mutation (e.g., substitution, insertion, deletion).

[0253] In some embodiments, the library of the Disclosure comprises all k-mer peptides which may originate from different genomes, proteomes, transcriptomes, ORFeomes, or any combination thereof, where two or more genomes, proteomes, transcriptomes, ORFeomes, or combinations thereof are difference sequences (e.g., different between them), which are compared to identify sequences that differ in, for example, nucleotide sequence, amino acid sequence, nucleotide abundance, or protein abundance. In some embodiments, difference sequences of genomes, proteomes, transcriptomes, or ORFeomes are generated by comparing the tissue of interest. In some embodiments, difference sequences of genomes, proteomes, transcriptomes, or ORFeomes are generated by comparing sequences derived from cells of interest (e.g., healthy cells versus cancer cells). In some embodiments, difference sequences of genomes, proteomes, transcriptomes, or ORFeomes are generated by comparing the sequences of the organism of interest. In some embodiments, differential sequences of a genome, proteome, transcriptome, or ORFeome may be generated by comparing the subject of interest (e.g., a diseased subject versus a healthy subject).

[0254] In some embodiments, the library of the Disclosure comprises all k-mer peptides that may originate from homologous sequences of a genome, proteome, transcriptome, ORFeome, or any combination thereof, where two or more genomes, proteomes, transcriptomes, ORFeomes, or combinations thereof are compared to identify homologous sequences (e.g., sequences sharing some degree of homology), such as homologous nucleotide sequences, homologous amino acid sequences, homologous nucleotide abundances, or homologous protein abundances. In some embodiments, homologous sequences of a genome, proteome, transcriptome, or ORFeome are generated by comparing them with a tissue of interest. In some embodiments, homologous sequences of a genome, proteome, transcriptome, or ORFeome are generated by comparing sequences from cells of interest (e.g., healthy cells versus cells involved in autoimmune cells, e.g., cells that induce autoimmunity or cells that are targeted during autoimmunity). In some embodiments, homologous sequences of a genome, proteome, transcriptome, or ORFeome are generated by comparing sequences from organisms of interest. In some embodiments, homologous sequences of a genome, proteome, transcriptome, or ORFeome are generated by comparing sequences from subjects of interest (e.g., diseased subjects versus healthy subjects).

[0255] In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from the polypeptide sequence of interest, e.g., all possible 9-mer peptides encompassing the complete protein sequence of a viral protein. In some embodiments, the library of the Disclosure includes k-mer peptides that can be generated from the polypeptide sequence of interest, where the polypeptide sequence of interest is modified, e.g., mutated in silico, to produce k-mer peptides containing mutations (e.g., substitutions, insertions, deletions).

[0256] In some embodiments, the library of the Disclosure includes all k-mer peptides that may originate from mutations in the sequence of interest, e.g., all 9-mer peptides that can be generated from a single nucleotide mutation in a polynucleotide sequence encoding an antigen or epitope. For example, the library of the Disclosure includes all 9-mer peptides that can be generated from mutations in 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides in a polynucleotide sequence encoding an antigen or epitope. In some embodiments, the library of the Disclosure includes all k-mer peptides that may originate from alanine substitutions, e.g., alanine substitutions at any position in any of the sequences described herein (e.g., proteins, a group of proteins, proteomes, in silico-transcribed and translated genomes). In some embodiments, the library of the Disclosure includes a site-scanning library, where selected amino acid residues are sequentially substituted with all other native amino acids. In some embodiments, the library of the Disclosure includes a combinatorial site-scanning library, where selected amino acid residues are sequentially substituted with all other native amino acids at two or more positions at a time. In some embodiments, the library of the Disclosure comprises a duplicate peptide library containing duplicate peptides from a template sequence (e.g., an in silicotranslated genome), wherein duplicate peptides of a set length are offset by a defined number of residues. In some embodiments, the library of the Disclosure comprises a T cell shortened peptide library, wherein each replica of the library contains an equimolar mixture of peptides having a shortening at one end (e.g., 8-mer, 9-mer, 10-mer, and 11-mer which can be derived from a C-terminal shortening of a nominal 11-mer). In some embodiments, the library of the Disclosure comprises a set of customized peptides, wherein the set of customized peptides is listed.

[0257] In some embodiments, the genome, exome, transcriptome, proteome, or ORFeome of the present disclosure is the genome, exome, transcriptome, proteome, or ORFeome of a virus. Non-limiting examples of viruses include adenovirus, adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, banana virus, vermaforest virus, bunyamwella virus, lacrosse bunyavirus, snowshoe rabbit bunyavirus, cercomoni herpesvirus, Chandipla virus, chikungunya virus, cosavirus A, cowpox virus, coxsackievirus, Crimean-Congo hemorrhagic fever virus, cytomegalovirus ( CMV), dengue virus, Dorivirus, Jugbe virus, Dobenhage virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus (EBV), European bat lyssavirus, GB virus C / G hepatitis virus, Hantan virus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis Delta virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronaviruses, human cytomegaloviruses, human endogenous retroviruses (HERVs), human enteroviruses, human herpesviruses (e.g., HHV-1, HHV-2, HHV-6A, HHV-6B, HHV-7, HHV-8), human immunodeficiency viruses (e.g., HIV-1, HIV-2), human papillomaviruses (e.g., HPV-1, HPV-2, HPV-16, HPV-18), human parainfluenza, human parvovirus B19, human polynuclear respiratory virus (RSV), human Rhinovirus, human SARS coronavirus, human spumaretrovirus, human T lymphotropic virus (HTLV, e.g., HTLV-1, HTLV-2, HTLV-3), human torovirus, influenza A virus, influenza B virus, influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunzin virus, Lagos bat virus, Lake Victoria Marburg virus,Langatt virus, Lassa virus, Rosedale virus, jumping disease virus, lymphocytic choriomeningitis virus, Machupo virus, Mayarovirus, MERS coronavirus, measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mocola virus, molluscum contagiosum virus, monkeypox virus, mumps virus, Marie Valley encephalitis virus, New York virus, Nipah virus, norovirus, Norwalk virus, Onyonnyon virus, Aarf virus, Oropushé virus, Pichinde virus, poliovirus, Puntatrophlevovirus, Pumara virus, rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, rotavirus (e.g., rotavirus) Examples include rotavirus A, rotavirus B, rotavirus C, rotavirus X, rubella virus, Sagiyama virus, Sarivirus A, Sicilian sandfly fever virus, Sapporo virus, Semlik Forest fever virus, Seoul virus, monkey foam virus, Simian virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, tick-borne Poissant virus, Torkutenovirus, Tuscany virus, Ukniemi virus, vaccinia virus, varicella-zoster virus, smallpox virus, Venezuelan horse encephalitis virus, vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yabasal tumor virus, Yabasal disease virus, yellow fever virus, and Zika virus.

[0258] In some embodiments, the genome, exome, transcriptome, proteome, or ORFeome of the Disclosure is the genome, exome, transcriptome, proteome, or ORFeome of a cancer. In some embodiments, the library of the Disclosure includes known cancer neoepitopes. In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from known cancer antigen proteins. In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from genes involved in epithelial-mesenchymal transition. In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from cancer-related genes. In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from mutagenic cancer driver genes. In some embodiments, the library of the Disclosure includes all k-mer peptides that may be derived from proto-oncogenes, oncogenes, or tumor suppressor genes. In some embodiments, the library of this disclosure comprises all k-mer peptides that may be derived from proto-oncogenes, oncogenes, or tumor suppressor genes, wherein the k-mer includes mutations described herein (e.g., amino acid substitutions, alanine substitutions, site scanning, combinatorial site scanning, etc.).

[0259] Non-specific examples of cancer include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, atypical malformation / rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, carcinoma of unknown primary origin, cardiac tumor, central nervous system cancer, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, and skin cancer. T-cell lymphoma, ductal carcinoma in situ, germ blastoma, endometrial cancer, epithelial carcinoma, ependymoma, esophageal cancer, sensory neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiocytoma of bone, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytic hyperplasia, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet tumor, Kaposi's sarcoma, renal (renal cell) carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip cancer and Oral cancer, liver cancer, lung cancer (non-small cell and small cell), lymphoma, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic cervical squamous cell carcinoma of unknown primary origin, midline cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, lip cancer and oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, papillomas, paragangliomas, para Nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma of the skin, cervical squamous cell carcinoma of unknown primary origin, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, ureteral and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, hemangioma, vulvar cancer,Wilms' tumor is another example.

[0260] In some embodiments, the genome, exome, transcriptome, proteome, or ORFeome of the Disclosure is an inflammatory or autoimmune genome, exome, transcriptome, proteome, or ORFeome. In some embodiments, the library of the Disclosure comprises known inflammatory or autoimmune neoepitopes or autoepitopes. In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from known inflammatory or autoimmune antigen proteins. In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from genes associated with inflammation or autoimmunity. In some embodiments, the library of the Disclosure comprises all k-mer peptides that may be derived from mutations in driver genes associated with inflammation or autoimmunity.

[0261] Non-exclusive examples of inflammatory diseases or conditions or autoimmune diseases or conditions include acute disseminated encephalomyelitis (ADEM); acute necrotizing hemorrhagic leukoencephalitis; Addison's disease; adjuvant-induced arthritis; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM / anti-TBM nephritis; antiphospholipid syndrome (APS); autoimmune angioedema; autoimmune aplastic anemia; autoimmune autonomic neuropathy; autoimmune gastric atrophy; autoimmune hemolytic anemia; autoimmune hepatitis; autoimmune Hyperlipidemia; autoimmune immunodeficiency; autoimmune inner ear disease (AIED); autoimmune myocarditis; autoimmune oophoritis; autoimmune pancreatitis; autoimmune retinopathy; autoimmune thrombocytopenic purpura (ATP); autoimmune thyroid disease; autoimmune urticaria; axonal and neurological neuropathy; Baro's disease; Behçet's disease; bullous pemphigoid; cardiomyopathy; Castleman disease; celiac disease; Chagas disease; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic recurrent multifocal osteomyelitis ostomyelitis (CRMO); Churg-Strauss syndrome; scarring pemphigoid / benign mucosal pemphigoid; Crohn's disease; Cogan's syndrome; collagen-induced arthritis; cold agglutinin disease; congenital heart block; coxsackie myocarditis; CREST disease; essential mixed cryoglobulinemia; demyelinating neuropathy; herpetiform dermatitis; dermatomyositis; Devic's disease (neuromyelitis optica); discoid lupus; Dressler syndrome; endometriosis; eosinophilic esophagitis; eosinophilic fasciitis; erythema nodosum; experimental allergic encephalomyelitis; experimental autoimmune encephalomyelitis; Evans syndrome; fibromyalgia; fibrotic alveolitis; giant cell arteritis (temporal arteritis); giant cell heart Myositis; glomerulonephritis; Goodpasture syndrome; granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis); Graves' disease; Guillain-Barré syndrome; Hashimoto's encephalitis; Hashimoto's thyroiditis; thyroiditis; hemolytic anemia; Henoch-Schönlein purpura; herpes zoster of pregnancy; hypogammaglobulinemia; idiopathic thrombocytopenic purpura (ITP); IgA nephropathy; IgG4-related sclerosing disease; immunomodulatory lipoprotein; inclusion body myositis; interstitial cystitis; inflammatory bowel disease; juvenile arthritis; juvenile oligoarthritis; juvenile diabetes mellitus (type 1 diabetes mellitus); juvenile myositis; Kawasaki syndrome; Lambert-Eaton syndrome; leukocytosis-destructive vasculitis; lichen planus; lichen sclerosing;Lung-like conjunctivitis; linear IgA disease (LAD); lupus (SLE); chronic Lyme disease; Meniere's disease; microscopic polyangiitis; mixed connective tissue disease (MCTD); Mollen's ulcer; Mucher-Habermann disease; multiple sclerosis; myasthenia gravis; myositis; narcolepsy; neuromyelitis optica (Devic); neutropenia; non-obese diabetes mellitus; ocular scarring pemphigoid; optic neuritis; relapsing rheumatoid arthritis; PANDAS (pediatric autoimmune neuropsychiatric disorder associated with Streptococcus); paraneoplastic cerebellar degeneration; paroxysmal nocturnal hemoglobinuria (PNH); Parry-Romberg syndrome; Personage-Turner syndrome; Ciliary body uterine ulcer (peripheral uveitis); Pemphigus; Pemphigus vulgaris; Perivenous encephalomyelitis; Pernicious anemia; POEMS syndrome; Polyarteritis nodosa; Type I, II, and III polyglandular autoimmune syndromes; Polymyalgia rheumatica; Polymyositis; Post-myocardial infarction syndrome; Post-pericardiotomy syndrome; Progesterone dermatitis; Primary biliary cirrhosis; Primary sclerosing cholangitis; Psoriasis; Psoriasis vulgaris; Psoriatic arthritis; Idiopathic pulmonary fibrosis; Pyoderma gangrene; Pure red cell aplasia; Raynaud's phenomenon; Reactive arthritis; Reflex sympathetic dystrophy; Reiter's syndrome; Relapsing polychondritis; Lower limb restlessness syndrome; Retroperitoneal fibrosis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Schmidt syndrome; Scleritis; Scleroderma; Sclerosing cholangitis; Sclerosing sialadenitis; Sjögren's syndrome; Sperm-testicular autoimmunity; Generalized rigidity syndrome; Subacute bacterial endocarditis (SBE); Suzak syndrome; Examples include sympathetic ophthalmitis; systemic lupus erythematosus (SLE); systemic sclerosis; Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome; transverse myelitis; type 1 diabetes mellitus; ulcerative colitis; undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vesicular dermatosis; vitiligo; and Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA)). Non-limiting examples of inflammatory diseases or conditions, or autoimmune diseases or conditions, include infections such as chronic infections, latent infections, delayed infections, persistent viral infections, bacterial infections, fungal infections, mycoplasma infections, or parasitic infections.

[0262] As described herein, for example, U.S. Provisional Application No. 62 / 791,601 is incorporated herein by reference in its entirety. B. Peptide production Peptides suitable for use in pMHC polymers are produced by methods known in the art, or synthetically by commercial suppliers or by using a peptide synthesizer in accordance with the manufacturer's instructions for use. For example, in some embodiments, peptides suitable for use in pMHC polymers can be produced by in silico production methods.

[0263] In other embodiments, peptides can be synthesized by chemical methods, such as tea bag synthesis, digital photolithography, pin synthesis, and SPOT synthesis. For example, a peptide array can be prepared via SPOT synthesis, where amino acid chains are constructed on a cellulose membrane by repeated cycles of amino acid addition and cleavage of side chain protecting groups.

[0264] In other embodiments, the peptide can be expressed using recombinant DNA technology, for example, by introducing an expression construct into bacterial, insect, or mammalian cells and purifying the recombinant protein from the cell extract.

[0265] In some embodiments, peptides can be synthesized by in vitro transcription and translation, where synthesis utilizes the biological principles of transcription and translation in a cell-free environment by providing, for example, nucleic acid templates, related building blocks (e.g., RNA, amino acids), enzymes (e.g., RNA polymerase, ribosomes), and conditions.

[0266] In some embodiments, in vitro transcription and translation may involve cell-free protein synthesis (CFPS). To obtain high yields by CFPS, the use of a bacterial system is required, where the first amino acid of the translated sequence is N-formylmethionine (fMet). This residue has a positively charged amino terminus (NH3). +It differs from methionine by containing a neutral formyl group (HCO) instead of ). The construct is engineered to include a gene encoding the enzyme cleavage domain and a library polypeptide described in the entirety of U.S. Provisional Application No. 62 / 791,601, which is incorporated herein by reference.

[0267] At least the removal of the first methionine amino acid ensures successful peptide folding and loading onto MHC proteins. In addition, the removal of the first methionine amino acid raises the upper limit of peptide library diversity, e.g., 20 x (where x is the length of the peptide) provides this, but the inclusion of this residue increases the diversity of the library by 20 (x-1) Restrict it to

[0268] In some embodiments, peptides are synthesized using an in vitro transcription / translation (IVTT) system capable of both, for example, transcribing a DNA construct into RNA and then translating the RNA into a protein. For example, the methods of this disclosure include methods for performing in vitro transcription / translation (IVTT) to produce a highly diverse peptide library and enable correct protein folding. IVTT can enable direct protein production in a cell-free environment from DNA or RNA templates.

[0269] The IVTT method used herein can be performed using, for example, a PCR product, a linear DNA plasmid, a circular DNA plasmid, or an mRNA template having a ribosome-binding site (RBS) sequence. After isolating a suitable template, transcription components, for example, containing ribonucleotide triphosphates and RNA polymerase, can be added to the template. After transcription is complete, translation components, for example, those found in rabbit reticulocyte hemolysis or wheat germ extract, can be added. In some methods, transcription and translation can be performed in a single step, where, for example, purified translation components found in rabbit reticulocyte hemolysis or wheat germ extract are added simultaneously with the addition of the transcription components to the nucleic acid template.

[0270] In some embodiments, the nucleotide sequence encoding the N-terminal methionine residue and cleavable portion of a peptide can be encoded in a DNA or RNA construct. The cleavable portion is positioned such that at least one N-terminal amino acid residue of the peptide precedes or is within the cleavable portion. In some embodiments, the method involves encoding a cleavable portion positioned such that one N-terminal amino acid residue of the peptide precedes or is within the cleavable portion. In some embodiments, the one N-terminal amino acid residue is a methionine residue. The cleavable portion can be cleaved using an enzyme, such as a cleavable portion-specific protease, which can also cleave the cleavable portion from the rest of the peptide.

[0271] Examples of cleavable regions that may be encoded in the DNA or RNA constructs described herein include any cleavable region that can be cleaved by an enzyme. In some embodiments, the cleavable region can be cleaved by a protease. The cleavable region can be used to cleave a peptide using an enzyme specific to the cleavable region. The enzyme may be, for example, factor Xa, human rhinovirus 3C protease, AcTEV® protease, WELQut protease, Genenase®, small ubiquitin-like modifier (SUMO) protein, Ulp1 protease, or enterokinase. Ulp1 protease can cleave the cleavable region in a specific manner by recognizing the tertiary structure rather than the amino acid sequence. Enterokinases (enteropeptidases) can also be used to cleave the cleavable region from a candidate peptide. Enterokinases can cleave after lysine at the following cleavage site: DDDDK (SEQ ID NO: 188). Enterokinases can also cleave at other basic residues depending on the sequence and conformation of the protein substrate.

[0272] In some embodiments, the cleavable portion may be a small ubiquitin-like modifier (SUMO) protein. The SUMO domain can cleave the peptide using a SUMO-specific protease. In some embodiments, the cleavable portion may be an enterokinase cleavage site: DDDDK (SEQ ID NO: 188). The protease may be, for example, Ulp1 protease or enterokinase. Ulp1 protease can cleave SUMO in a specific manner by recognizing the tertiary structure of SUMO rather than its amino acid sequence. Enterokinase (enteropeptidase) can also be used to cleave after lysine at the following cleavage site: DDDDK (SEQ ID NO: 188). Depending on the sequence of the protein substrate, enterokinase may also cleave at other basic residues.

[0273] During or after the translation of a peptide-encoding construct, the N-terminal amino acid residue (e.g., the SUMO domain) can be efficiently cleaved to produce a properly folded peptide. In some embodiments, at least one N-terminal amino acid residue is cleaved to produce a peptide. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-terminal amino acid residues are cleaved to produce a peptide. The N-terminal amino acid can be any amino acid residue. The N-terminal amino acid residue can be a methionine amino acid residue. Therefore, this properly folded peptide may not be constructed with an N-terminal methionine and may be part of a highly diverse peptide library produced by a cell-free in vitro method.

[0274] After translation of a peptide-encoding construct, the N-terminal amino acid residue can be cleaved to produce peptides for a high-diversity peptide library. In some embodiments, at least one N-terminal amino acid residue is cleaved to produce a peptide. In some embodiments, one or more N-terminal amino acids are cleaved, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 140, 150, 160, 170, 180, 190, 200, 250, or more N-terminal amino acid residues are cleaved to produce a peptide. The N-terminal amino acid can be any amino acid residue. The N-terminal amino acid residue can be a methionine amino acid residue.

[0275] In some embodiments, the DNA or RNA construct comprises puromycin. In some embodiments, the DNA or RNA construct comprises a spacer sequence lacking a stop codon. In some embodiments, the peptide is purified by affinity tag purification (e.g., using a FLAG tag). In some embodiments, the peptide comprises a HaloTag enzyme sequence. In some embodiments, the peptide comprises avidin or streptavidin.

[0276] For mammalian expression, constructs encoding the CMV peptide were designed using a C-terminal Flag tag with or without a C-terminal His tag in the mammalian expression vector. The peptide was expressed in Expi293F or ExpiCHO-S cells (Life Technologies) by transient transfection, as recommended by the manufacturer.

[0277] Peptides were purified from cell culture supernatant by anti-Flag affinity chromatography (Genscript) or Ni affinity chromatography. Size exclusion chromatography (SEC) was performed on hydrophilic resin (GE Life Sciences) pre-equilibriumized with 20 mM HEPES, 150 mM NaCl, and pH 7.2.

[0278] Alternatively, the peptides were purified by Ni affinity chromatography without SEC purification using a column buffer of 23 mM sodium phosphate, 500 mM sodium chloride, 500 mM imidazole, and pH 7.4.

[0279] Peptides produced in mammalian cells were quantified using 280 nm UV light, while peptides produced in CFPS were quantified by sandwich ELISA compared to a standard protein. V. Peptide exchange p * Using MHC polymers, libraries or microarrays of diverse and unique peptide epitope-producing pMHC polymers are generated by in situ or in vitro peptide exchange reactions described herein. In some embodiments, the peptide exchange reactions are carried out in a multi-well format under natural conditions. Binding is determined by several techniques, such as ELISA, which monitors the stability of the MHC structure, or by biophysical techniques, such as fluorescence polarization, which monitor peptide binding. Non-limiting examples of peptide exchange via dipeptide exchange or UV-mediated exchange are described in detail in Example 4.

[0280] In some embodiments, fluorescently labeled placeholder peptides are used in exchange reactions in the presence of unlabeled exchange peptides to measure the dissociation efficiency of placeholder peptides or peptide fragments. * Aliquots of MHC polymers are left untreated or exposed to peptide exchange conditions (e.g., UV exposure) for different periods. *The amount of MHC-containing placeholder peptide is p * The reduction of MHC complexes is monitored by fluorescence analysis.

[0281] In some embodiments, the placeholder peptide has a lower affinity for the MHC peptide binding groove than the exchanged peptide epitope, and step (d) is performed in a competitive assay, p * This involves contacting an MHC monomer with an excess peptide epitope. In some embodiments, the placeholder peptide has a KD about 10 times lower than the exchanged peptide epitope.

[0282] Peptides that bind to the peptide binding groove of an MHC molecule may be naturally occurring peptides, but they can also be synthetically produced using knowledge of the binding specificity of the B and F pockets of a particular MHC molecule or the supertype family to which it belongs. Suitable ligands can be generated using knowledge of the available 3D structures of the MHC complex and the binding pocket specificity of individual MHC molecules.

[0283] The peptide bond specificity of MHC I polypeptides is primarily governed by the physiochemical properties of the B and F binding pockets in their coupled configuration. The B and F binding pockets typically bind to "anchor residues" in the peptide that define peptide binding in the MHC peptide bond groove. The diversity observed in amino acid residues within the MHC molecule's peptide bond groove defines the peptide bond and presentation repertoire of individual MHC molecules (Chang et al. 2011; Frontiers in Bioscience, Landmark Edition, Vol. 16:3014-3035). The specificity of the pockets to anchor residues has been elucidated for numerous MHC molecules, as described, for example, by Sidney et al. (BMC Immunology Vol. 9:1, 2008).

[0284] This disclosure is p *A step of producing an MHC multimer, wherein the peptide in the binding groove is a placeholder peptide; p * A step of contacting the MHC polymer with a reducing agent to remove the placeholder peptide; and a step of applying sufficient conditions for the binding of the peptide epitope in the MHC peptide binding groove. * The step includes contacting an MHC polymer with an MHC peptide epitope, p * Further methods for producing MHC multimers are provided.

[0285] The two contact steps are preferably carried out by providing a sample containing an MHC molecule having an MHC peptide epitope and a reducing agent. The MHC peptide epitope is preferably present when the reducing agent is added. In some embodiments, one MHC peptide epitope is added per reaction. In some embodiments, two or more peptide epitopes are added to the reaction.

[0286] In some embodiments, peptide exchange is induced by raising the temperature of the mixture between approximately 30°C and 37°C. In some embodiments, the mixture is raised to 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.

[0287] In some embodiments, peptide exchange is induced by reducing the pH of the mixture to between approximately pH 2.5 and 5.5. In some embodiments, peptide exchange is induced by increasing the pH of the mixture to between approximately pH 9 and 11.

[0288] In some embodiments, the placeholder peptide includes a photocleavable moiety for forming the described pMHC complex (e.g., Toebes et al. Nat. Med. 12:246-251, 2006; Bakker et al. PNAS 105:3825-383, 2008; Frosig et al., Cytometry Part A, 87A:967-975, 2015; Chang et al., Eur. J. Immunol. 43:1109-1120, 2013). In some embodiments, the placeholder peptide includes a non-natural amino acid containing a (2-nitro)phenyl side chain. In some embodiments, the amino acid is a UV-sensitive β-amino acid containing 3-amino-3-(2-nitro)phenyl-propionic acid. In some embodiments, the UV-sensitive amino acid is (2-nitro)phenylglycine.

[0289] In some embodiments, the placeholder peptide is an HLA-A2 peptide. In some embodiments, the HLA-A2 placeholder peptide is p * A2, KILGCVFJV (SEQ ID NO: 15), or GILGFVFJL (SEQ ID NO: 7) (where J is 3-amino-3-(2-nitro)phenyl-propionic acid).

[0290] In some embodiments, the placeholder peptide is an HLA-A1, -A3, A11, or -B7 peptide containing a photocleavable portion. In some embodiments, the placeholder peptide is A*01:01, STAPGJLEY (SEQ ID NO: 16); A*03:01, RIYRJGATR (SEQ ID NO: 17); A*11:01, RVFAJSFIK (SEQ ID NO: 18); A*24:02, VYGJVRACL (SEQ ID NO: 11); B*07:02, AARGJTLAM (SEQ ID NO: 14); B*35:01, KPIVVLJGY (SEQ ID NO: 19); C*03 :04, FVYGJSKTSL (sequence number 20), B*08:01, FLRGRAJGL (sequence number 21); C*07:02, VRIJHLYIL (sequence number 22); C*04:01, QYDJAVYKL (sequence number 23); B*15:01, ILGPJGSVY (sequence number 24); B*40:01, TEADVQJWL (sequence number 25); B*58:01, ISARGQJLF (sequence number 26); and C * 08:01, selected from the group consisting of KAAJDLSHFL (SEQ ID NO: 27) (where J is 3-amino-3-(2-nitro)phenyl-propionic acid). In other embodiments, the placeholder peptide has the sequence shown in any one of SEQ ID NOs: 7-27 or 271-279.

[0291] In some embodiments, the placeholder peptide further includes a fluorescent label. In such embodiments, the fluorescent label is attached to a cysteine ​​residue in the placeholder peptide.

[0292] Upon irradiation with long-wavelength UV, peptides are cleaved in the presence of one or more peptides and dissociate from the MHC complex to facilitate the formation of stable pMHC monomers or polymers. Typically, MHC peptide exchange is performed in a multi-well format for high-throughput screening of peptide ligands as described herein. Only peptide candidates that efficiently bind to and stabilize peptide-acceptable MHC molecules prevent dissociation of the MHC complex. Peptide exchange can be monitored by several techniques, such as ELISA or fluorescence polarization, as commonly described, for example, in Rodenko et al. (Nat. Protocol. 1:1120-1132, 2006).

[0293] The resulting pMHC polymers are then analyzed by gel filtration HPLC and MHC ELISA to determine three parameters: the efficiency of MHC refolding, the stability of the pMHC complex in the absence of UV exposure, and the UV sensitivity of the complex.

[0294] Certain dipeptides can assist in the folding and peptide exchange of MHC class I molecules. These dipeptides specifically bind to the F-pocket of MHC class I molecules, promoting peptide exchange and HLA-A * 02:01, HLA-B * Peptide exchange in 27:05 and H-2Kb molecules has been previously described and validated (Saini et al. Proc Natl Acad Sci US A. 2013 Sep 17; 110(38):15383-8).

[0295] Therefore, in some embodiments, peptide exchange of a placeholder peptide with a peptide or the peptide of interest is catalyzed by a dipeptide that catalyzes rapid peptide exchange in MHC class I molecules (see, for example, Saini et al., Proc Natl Acad Sci US A. 2015 Jan 6; 112(1):202). Preferred dipeptides are those having a hydrophobic second residue. In some embodiments, the dipeptide is glycyl-leucine (GL), glycyl-valine (GV), glycyl-methionine (GM), glycyl-cyclohexylalanine (GCha), glycyl-homoleucine (GHle), or glycyl-phenylalanine (GF).

[0296] In another embodiment, peptide exchange of a placeholder peptide with a peptide or the peptide of interest (peotides) is achieved by chaperone-mediated peptide exchange, for example, using the molecular chaperone TAPBPR, as described in Overall et al. (2020) Nature Comm. 11:1909. Production of the VI.pMHC library In one embodiment, a method for producing a library of pMHC polymers containing diverse load peptide epitopes is provided herein. The various steps in the preparation of the peptide-exchanged barcoded pMHC library are schematically shown in Figure 18. These steps utilize standard methods known in the art for preparing barcoded libraries and include the use of single-cell sequencing, the use of porous hydrogels, the use of single-template PCR for generating peptide-coded amplicons (barcodes), and the use of in vitro transcription / translation (IVTT) in droplets.

[0297] A non-limiting example of single-cell sequencing using pooled barcoded UV peptide-exchanged MHC tetramers is described in Example 9. A non-limiting example of the production of a porous hydrogel for high-throughput production of a pool of barcoded UV peptide-exchanged MHC tetramers is described in detail in Example 10. A non-limiting example of the use of single-template PCR to generate peptide-coded amplicons is described in detail in Example 11. A non-limiting example of loading barcoded, exchange-ready MHC tetramers onto a hydrogel is described in Example 12. A non-limiting example of in vitro transcription / translation (IVTT) in peptide droplets and UV exchange to loaded MHC tetramers is described in detail in Example 13. A non-limiting example of the release of UV-peptide-exchanged barcoded pMHC tetramers from a hydrogel is described in detail in Example 14.

[0298] In some embodiments, the method comprises a plurality of placeholder peptide loads of MHCI(p) each containing (a)(i) an MHCI heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a conjugation moiety, and (iv) a placeholder peptide bound in the peptide bond groove of each MHCI monomer. * (b) a step of providing a monomer; (c) a step of providing multiple polymerizing domains, wherein each subunit of the polymerizing domain includes a conjugation moiety; (d) two or more p * For covalent conjugation between the MHCI monomer and the polymerizing domain, p under sufficient conditions * By combining the MHCI monomer and the polymerizing domain, p * (d) a step of producing MHCI multimers; and (d) multiple p * The method includes the step of producing multiple peptide-loaded MHCI (pMHCI) multimers by replacing placeholder peptides in an MHCI multimer with a peptide library containing multiple unique MHCI peptide epitopes.

[0299] In some embodiments, the method comprises a plurality of placeholder peptide loads of MHCI(p) each containing (a)(i) an MHCI heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a conjugation moiety, and (iv) a placeholder peptide bound in the peptide bond groove of each MHCI monomer. * (b) a step of providing a monomer; (c) a step of providing a plurality of polymerizing domains, wherein each subunit of the polymerizing domain comprises a conjugation moiety and the polymerizing domain comprises at least one non-covalent site; (d) two or more p * Multiple p under sufficient conditions for covalent conjugation between MHCI monomers and polymerizing domains * By combining MHCI monomers and multiple polymerizing domains, multiple p * Steps to produce MHCI multimers; (d)p * The method comprises the steps of (e) replacing a placeholder peptide bound in the peptide bond groove of an MHCI multimer with a plurality of unique rescue peptide epitopes to produce a plurality of pMHCI multimers, and (e) binding an oligonucleotide barcode to a non-covalent site on the multimerization domain.

[0300] In some embodiments, the method comprises a plurality of placeholder peptide loads MHCI(p * (b) Providing a monomer; (c) Providing multiple polymer domains, each containing a peptide linker with a conjugation moiety at the N-terminus of each subunit of the polymerizing domain; (d) Providing two or more p *Multiple p under sufficient conditions for covalent conjugation between MHCI monomers * By combining MHCI monomers and multiple polymerizing domains, multiple p * Steps to produce MHCI multimers; and (d)p * The process includes the step of producing multiple pMHCI multimers by replacing a placeholder peptide bound in the peptide bond groove of an MHCI multimer with multiple unique rescue peptide epitopes. VII. Labeling pMHC multimers can be conjugated with fluorescent labels that enable the identification of T cells binding to the peptide-MHC multimer, for example, via flow cytometry or microscopy. T cells can also be selected based on the fluorescent label, for example, by fluorescently labeled cell sorting.

[0301] In some embodiments, one or more detectable labels are conjugated to the linker. According to the present invention, a “detectable label” is any molecule or functional group that enables the detection of a biological or chemical feature or change in a system, such as the presence of a target substance in a sample.

[0302] Examples of detectable labels that may be used include fluorophores, chromophores, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, beads or other solid surfaces, gold or other metal particles or heavy atoms, spin labels, radioisotopes, enzyme substrates, haptens, antigens, quantum dots, aminohexyl, pyrene, nucleic acids or nucleic acid analogs, or proteins, such as receptors, peptide ligands or substrates, enzymes, and antibodies (including antibody fragments).

[0303] Examples of polymer particle labeling that can be used include polystyrene, PMMA, or silica microparticles, beads, or latex particles, which may be embedded with fluorescent dyes, polymer micelles, or capsules containing dyes, enzymes, or substrates. Examples of metal particles that can be used include gold particles and coated gold particles, which may be converted by silver staining. Examples of haptens that can be conjugated include, in some embodiments, fluorophores, myc, nitrotyrosine, biotin, avidin, streptavidin, 2,4-dinitrophenyl, digoxigenin, bromodeoxyuridine, sulfonates, acetylaminoflurene, trinitrophenol mercury, and estradiol.

[0304] Examples of enzymes that may be used include horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO). Examples of substrates commonly used for horseradish peroxidase (HRP) include 3,3'-diaminobenzidine (DAB), nickel-enhanced diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), Indofan blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), and alpha-naphtholpyronin (alpha-naphtholpyronin). Examples include .-NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolylphosphate (BCIP), nitrobluetetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT), tetranitrobluetetrazolium (TNBT), and delta-bromo-chloro-S-indoxyl-beta-D-galactoside / ferro-ferricyanide (BCIG / FF). Examples of substrates commonly used for alkaline phosphatases include naphthol-AS-B1-phosphate / Fastred TR (NABP / FR), naphthol-AS-MX-phosphate / Fastred TR (NAMP / FR), naphthol-AS-B1-phosphate / new fuschin (NABP / NF), bromochloroindolyl phosphate / nitrobluetetrazolium (BCIP / NBT), and β-bromo-chloro-S-indolyl-beta-delta-galactopyranoside (BCIG).

[0305] Examples of luminescent labels that may be used include luminol, isoluminol, acridinium esters, 1,2-dioxetane, and pyridopyridazine. Examples of electrochemiluminescent labels include ruthenium derivatives. Examples of radioactive labels that may be used include radioisotopes of iodine, cobalt, selenium, hydrogen, carbon, sulfur, and phosphorus.

[0306] Some "detectable labels" also include "color labels," where a biological change or event in a system can be assayed by the presence or change of color. Examples of "color labels" are chromophores, fluorophores, chemiluminescent compounds, electrochemiluminescent labels, bioluminescent labels, and enzymes that catalyze a change in the color of a substrate.

[0307] As used herein, a "fluorophore" is a molecule that emits detectable electromagnetic radiation upon excitation by electromagnetic radiation at one or more wavelengths. A wide variety of fluorophores are known in the art, have been developed by chemists for use as detectable molecular labels, and can be conjugated to pMHC polymers provided herein. Examples include FLUORESCEIN® or its derivatives, e.g., FLUORESCEIN®-5-isothiocyanate (FITC), 5-(and 6)-carboxyFLUORESCEIN®, 5- or 6-carboxyFLUORESCEIN®, 6-(FLUORESCEIN®)-5-(and 6)-carboxamidehexanoic acid, FLUORESCEIN® isothiocyanate, rhodamine or its derivatives, e.g., tetramethylrhodamine and tetramethylrhodamine-5-(and -6) isothiocyanate (TRITC).Other fluorophores include coumarin dyes, e.g., (diethyl-amino)coumarin or 7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester (AMCA); sulforhodamine 101 sulfonyl chloride (TexasRed® or TexasRed® sulfonyl chloride); 5-(and-6)-carboxyrhodamine 101, succinimidyl ester, also known as 5-(and-6)-carboxy-X-rhodamine, succinimidyl ester (CXR); lisamin or lisamin derivatives, e.g., lisaminrhodamine B sulfonyl chloride (LisR); 5-(and-6)-carboxyfluorescein®, succinimidyl ester (CFI); fluorescein® 5-isothiocyanate (FITC); 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester (DECCA); 5-(and-6)-carb Xytetramethyl-rhodamine, succinimidyl ester (CTMR); 7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester (HCCA); 6->FLUORESCEIN®,-5-(and-6)-carboxamidehexanoic acid (FCHA); N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid, succinimidyl ester; 5,7-dimethylBODIPY® ) Also known as propionic acid, succinimidyl ester (DMBP); available from "Activated FLUORESCEIN® Derivatives" (FAP), Probes, Inc.; eosin-5-isothiocyanate (EITC); erythrosine-5-isothiocyanate (ErlTC); and Cascade® blue acetyl azide (CBAA) (O-acetylated derivative of 1-hydroxy-3,6,8-pyrene-trisulfonic acid).Further possible fluorophores useful in the present invention include fluorescent proteins, such as green fluorescent protein and its analogues or derivatives; fluorescent amino acids, such as tyrosine and tryptophan, and their analogues; fluorescent nucleosides; and other fluorescent molecules, such as Cy2, Cy3, Cy3.5, CY5.TM, CY5.TM.5, Cy7; IR dyes; Dyomics dyes; phycoerythrin; Oregon Green 488; Pacific Blue; Rhodamine Green; and Alexa dyes. Further examples of fluorescent labeling include R-phycoerythrin, oralophycoerythrin; and inorganic fluorescent labeling, such as conjugates of particles based on semiconductor materials, such as coated CdSe nanocrystals.

[0308] Some of the fluorophores and others mentioned above are commercially available from companies such as Probes, Inc. (Eugene, Oregon), Pierce Chemical Co. (Rockford, Ill.), or Sigma-Aldrich Co. (St. Louis, Mo.).

[0309] Detectable labels can be detected by a number of methods, including, for example, in the case of optical labels, by reflectance, transmittance, light scattering, optical rotation, and fluorescence, or a combination thereof, or in the case of radioactive labels, by film, scintillation counting, or phosphoimaging. See, for example, Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, Fla.); Methods in Molecular Biology, vol. 80 1998, John D. Pound (ed.) (Humana Press, Totowa, NJ). In some embodiments, two or more detectable labels were used. VIII. Identifiers / Barcoding In certain embodiments, the conjugated polymer of the Disclosure includes an identifier tag or label, such as an oligonucleotide barcode, which facilitates the identification of the conjugated polymer. Typically, the identifier tag, such as an oligonucleotide barcode, is attached to the polymerizing domain of the conjugated polymer, for example, through a binding site on the polymerizing domain, such as an oligonucleotide barcode. For example, if the polymerizing domain is streptavidin or avidin, the pMHCI monomer is conjugated to the polymerizing domain at a site other than the biotin-binding site, so the conjugated polymer can be labeled with an identifier tag, such as an oligonucleotide barcode, using a biotinylated form of the identifier tag, such as a biotinylated oligonucleotide barcode. The labeling of the conjugated polymer is then easily achieved by incubation of the conjugated polymer with a biotinylated identifier tag, such as a biotinylated oligonucleotide barcode. A non-limiting example of barcoding conjugated polymers using biotinylated oligonucleotides is described in detail in Example 8.

[0310] In another embodiment, the conjugated polymer is labeled with an identifier tag, such as an oligonucleotide barcode, at the peptide portion of the polymer. That is, a barcode-labeled MHC-binding peptide can be used in the exchange reaction described herein for loading the conjugated polymer with the barcode-labeled peptide.

[0311] Typically, an oligonucleotide barcode is a unique oligonucleotide sequence ranging from 10 to more than 50 nucleotides. The barcode has shared amplification sequences at the 3' and 5' ends, as well as a unique sequence in the middle. This sequence can be revealed by sequencing and can function as a specific barcode for a given molecule.

[0312] In one embodiment, the nucleic acid component (typically DNA) of the barcode has a specific structure. Therefore, in one embodiment, at least one nucleic acid molecule consists of at least a 5' first primer region, a central region (barcode region), and a 3' second primer region. In this method, the central region (barcode region) can be amplified by the primer set. The length of the nucleic acid molecule can also vary. Therefore, in other embodiments, at least one nucleic acid molecule has a length in the range of 20 to 100 nucleotides, e.g., 30 to 100, e.g., 30 to 80, e.g., 30 to 50 nucleotides. In one embodiment, the nucleic acid identifier is 40 to 120 nucleotides long. The coupling of the oligonucleotide barcode to the conjugated multimer can also vary. Therefore, in one embodiment, at least one oligonucleotide barcode is ligated to the conjugated multimer via a biotin-binding domain that interacts with streptavidin or avidin within the conjugated multimer. Other coupling sites may also be used depending on the availability of suitable binding sites with the conjugated polymer (e.g., within the polymerization domain of the conjugated polymer) and suitable corresponding binding domains that can be attached to the oligonucleotide barcode molecule to facilitate attachment.

[0313] In further embodiments, at least the oligonucleotide barcode molecule includes or consists of DNA, RNA, and / or artificial nucleotides, such as PLA or LNA. Preferably, other nucleotides, but DNA may be included, for example, to increase stability.

[0314] The use of barcode technology is well known in the art; see, for example, Shiroguchi et al., Proc. Natl. Acad. Sci. USA., 2012 Jan. 24; 109(4):1347-52; and Smith et al., Nucleic Acids Research, 2010 July; 38(13)11:e142. Further methods and compositions for using barcode technology are described in US2016 / 0060621. The use of barcode technology for specifically labeling MHC polymers has also been described; see, for example, Bentzen et al., Nature Biotech. 34:10: 1037-1045, 2016; and Bentzen and Hadrup, Cancer Immunol. Immunotherap. 66:657-666, 2017. Standard methods for preparing barcode oligonucleotides, including their conjugation (e.g., biotinylation) with a suitable binding moiety capable of binding to a conjugated polymer, are known in the art and can be applied to prepare barcode oligonucleotides for labeling conjugated polymers.

[0315] Methods for generating customizable DNA barcode libraries are publicly available. These include Generator and nxCode, each consisting of 96 to 587 barcodes, as well as The DNA Barcodes Package and TagD software (which reports generating libraries of 100,000 barcodes).

[0316] The preparation of various large-scale barcode libraries has been described in the Art, and the methods can be used to obtain barcode libraries for labeling pMHC-conjugated multimer libraries. For example, Xu et al. described a set of 240,000 unique 25-mer oligonucleotides having sequences with similar amplification characteristics while maintaining the greatest diversity of their identification motifs (Xu et al. PNAS 106:2289-2294, 2008). Wang et al. described the construction of a barcode set using particle ensemble optimization (Wang et al. IEEE / ACM Trans. Comput. Biol. Bioinform. 15:999-1002). Lyons described the generation of a large-scale library of DNA barcodes with up to 1 million members (Lyons, Sci. Reports 7:13899, 2017).

[0317] In some cases, a unique molecular identifier (UMI) barcode is encoded by a continuous sequence of tagged nucleotides at one end of a target nucleic acid. In other cases, a unique molecular identifier (UMI) barcode is encoded by a discontinuous sequence. A discontinuous UMI may have a portion of the barcode at a first end of the target nucleic acid and a portion of the barcode at a second end of the target nucleic acid. In some cases, the UMI is a discontinuous barcode containing a variable-length barcode sequence at the first end of the target nucleic acid and a second identifier sequence at the second end. In some cases, the UMI is a discontinuous barcode having a variable-length barcode sequence at the first end of the target nucleic acid and a second identifier sequence at the second end, where the second identifier sequence is determined by the location of transposase fragmentation events, e.g., transposase fragmentation sites and transposase terminal insertion events.

[0318] In some cases, the barcode is a “variable-length barcode.” As used herein, a variable-length barcode is an oligonucleotide that differs from other variable-length barcode oligonucleotides in a population by length, which can be identified by the number of consecutive nucleotides in the barcode. In some cases, in addition to variable length, the barcode complexity for variable-length barcodes, as described in the paragraph above, may be provided by the use of a variable nucleotide sequence.

[0319] In exemplary embodiments, a variable-length barcode may have a length of 0 to 5 nucleotides. Such a variable-length barcode may be represented by the term "[0-5]". In such embodiments, it is understood that the population of target nucleic acids to be attached to such a variable-length barcode is expected to include at least one target nucleic acid attached to a variable-length barcode having at least one nucleotide (e.g., attached to a variable-length barcode having only one, two, three, four, or five nucleotides). In such embodiments, it is further understood that the population of target nucleic acids attached to such variable-length barcodes may include at least one target nucleic acid that does not contain a variable-length barcode (i.e., a variable-length barcode having a length of 0), and / or at least one target nucleic acid that contains a variable-length barcode having only one nucleotide, and / or at least one target nucleic acid that contains a variable-length barcode having only two nucleotides, and / or at least one target nucleic acid that contains a variable-length barcode having only three nucleotides, and / or at least one target nucleic acid that contains a variable-length barcode having only four nucleotides, and / or at least one target nucleic acid that contains a variable-length barcode having only five nucleotides. In such embodiments, the [0-5] variable-length barcode can, by itself, uniquely identify (distinguish) five different target nucleic acid molecules of the same sequence. Furthermore, in such embodiments, the [0-5] variable-length barcode can uniquely identify (distinguish) five different target nucleic acid molecules of a first sequence, five different target nucleic acid molecules of a second sequence, and so on, for each different target nucleic acid sequence. Furthermore, barcode-labeled MHC multimers can be used in combination with single-cell sorting and TCR sequencing, where TCR specificity can be determined by the co-adhered barcode. This allows us to identify TCR specificity for 1000+ potentially different antigen-reactive T cells in parallel from the same sample and match TCR sequences with antigen specificity. The future potential of this technology lies in its ability to predict antigen responsiveness based on TCR sequences.

[0320] The complexity of barcode-labeled MHC multimer libraries allows for the individualized selection of relevant TCRs within a given organism.

[0321] The barcodes are co-attached to multimers and serve as specific labels for particular peptide-MHC complexes. This method allows for the mixing of at least 1000–1000 or more different peptide-MHC multimers, enabling specific interactions with T cells from blood or other biological specimens, flushing out unbound MHC multimers, and determining the sequencing of DNA barcodes. When selecting a target cell population, the barcode sequences present above background levels provide a fingerprint for identifying antigen-responsive cells within a given cell population. The number of sequence reads for each specific barcode correlates with the frequency of specific T cells, which can be estimated by comparing the read frequency to the T cell input frequency.

[0322] DNA barcoding serves as a specific label for antigen-specific T cells and can be used to determine T cell specificity, for example, after single-cell sorting, functional analysis, or phenotypic evaluation. In this method, antigen specificity may be related to both the T cell receptor sequence (which can be revealed by single-cell sequencing) and the functional and phenotypic characteristics of the antigen-specific cell.

[0323] Barcode-labeled MHC multimer libraries can be used for the quantitative evaluation of MHC multimer binding to a given T cell clone or TCR transfected / transfected cells. Since sequencing of barcode labels allows several different labels to be determined simultaneously in the same cell population, this strategy can be used to determine the avidity of a given TCR compared to a library of relevant peptide-MHC multimers. The relative contribution of different DNA-barcode sequences in the final read is determined based on the quantitative contribution of each different peptide-MHC multimer in the library to TCR binding. Titration-based analysis makes it possible to determine the quantitative binding characteristics of TCRs in relation to a large library of peptide-MHC multimers, all merged into a single sample. For this particular purpose, the MHC multimer library may specifically hold relevant peptide sequences or alanine-substituted peptide libraries.

[0324] In some embodiments, a unique identifier can be used for each sample of multiple samples. In some embodiments, the identifier can be shared among two or more samples. In some embodiments, the identifier may include some sequences shared among all samples and other sequences unique to one sample. In some embodiments, the identifier may include sequences shared among all samples and sequences unique to one sample. In some embodiments, sequences shared among samples can be used as identifiers for amplification (e.g., PCR amplification with suitable primers). In some embodiments, sequences unique to one sample or shared among subsets of samples can be used for detection or quantification by qPCR (e.g., sequences for hydrolysis probes, e.g., TaqMan probes). In some embodiments, sequences unique to one sample or shared among subsets of samples can be used for detection or quantification via sequencing.

[0325] In some embodiments, identifiers may include sequences generated in a unique in silico, each identifier sequence may be assigned to one of several samples, and the identifier-sample assignments may be stored in a database. In some embodiments, identifiers may include nucleotide sequences encoding all or part of a peptide or protein. In some embodiments, identifiers may include nucleotide sequences encoding an open reading frame. In some embodiments, identifiers may include nucleotide sequences containing a promoter sequence. In some embodiments, identifiers may include nucleotide sequences containing a binding site for a DNA-binding protein, such as a transcription factor or polymerase enzyme. In some embodiments, identifiers may include one or more sequences targeted by a nuclease, such as a restriction enzyme. In some embodiments, identifiers may include all sequence elements necessary for in vitro transcription and translation of the sequence. In some embodiments, identifiers may not include all sequence elements necessary for in vitro transcription and translation of the sequence.

[0326] In some embodiments, the identifier may include a biotinylated nucleotide sequence. In some embodiments, the identifier may be biotinylated by PCR amplification with a biotinylated primer. In some embodiments, the identifier may be biotinylated by enzymatic incorporation of biotinylated labeling, such as biotin dUTP labeling, using a mixed primer that labels an RNA polymerase, including Klenow DNA polymerase enzyme, nick translation, or RNA polymerases including T7, T3, and SP6 RNA polymerases. In some embodiments, the identifier may be biotinylated by photobiotination, for example, by adding photoactivatable biotin to the sample and irradiating the sample with UV light.

[0327] In some embodiments, identifiers can be generated from a template polynucleotide, for example, via PCR amplification of template DNA. In some embodiments, the template polynucleotide may include a nucleotide sequence encoding an open reading frame. In some embodiments, the template polynucleotide may include a nucleotide sequence containing a promoter sequence. In some embodiments, the template polynucleotide may include a nucleotide sequence containing a binding site for a DNA-binding protein, such as a transcription factor or polymerase enzyme. In some embodiments, the template polynucleotide may include one or more sequences targeted by a nuclease, such as a restriction enzyme. In some embodiments, the template polynucleotide may include all the sequence elements necessary for in vitro transcription and translation of the sequence. In some embodiments, the template polynucleotide may not include all the sequence elements necessary for in vitro transcription and translation of the sequence.

[0328] pMHC multimers with attached identifiers (e.g., oligonucleotide barcodes) can be incubated with multiple T cells, followed by sorting of the T cells into single-cell compartments. The T cells are lysed, and nucleic acids are produced from the lysed T cells containing the identifiers. The nucleic acids are pooled and sequenced. The identifiers allow for matching peptide identifiers with T cell sequences from the same compartment. The TCR antigen specificity profile is determined by identifying TCR sequences (e.g., variable regions, hypervariable regions, or CDRs) from the compartment and quantifying peptide identifier reads from the same compartment.

[0329] It is possible to identify numerous TCRs that show binding affinity to peptides in a peptide library, and to identify numerous peptides that show binding affinity to specific TCRs.

[0330] Epitope mutations in the antigens of identified TCR-antigen pairs that result in increased TCR binding affinity can be identified.

[0331] It is possible to identify disease-related proteins as controls and associated peptides and TCR sequences, which can then be used to design vaccines and cell therapies.

[0332] To evaluate the response to treatment, the corresponding TCR sequence is identified for each sequenced peptide identifier. Numerous TCRs showing binding affinity to some peptides in the peptide library are identified, and numerous peptides showing binding affinity to some TCRs are identified. Subjects are followed over the long term, and assay results are compared to identify peptides and TCR sequences associated with a successful response to immunotherapy. IX. Vectors and Polynucleotides Nucleic acid sequences encoding any of the proteins described herein are also included in this disclosure. As will be apparent to those skilled in the art, due to third base degeneracy, almost all amino acids can be represented by two or more triplet codons in the encoding nucleotide sequence. In addition, minor base pair changes may result in conserved substitutions in the encoded amino acid sequence, but are not expected to substantially alter the biological activity of the gene product. Thus, the nucleic acid sequences encoding the proteins described herein may be slightly modified in the sequence and still still encode their respective gene products.

[0333] Nucleic acids encoding any of the various proteins or polypeptides described herein may be chemically synthesized. Codon use frequencies may be selected to improve expression in cells. Such codon use frequencies depend on the selected cell type. Specific codon use frequency patterns have been developed for E. coli and other bacteria, as well as for mammalian cells, plant cells, yeast cells, and insect cells. For example, see Mayfield et al., Proc. Natl. Acad. Sci. USA, 100(2):438-442 (Jan. 21, 2003); Sinclair et al., Protein Expr. Purif., 26(I):96-105 (October 2002); Connell, ND, Curr. Opin. Biotechnol., 12(5):446-449 (October 2001); Makrides et al., Microbiol. Rev., 60(3):512-538 (September 1996); and Sharp et al., Yeast, 7(7):657-678 (October 1991).

[0334] General techniques for nucleic acid manipulation are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), which are incorporated herein by reference, or in Ausubel, F. et al., Current Protocols in Molecular Biology, Green Publishing and Wiley-Interscience, New York (1987) and periodic updates. Generally, polypeptide-encoding DNA is operably ligated to suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include transcriptional promoters, optional operator sequences to control transcription, sequences encoding suitable mRNA-ribosome binding sites, and sequences to control transcription and translation termination. Additionally, the ability to replicate in a host, usually conferred by the origin of replication, and select genes to facilitate transformant recognition are incorporated.

[0335] The proteins described herein may be produced not only directly, but preferably recombinantly, as fusion polypeptides with heterologous polypeptides, which are signal sequences or other polypeptides having a specific cleavage site at the N-terminus of a mature protein or polypeptide. The selected heterologous signal sequence is preferably recognized and processed by the host cell (i.e., cleaved by a signal peptidase).

[0336] For prokaryotic host cells that do not recognize and process the native signal sequence, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, 1pp, or a thermostable enterotoxin II reader.

[0337] For yeast secretion, the native signal sequence may be replaced with, for example, a yeast invertase reader, a factor reader (including Saccharomyces and Kluyveromyces alpha factor readers), or an acid phosphatase reader, a C. albicans glucoamylase reader, or a signal sequence described in U.S. Patent No. 5,631,144. For mammalian cell expression, mammalian signal sequences and viral secretion readers, such as the herpes simplex gD signal, are available. The DNA for such precursor regions may be ligated to the protein-coding DNA in the reading frame.

[0338] Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors, these sequences include origins of replication or self-replicating sequences that allow the vector to replicate independently of host chromosomal DNA. Such sequences are well known for various bacteria, yeasts, and viruses. Origins of replication derived from plasmid pBR322 are suitable for most Gram-negative bacteria, 2-micron plasmid origins are suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. Generally, components of the origin of replication are not required for mammalian expression vectors (the SV40 origin may typically be used only because it contains an initial promoter).

[0339] Expression and cloning vectors may contain select genes, also known as select markers. Typical select genes include (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that compensate for nutritional deficiencies; or (c) proteins that supply essential nutrients not available from the complex medium, such as the gene encoding Bacilli's D-alanine racemase.

[0340] Expression and cloning vectors typically contain a promoter that is recognized by the host organism and operably ligated to a nucleic acid encoding a protein described herein, such as a fibronectin-based scaffold protein. Suitable promoters for use in prokaryotic hosts include the phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters, such as the tan promoter. However, other known bacterial promoters are also preferred. Promoters for use in bacterial systems also contain a Shine-Dalgarno (SD) sequence operably ligated to the DNA encoding a protein described herein. Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25–30 bases upstream from the transcription initiation site. Another sequence found 70–80 bases upstream from the transcription initiation of many genes is the CNCAAT region (where N can be any nucleotide). Most eukaryotic genes have an AATAAA sequence at their 3' end, which can be a signal for the addition of a polyA tail at the 3' end of the coding sequence. Preferably, all of these sequences are inserted into eukaryotic expression vectors.

[0341] Examples of sequences suitable for use in yeast hosts include promoters for 3-phosphoglycerate kinases or other glycosphagases, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0342] Transcription from a vector in mammalian host cells may be controlled by a promoter derived from the genome of a virus, such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, most preferably Simianvirus 40 (SV40), or by a heterogeneous mammalian promoter, such as an actin promoter or immunoglobulin promoter derived from a heat shock promoter, provided that such promoter is compatible with the host cell system.

[0343] The transcription of DNA encoding the proteins described herein by higher eukaryotes is often enhanced by the insertion of enhancer sequences into vectors. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, enhancers derived from eukaryotic viruses are used. Examples include the SV40 enhancer located post-origin (bp100-270), the cytomegalovirus early promoter enhancer, the post-origin polyoma enhancer, and the adenovirus enhancer. For elements that enhance the activation of eukaryotic promoters, see also Yaniv, Nature, 297:17-18 (1982). Enhancers may be spliced ​​into the vector at the 5' or 3' position relative to the peptide coding sequence, but preferably located 5' from the promoter.

[0344] Expression vectors used in eukaryotic host cells (e.g., nucleated cells from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are typically available from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of mRNA encoding the proteins described herein. One useful transcription termination component is the polyadenylated region of bovine growth hormone. See WO94 / 11026 and the expression vectors disclosed therein.

[0345] Recombinant DNA may also contain any type of protein tag sequence that may be useful for purifying proteins. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, New York (1985)), the relevant disclosures of which are incorporated herein by reference.

[0346] The expression construct is introduced into the host cell using a method appropriate to the host cell, as will be apparent to those skilled in the art. Various methods for introducing nucleic acids into host cells are known in the art and are not limited to, but include electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; particulate guns; lipofection; and infection (when the vector is an infectious pathogen).

[0347] Suitable host cells include prokaryotes, yeasts, mammalian cells, or bacterial cells. Suitable bacteria include Gram-negative or Gram-positive organisms, such as E. coli or Bacillus spp. Yeast, preferably from the Saccharomyces species, such as S. cerevisiae, can also be used for polypeptide production. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. Baculovirus systems for heterologous protein production in insect cells have been outlined by Luckow et al. (Bio / Technology, 6:47 (1988)). Examples of suitable mammalian host cell systems include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell systems. Purified polypeptides are prepared by culturing suitable host / vector systems to express recombinant proteins. For many applications, the small-sized polypeptides described herein will be expressed in E. coli as a preferred method for expression. The proteins are then purified from culture medium or cell extracts.

[0348] The host cells used to produce the proteins of the present invention can be cultured in various culture media. Commercially available media, such as Ham F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma)), are suitable for culturing host cells. In addition, many media described in Ham et al., Meth. Enzymol., 58:44 (1979), Barites et al., Anal. Biochem., 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, 6,048,728, 5,672,502, or U.S. Patent No. RE30,985 can be used as culture media for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar concentration range), and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations that would be known to those skilled in the art. Culture conditions, e.g., temperature, pH, etc., are those previously used in host cells selected for expression and would be apparent to those skilled in the art.

[0349] The proteins described herein may also be produced using cell-free translation systems. For such purposes, the nucleic acids encoding polypeptides must be modified to enable in vitro transcription to produce mRNA and to enable cell-free translation of mRNA in the specific cell-free system used (eukaryotes such as mammals or yeast cell-free translation systems, or prokaryotes such as bacteria cell-free translation systems).

[0350] The proteins described herein can also be produced by chemical synthesis (for example, by the method described in Solid Phase Peptide Synthesis, 2nd Edition, The Pierce Chemical Co., Rockford, Ill. (1984)). Modifications to the proteins can also be produced by chemical synthesis.

[0351] The proteins of the present invention can be purified by isolation / purification methods for proteins that are generally known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., using ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal-phase chromatography, reverse-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent partitioning, or any combination thereof. After purification, the polypeptides may be exchanged in different buffers and / or concentrated by any of the various methods known in the art, including filtration and dialysis, but are not limited to these.

[0352] The purified polypeptide is preferably at least 85% pure, preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact purity level, the polypeptide is pure enough for its intended use. X. How to use Another aspect of the present invention relates to a method for detecting antigen-responsive T cells in, for example, a sample. Generally, the method includes the steps of: providing a plurality of pMHC conjugated multimers of the present disclosure; contacting the conjugated multimers with the sample; and detecting the binding of the conjugated multimers to antigen-responsive T cells in the sample, thereby detecting the T cell response to antigen peptides present in the plurality of conjugated multimers. In one embodiment, the binding is detected by amplifying the barcode region of an oligonucleotide barcode linked to the conjugated multimer. Typically, for pMHCI conjugated multimers, the antigen-responsive T cells are CD8+ T cells whose TCR recognizes MHC class I molecules to which the peptide is bound, while for pMHCII conjugated multimers, the antigen-responsive T cells are CD4+ T cells whose TCR recognizes MHC class II molecules to which the peptide is bound.

[0353] This conjugated multimer technology enables the detection of a large number (potentially >1000) of different antigen-specific T cells in a single sample. The technology can be used, for example, for T cell epitope mapping, detection of immune recognition, diagnostic testing, and measurement of immunoreactivity after vaccination or immunotherapy. For therapeutic use, pMHC conjugated multimers enable the identification and selection of antigen-specific T cells administered for therapeutic purposes, such as in adoptive T cell transfer therapy. A. assay In one embodiment of the present invention, MHC multimers can be used for the detection of individual T cells in a liquid sample using flow cytometry or flow cytometry-like analysis.

[0354] Liquid cell samples can be analyzed using a flow cytometer, where a laser beam passes through a stream, allowing for the detection and counting of individual cells. For the identification of specific T cells using MHC multimers, cells are stained with fluorescently labeled MHC multimers by incubating them with MHC multimers and then forcibly passing them through a nozzle that creates a stream of detached cells in a large volume of liquid. Each cell passes through the laser beam, exciting any fluorescent dye bound to the cell, thereby emitting fluorescence. A sensitive photomultiplier tube detects the emitted fluorescence, providing information about the amount of MHC multimers bound to the cells. By this method, MHC multimers can be used to identify individual T cells and / or specific T cell populations in a liquid sample.

[0355] Cell samples that can be analyzed by MHC multimers in flow cytometry include, but are not limited to, blood samples or fractions thereof, T cell lines (hybridomas, transfected cells), and homogenized tissues such as the spleen, lymph nodes, tumors, brain, or any other tissue containing T cells.

[0356] When analyzing blood samples, whole blood can be used with or without erythrocyte lysis prior to analysis in a flow cytometer. Lysis reagents can be added before or after staining with MHC polymers. When analyzing blood samples without erythrocyte lysis, one or more gating reagents may be included to distinguish lymphocytes from erythrocytes. Preferred gating reagents are marker molecules specific to surface proteins on erythrocytes, allowing for the subtraction of this cell population from the remaining cells in the sample. As an example, fluorescently labeled CD45-specific marker molecules, such as antibodies, can be used to establish trigger identifiers that allow the flow cytometer to distinguish between erythrocytes and stained leukocytes.

[0357] As an alternative to whole blood analysis, lymphocytes can be purified before flow cytometry analysis using standard procedures such as the FICOLL®-Hypaque gradient. Another possibility is to isolate T cells from a blood sample by adding the sample to an antibody or other T cell-specific marker immobilized on a solid support, for example. The T cell-specific marker can then be attached to the solid support, and after washing, the specific T cells can be eluted. This purified T cell population, along with MHC multimers, can then be used for flow cytometry analysis.

[0358] T cells can also be purified from other lymphocytes or blood cells by rosette formation. Human T cells form spontaneous rosettes with sheep erythrocytes, also known as E-rosette formation. E-rosette formation can be performed by incubating lymphocytes with sheep erythrocytes and then purifying them on a density gradient, such as the FICOLL® Hypaque gradient.

[0359] Instead of actively isolating T cells, unwanted cells such as B cells, NK cells, or other cell populations can be removed before analysis. A preferred method for removing unwanted cells is to incubate the sample with a marker molecule specific to the unwanted cells, or one or more surface proteins on it, immobilized on a solid support. An example is the use of beads coated with antibodies or other marker molecules specific to the surface receptors on the unwanted cells, e.g., markers for CD19, CD56, CD14, CD15, or others. Briefly, beads coated with specific surface markers are added to the cell sample. Cells different from the desired T cells, which have the appropriate surface receptors, are bound to the beads. The beads are removed, for example, by centrifugation or magnetic extraction (if magnetic beads are used), and the remaining cells are enriched with T cells.

[0360] Another example is affinity chromatography, which uses columns containing material coated with antibodies or other markers specific to unwanted cells.

[0361] Alternatively, a specific antibody or marker can be added to a blood sample along with complement, thereby killing the cells recognized by the antibody or marker.

[0362] Various gating reagents can be included in the analysis. Here, gating reagents refer to labeled antibodies or other labeled marker molecules that identify a subset of cells by binding to specific surface proteins or intracellular components or intracellular secretory components. Preferred gating reagents when using MHC multimers are antibodies and marker molecules against CD2, CD3, CD4, and CD8 that identify major subsets of T cells. Other preferred gating reagents include antibodies and markers against CD11a, CD14, CD15, CD19, CD25, CD30, CD37, CD49a, CD49e, CD56, CD27, CD28, CD45, CD45RA, CD45RO, CD45RB, CCR7, CCR5, CD62L, CD75, CD94, CD99, CD107b, CD109, CD152, CD153, CD154, CD160, CD161, CD178, CDw197, CDw217, Cd229, CD245, CD247, Foxp3, or other antibodies or marker molecules that recognize specific proteins unique to different lymphocytes, lymphocyte populations, or other cell populations. Examples include antibodies and markers against interleukins, such as IL-2, IL-4, IL-6, IL-10, IL-12, and IL-21; interferons, such as INFγ, TNFα, and TNFβ; or other cytokines or chemokines.

[0363] Gating reagents can be added before, after, or simultaneously with the addition of MHC polymers to the sample. After labeling with MHC polymers and before analysis in a flow cytometer, stained cells can be treated with a fixative (e.g., formaldehyde, ethanol, or methanol) to crosslink the MHC polymers on the cell surface. Stained cells can also be analyzed directly without fixation.

[0364] In one embodiment, a flow cytometer may be provided for separating and collecting specific cell types. This is called cell sorting. MHC multimers combined with sorting in the flow cytometer can be used to isolate antigen-specific T cell populations. The gating reagents described above may include further identification of the T cell population to be iso...

Claims

1. (a) Two or more MHC monomers; (b) streptavidin or avidin comprising four subunits and having at least one biotin-binding site; and (c) Oligonucleotide barcode A barcode-labeled MHC polymer containing, Each MHC monomer is bound to one of the streptavidin or avidin subunits through covalent linkage. The oligonucleotide barcode includes a biotin portion and is non-covalently bound to the biotin binding site on the streptavidin or avidin. MHC multimer.

2. The MHC polymer according to claim 1, wherein the MHC polymer further comprises an MHC-binding peptide loaded onto each MHC monomer of the polymer.

3. The MHC polymer according to claim 1 or 2, wherein the MHC polymer comprises at least three MHC monomers.

4. The MHC polymer according to any one of claims 1 to 3, wherein the MHC polymer comprises four MHC monomers.

5. The MHC polymer according to any one of claims 1 to 4, wherein each MHC monomer and each subunit of streptavidin or avidin includes a conjugation moiety, and each conjugation moiety includes a sortag motif.

6. The MHC polymer according to any one of claims 1 to 5, wherein the MHC monomer is an MHC class I (MHCI) monomer.

7. The MHC polymer according to claim 6, wherein each MHCI monomer comprises a human MHCI heavy chain polypeptide or a functional fragment thereof, and a human β2-microglobulin polypeptide or a functional fragment thereof.

8. The MHC polymer according to claim 7, wherein each MHCI monomer is a fusion protein comprising a human MHCI heavy chain polypeptide or a functional fragment thereof, and human β2-microglobulin or a functional fragment thereof.

9. A method for producing barcode peptide-loaded major histocompatibility complex class I (pMHCI) multimers, (a) Two or more placeholder peptide-loaded MHCI (p) each comprising (i) an MHCI heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a conjugation moiety, and (iv) a placeholder peptide bound in the peptide bond groove of each MHCI monomer. ＊ Steps to provide MHCI monomers; (b) A step of providing streptavidin or avidin, wherein each subunit of the streptavidin or avidin includes a conjugation moiety, and the streptavidin or avidin includes at least one biotin-binding site; (c) Two or more p ＊ Under conditions sufficient for covalent conjugation between the MHCI monomer and the streptavidin or avidin, the p ＊ A combination of the MHCI monomer and the streptavidin or avidin is used to obtain p ＊ A step to produce MHCI multimers; (d) the above p ＊ The p in the MHCI polymer ＊ A step of producing a pMHCI polymer by replacing the placeholder peptide bound in the peptide bond groove of each MHCI monomer with a rescue peptide epitope; and (e) The step of non-covalently binding an oligonucleotide barcode containing a biotin portion to the biotin binding site on streptavidin or avidin. Methods that include...

10. The method according to claim 9, wherein the pMHCI polymer comprises at least three pMHCI monomers.

11. The method according to claim 9 or 10, wherein the pMHCI polymer comprises four pMHCI monomers.

12. each p ＊ The conjugation portion of the MHCI monomer includes X, and the conjugation portion of each subunit of streptavidin or avidin includes Y. (i) X is a distorted alkyne, and Y is an azid; (ii) X is a terminal alkyne and Y is an azide; (iii) X is an azid, and Y is a terminal alkyne; (iv) X is Azid, and Y is a distorted Alkyne; (v) X is a diene and Y is a dienophil; (vi) X is a dienophile and Y is a diene; (vii) X is a thiol and Y is an alkene; or (viiii) X is an alkene and Y is a thiol. The method according to any one of claims 9 to 11.

13. The method according to claim 12, wherein the azide is a copper chelated azide.

14. The method according to claim 13, wherein the copper chelated azide is picolyl azide.

15. The method according to any one of claims 9 to 14, wherein each conjugation portion includes a sortag motif.

16. each p ＊ The method according to any one of claims 9 to 15, wherein the MHCI monomer comprises a human MHCI heavy chain polypeptide or a functional fragment thereof, and a human β2-microglobulin polypeptide or a functional fragment thereof.

17. A polypeptide library comprising a plurality of MHC polymers according to any one of claims 6 to 8, wherein each of the MHC polymers is a peptide-loaded MHC class I (pMHCI) polymer.

18. A method for isolating lymphocytes bound to MHC multimers, wherein the method is (a) the step of contacting a plurality of lymphocytes with the polypeptide library described in claim 17; and (b) A step of generating a plurality of compartments, each compartment comprising lymphocytes bound to the pMHCI polymer of the polypeptide library and a capture support. Methods that include...

19. The method according to claim 18, wherein the plurality of lymphocytes are T cells, B cells, or NK cells.

20. A method for identifying T cells bound to pMHC multimers, wherein the method is (a) the step of contacting a plurality of lymphocytes with the polypeptide library of claim 17; (b) the step of compartmentalizing one of the plurality of lymphocytes bound to a pMHCI multimer of the polypeptide library into a single compartment, wherein the pMHCI multimer includes a unique identifier; and (c) The step of determining the unique identifier for the pMHCI bound to the compartmentalized lymphocyte. Methods that include...

21. A method for producing the library described in claim 17, (a)(i) An MHC I heavy chain polypeptide or a functional fragment thereof, (ii) a β2-microglobulin polypeptide or a functional fragment thereof, (iii) a conjugation moiety, and (iv) a plurality of placeholder peptide-loaded MHC I (p ＊ MHC I) monomers each containing a placeholder peptide bound in the peptide-binding groove of each MHC I monomer; (b) A step of providing a plurality of streptavidins or avidins, wherein each subunit of the streptavidin or avidin includes a conjugation moiety, and each streptavidin or avidin includes at least one biotin-binding site; (c) the above p ＊ The plurality of p under conditions sufficient for covalent conjugation between the MHCI monomer and the streptavidin or avidin ＊ The MHCI monomer is brought into contact with the plurality of streptavidins or avidins, and a plurality of p ＊ A step to produce MHCI multimers; (d) the above p ＊ A step of producing multiple pMHCI multimers by replacing the placeholder peptide bound in the peptide binding groove of the MHCI multimer with a plurality of unique rescue peptide epitopes; and (e) The step of non-covalently binding an oligonucleotide barcode containing a biotin portion to the biotin binding site of streptavidin or avidin. Methods that include...

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