Patent Application
20210155670
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 8, 2021, is named 116550-01-5011-US_ST25.txt and is 38 kilobytes in size.
The class I molecules of the Major Histocompatibility Complex (MHC) play a pivotal role in orchestrating an adaptive immune response by alerting the immune system to the presence of developing infections and tumors in the body. Immune surveillance is achieved through the display of short (8-11 residue long) peptides derived from viral proteins or mutated oncogenes via a tight interaction with the MHC-I peptide-binding groove. Such peptide/MHC-I protein complexes are assembled inside the cell and displayed on the surface of all antigen-presenting cells where they can interact with specialized receptors on T cells and natural killer (NK) cells. The MHC-I proteins are extremely polymorphic (more than 13,000 different alleles have been identified in the human population to date), and each allele can display an estimated 1,000-10,000 different peptides, which makes the characterization of specific T cell responses against a panel of known peptide epitopes a daunting task. Further adding to the challenge of characterizing such T cell responses is the fact that typical T cell receptor affinities for their cross-reactive pMHC (peptide loaded MHC) ligands are low (in the micromolar range).
Multivalent, fluorescent pMHC-I multimers (Altman J D et al., Science 274, 94-96 (1996); incorporated by reference herein) were developed and used to stain T cells (Altman and Davis, Curr Protoc Immunol Ch. 17 Unit 17.3 (2003); incorporated by reference herein). T cells that recognize a specific peptide/MHC multimer can be identified and sorted using flow cytometry, and their receptors can be identified in subsequent steps. Peptide loaded MHC-I tetramers have revolutionized experimental immunology and the development of new therapies, leading to a breadth of discoveries (Doherty, J Immunol 187, 5-6 (2011); incorporated by reference herein). However, the preparation of properly conformed pMHC molecules via in vitro refolding of inclusion bodies expressed in E. coli (Garboczi D N et al., PNAS 89, 3429-3433 (1992); incorporated by reference herein), requires a laborious, multi-step process that is highly inefficient (typical refolding yields are <5% by weight).
Moreover, all MHC molecules expressed in E. coli lack the functionally relevant post-translational glycosylation that is required for proper immune surveillance function (Barber L D et al., J Immunol 156, 3275-3284 (1996); incorporated by reference herein). For example, in E. coli expressed MHC molecules, a conserved glycan at residue N86, which is present in all human HLA-A, HLA-B, and HLA-C alleles, and is located at a site near the TCR recognition surface, is completely missing. Therefore, E. coli expressed refolded tetramers will fail to identify high-affinity T cell receptors and natural killer cell receptors that have binding to an MHC molecule, where that binding is dependent on glycosylation. Such TCRs can be important targets for both understanding antigen recognition processes, and the development of immunotherapies to combat bacterial and viral infections and cancer.
Thus, there remains a need for novel MHC-I tetramer compositions that allow for the identification of such previously unidentified T cell and NK receptors, as well as methods for making such tetramers in an efficient manner.
Disclosed herein are novel glycosylated peptide receptive MHC-I complexes that allow for efficient production of pMHC-I multimers that can be used, for example, as T cell/NK cell staining reagents and drug delivery vehicles. Such glycosylated peptide receptive MHC-I complexes include an MHC-I (e.g., a single-chain MHC-I) and are produced in mammalian expression systems (e.g., CHO and HEK cells) that allow for the glycosylation of the complexes at one or more native amino acid positions (e.g., at the conserved N86 in HLA-A, HLA-B, and HLA-C). The protein constructs used to make such glycosylated peptide receptive MHC-I complexes include leucine zipper domains and one or more purification tags that facilitate purification of the complexes. The subject peptide receptive MHC-I/TAPBPR complexes are capable of binding high-affinity peptides with the correct peptide specificity (
In a first aspect, provided herein are MHC-I protein constructs. In some embodiments, the MHC-I protein constructs include: (a) a first polypeptide that includes a MHC Class I heavy chain that is glycosylated at least one native glycosylation position; and (b) a second polypeptide that includes a β2 microglobulin. In some embodiments, the constructs further include: (c) a third polypeptide that includes a leucine zipper domain; and (d) a fourth polypeptide that includes a protease cleavage site. In exemplary embodiments, (a), (b), (c), and (d), are covalently linked from N-to C-terminus orientation according to the following order: (b)-(a)-(d)-(c).
In some embodiments, the β2 microglobulin is N-terminal to the MHC Class I heavy chain, and the MHC-I protein construct further includes a first peptide linker between the β2 microglobulin and the MHC Class I heavy chain.
In some embodiments, the leucine zipper domain is C-terminal to the MHC Class I heavy chain, and the protease cleavage site is between the leucine zipper domain and the MHC Class I heavy chain. In certain embodiments, the MHC-I protein construct further includes a second peptide linker between the MHC Class I heavy chain and the protease cleavage site and a third peptide linker between the protease cleavage site and the leucine zipper domain.
In exemplary embodiments, the MHC-I protein construct further includes a (e) multimerization tag (e.g., an AviTag). In some embodiments, the multimerization tag is C-terminal to the MHC Class I heavy chain. In certain embodiments, the multimerization tag is C-terminal to the MHC Class I heavy chain and N-terminal to the protease cleavage site such that the tag remains bound to the MHC Class I heavy chain after protease cleavage. In further embodiments, the MHC-I protein construct further includes a fourth peptide linker between the MHC Class I heavy chain and the multimerization tag.
In some embodiments, the MHC-I protein constructs include: (a) a first polypeptide that includes a MHC Class I heavy chain, (b) a second polypeptide that includes a β2 microglobulin; (c) a third polypeptide that includes a leucine zipper domain; (d) a fourth polypeptide that includes a protease cleavage site; and (e) a multimerization tag, where (a), (b), (c), (d) and (e) are covalently linked from N-to C-terminus orientation according to the following order: (b)-(a)-(e)-(d)-(c). In an exemplary, the MHC-I protein constructs further include (f) one or more purification tags. In some embodiments that include the (f) one or more purification tags, (a), (b), (c), (d) and (f) are covalently linked from N-to C-terminus orientation according to the following order: (b)-(a)-(d)-(c)-(f).
In some embodiments, the MHC Class I heavy chain of the MHC-I protein construct is a human HLA-A, HLA-B, or HLA-C or a mouse H-2D or H-2L. In exemplary embodiments, the MHC Class I heavy chain is an HLA-A*02:01, HLA-A*24:02, HLA-A*68:01 or HLA-A*68:02 allele heavy chain. In certain embodiments, MHC Class I heavy chain has one or more mutations in the α3 domain of the heavy chain.
In exemplary embodiments, the protease cleavage site is a TEV protease cleavage site. In some embodiments, the multimerization tag of the MHC protein construct is an AviTag. In particular embodiments, the AviTag includes a biotinylated lysine.
In exemplary embodiments of the MHC-I protein construct, the MHC class I heavy chain is glycosylated at residue N86.
In another aspect, provided herein is a TAPBPR protein construct that includes: (a) a first polypeptide that includes a TAPBPR; (b) a second polypeptide that includes a leucine zipper domain; and (c) a third polypeptide that includes a protease cleavage site. In exemplary embodiments, (a), (b), and (c) are covalently linked from N-to C-terminus orientation according to the following order: (a)-(c)-(b).
In some embodiments, the leucine zipper domain is C-terminal to the TAPBPR and the protease cleavage site is between the TAPBPR and the leucine zipper domain. In certain embodiments, the TAPBPR protein construct includes a first peptide linker between the TAPBPR and the protease cleavage site and a second peptide linker between the protease cleavage site and the leucine zipper domain.
In some embodiments, the TAPBPR protein construct further includes: (d) one or more purification tags. In certain embodiments, the purification are C-terminal to the leucine zipper domain such that the purification tag remains bound to the leucine zipper domain after protease cleavage.
In exemplary embodiments, the TAPBPR protein construct includes: (a) a first polypeptide that includes a TAPBPR; (b) a second polypeptide that includes a leucine zipper domain; and (c) a third polypeptide that includes a protease cleavage site; and (d) one or more purification tag, wherein (a), (b), (c), and (d) are covalently linked from N-to C-terminus orientation according to the following order: (a)-(c)-(b)-(d).
In some embodiments, the protease cleavage site is specific for a TEV protease.
In certain embodiments, the one or more purification tags include a first Strep-tag II® tag. In certain embodiments, the one or more purification tags also includes a second Strep-tag II® tag C-terminal to the first Strep-tag II® tag and a third peptide linker between the first Strep-tag II® tag and the second Strep-tag II® tag.
In another aspect, provided herein are polynucleotides encoding the MHC-I protein constructs and TAPBPR protein constructs provided herein.
In one aspect, provided herein are expression vectors that include polynucleotides encoding the MHC-I protein constructs and/or TAPBPR protein constructs provided herein. In some embodiments, the polynucleotide expression vector includes a first polynucleotide that encodes any one of the subject MHC-I protein constructs described herein and a second polynucleotide that encodes any one of the TAPBPR protein construct described herein. In particular embodiments, the expression vector further includes a CMV promoter.
In some embodiments, the expression vector is an expression vector composition that includes two expression vectors. The first expression vector includes a first polynucleotide that encodes any one of the subject MHC-I protein constructs described herein. The second expression vector includes a second polynucleotide that encodes any one of the TAPBPR protein construct described herein. In certain embodiments, the first and second polynucleotide expression vector each includes a CMV promoter.
In another aspect, provided herein are mammalian host cells that include any of the expression vectors provided herein. In certain embodiments, the host cell is a CHO or HEK cell. In particular embodiments, the host cell is a CHO-K1 cell.
In another aspect, provided herein are methods of making peptide receptive MHC-I complexes. In some embodiments, the method includes the steps of: a) providing a mammalian host cell that includes: i) a first polynucleotide that encodes for one of the MHC protein constructs provided herein, and ii) a second polynucleotide that encodes for one of the TAPBPR constructs provided herein, where the leucine zipper domain of the first protein construct specifically binds the leucine zipper domain of the second protein construct and where the protease cleavage site in the first protein construct is the same protease cleavage site as in the second protein construct; b) culturing the mammalian host cell in a culture medium under conditions where the MHC protein construct and TAPBPR construct are expressed; c) collecting the culture medium after culturing; d) applying the culture medium to a column that includes an agent that binds the first protein construct or second protein construct, thereby forming a zippered MHC-I/TAPBPR complex bound to the column, wherein the MHC-I/TAPBPR complex includes an MHC-I heavy chain that is glycosylated at least one native glycosylation position; e) eluting the zippered MHC-I/TAPBPR complex from the column; and f) contacting the zippered MHC-I/TAPBPR complex with a protease specific for the protease cleavage site of the first protein construct and the protease cleavage site of the second protein construct, thereby creating a purified peptide-receptive MHC-I complex. In some embodiments, the peptide receptive MHC-I complexes further comprise a TAPBPR protein.
In some embodiments, the mammalian host cell includes an expression vector or expression vector composition provided herein. In certain embodiments, the column includes streptavidin or Strep-Tactin®. In some embodiments, the protease is TEV.
In exemplary embodiments, the leucine zipper domain of the first protein construct is Fos and the leucine zipper domain of the second protein construct is Jun. In other embodiments, the leucine zipper domain of the first protein construct is Jun and the leucine zipper domain of the second protein construct is Fos.
In another aspect, provided herein is a method of making a purified peptide receptive MHC-I complex. This method includes the steps of: a) providing a mammalian host cell that includes: i) a first polynucleotide that encodes for one of the MHC protein constructs provided herein, and ii) a second polynucleotide that encodes for a TAPBPR; b) culturing the mammalian host cell in a culture medium under conditions where the MHC protein construct and TAPBPR are co-expressed; and c) collecting the protein construct and TAPBPR.
In another aspect, provided herein is a method of making a tetrameric peptide MHC-I complex. The method includes the steps of: (a) contacting a plurality of purified peptide receptive MHC-I complexes with streptavidin, where the purified peptide receptive complexes include at least one biotinylated residue and an MHC-I heavy chain that is glycosylated at least one native glycosylation position, thereby making a tetrameric peptide receptive MHC-I complex; and (b) contacting the tetrameric peptide receptive MHC-I complex with a plurality of peptides of interest, thereby forming the tetrameric peptide-MHC-I complex. In some embodiments, the purified peptide receptive MHC-I complexes each include exactly one biotinylated residue.
In exemplary embodiments, the purified peptide receptive MHC-I complexes each include an AviTag that includes one lysine residue. In some embodiments, the method further includes the step of biotinylating the lysine residue in the AviTag. In certain embodiments, biotinylating the lysine residue in the AviTag includes contacting the purified peptide receptive MHC-I complexes with biotin in the presence of a biotin ligase enzyme (e.g., BirA). In particular embodiments, the streptavidin includes a fluorescent tag. In certain embodiments, at least one of the peptide receptive MHC-I complexes of the plurality of peptide receptive MHC-I complexes comprises a TAPBPR.
In another aspect, provided herein are tetrameric peptide-MHC class I complexes that include: a) a tetrameric streptavidin molecule comprised of four streptavidin subunits; and b) four peptide-MHC Class I complexes, where at least one of the pMHC-I is glycosylated at at least one native glycosylation position, and where each streptavidin subunit is bound via its biotin binding site to one of the four peptide-MHC Class I complexes.
In some embodiments, each of the peptide-MHC Class I complexes is glycosylated at residue N86 of the MHC Class I heavy chain. In certain embodiments, each of the four peptide-MHC Class I complexes includes a single-chain MHC-I, wherein the single-chain MHC-I comprises a MCH-I heavy chain covalently linked to a β2 microglobulin. In certain embodiments, the peptide-MHC Class I complexes further comprises a fluorescent tag. In particular embodiments, the fluorescent tag is attached to the tetrameric streptavidin molecule.
Some of the Figures are better understood when presented in color. Applicant's original submission included color figures and therefore, Applicant considers the color versions of the figures to be part of the original disclosure. Applicant reserves the right to present color versions of the figures in later proceedings.
A. Overview
Disclosed herein are novel glycosylated peptide receptive MHC-I complexes that allow for efficient production of high affinity peptide (peptide of interest) MHC-I multimers (pMHC-I multimers). Such glycosylated peptide receptive MHC-I complexes include an MHC-I (e.g., a single-chain MHC-I) and are produced in mammalian expression systems (e.g., CHO and HEK cells) that allow for the glycosylation of the complexes at one or more amino acid positions (e.g., conserved N86 in HLA-A, HLA-B, and HLA-C). In certain embodiments, the glycosylated peptide receptive MHC-I complex further includes a TAPBPR. In some embodiments, the peptide receptive MHC-I complex includes a endogenous peptides, chaperones, or other proteins/peptides associated with the peptide receptive complex. In certain embodiments, the glycosylated peptide receptive MHC-I complex is a glycosylated single chain MHC-I molecule capable of accepting a peptide of interest that is not associated with any other protein or peptide. The protein constructs used to make such glycosylated peptide receptive MHC-I complexes can include heterodimerization domains (e.g., leucine zipper domains) and/or one or more purification tags that facilitate purification of the complexes. The subject peptide receptive MHC-I complexes are capable of binding high-affinity peptides with the correct peptide specificity (
The compositions and methods described herein provide an efficient process for producing MHC tetramers than previous labor-intensive and inefficient methods.
Moreover, the use of glycosylated MHC-I molecules coexpressed in mammalian cells with the molecular chaperone TAPBPR results in a number of advantages. It provides native, peptide-receptive MHC-I complexes containing MHC-I molecules that are glycosylated at one or more native positions (e.g., the conserved N86). Upon multimerization and loading with high-affinity peptide, glycosylated peptide receptive MHC-I complexes allow stable antigen presentation in a physiologically relevant form of the MHC-I molecule.
Further, the multimers (e.g., tetramers) produced from the glycosylated peptide receptive MHC-I complexes provided herein advantageously allow for the identification of high-affinity T cell and natural killer cell receptors previously unidentified using traditional unglycosylated MHC tetramers, such as those produced in non-mammalian expression systems (e.g., Drosophila S2 or E. coli expression systems). TCRs identified using the MHC-I tetramers made using the complexes provided herein can provide important targets for both understanding antigen recognition processes, and the development of immunotherapies to combat bacterial and viral infections and cancers. Aspects of the glycosylated peptide receptive MHC-I complexes are further described in detail below.
B. MHC Protein Constructs
In one aspect, provided herein are MHC-I protein constructs that include: a) a MHC-I that includes a MHC-I heavy chain and a β2 microglobulin; b) a heterodimerization domain; and c) a protease cleavage site (see, e.g.,
The a) MHC-I, b) heterodimerization domain, and c) protease cleavage site of the MHC protein constructs provided herein are covalently linked from N- to C-terminus according to the following order: a) MHC-I, c) protease cleavage site, and b) heterodimerization domain. Any suitable linkers can be used to link the various parts of the MHC protein construct together, including those provided herein.
In related aspects, the MHC-I protein construct lacks a heterodimerization domain.
In some embodiments, the MHC-I protein construct further includes a d) multimerization tag that facilitates the formation of multimers (e.g., tetramers). Exemplary multimerization tags include, for example, tags that facilitate biotinylation such as AviTags. Biotinylated MHC-I protein constructs can be attached to a backbone (e.g., streptavidin) to form MHC multimers. In such embodiments, the parts of the MHC-I protein construct are covalently linked from N- to C-terminus according to the following order: a) single-chain MHC-I, d) multimerization tag, c) protease cleavage site, and b) heterodimerization domain (see, e.g.,
Subject MHC-I protein constructs provided herein are made using any suitable technique including standard molecule biology and cloning techniques as described by Maniatis et al., “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory, 1982, CSH, New York.
Nucleic acids encoding the MHC-I protein constructs and chaperone protein constructs described herein are coexpressed in a mammalian expression system (e.g., CHO or HEK cells). Expression in mammalian cells allow for the glycosylation of the single-chain MHC-I at one or more native glycosylation positions (e.g., N86). As used herein, a native glycosylation position refer to an amino acid position that is glycosylated in wild-type MHC-I. Such positions are referred to by a numbering convention based on the mature MHC-I molecule (i.e., without signal peptide) wherein amino acid position 1 is the first amino acid at the N-terminal of the mature MHC-I molecule, amino acid position 2 is the second amino acid from the N-terminal of the mature MHC-I molecule, etc.
As used herein, an “MHC class I,” “Major Histocompatibility Complex class I,” “MHC-I,” MHC I, and the like all refer to a member of one of two primary classes of major histocompatibility complex (MHC) molecules (the other being MHC class II) that are found on the cell surface of all nucleated cells in the bodies of j awed vertebrates. MHC class I molecules function to display peptide fragments of antigen to cytotoxic T cells, resulting in an immediate response from the immune system against a particular peptide antigen displayed within the peptide-binding groove of an MHC-I molecule.
MHC-I molecules are heterodimers that consist of two polypeptide chains: an a (heavy chain) and a β2-microglobulin (light chain). The two chains are typically linked noncovalently via interactions between the light chain and the α3 domain of the heavy chain and the floor of the α1/α2 domain. The heavy chain is polymorphic and encoded by an HLA gene, while the light chain is species-invariant and encoded by the Beta-2 microglobulin gene. The α3 domain is plasma membrane-spanning and interacts with the CD8 co-receptor of T cells. The α3-CD8 interaction holds the MHC-I molecule in place while the T cell receptor (TCR) on the surface of the cytotoxic T cell binds its syngeneic ligand (or matched, in the sense that both the TCR and MHC-I are encoded in the same germline), and checks the displayed peptide for antigenicity. The α1 and α2 domains of the heavy chain fold to make up a groove for peptides to bind. MHC class I molecules bind peptides that, in most cases, are 8-10 amino acid in length.
In mice, MEW class I is called the “H-2 complex” or “H-2” and include the H-2D, H-2K and H-2L subclasses. In humans, MEW class I molecules include the highly polymorphic human leukocyte antigens HLA-A, HLA-B, HLA-C and the less polymorphic HLA-E, HLA-F, HLA-G, HLA-K and HLA-L. Each human leukocyte antigen (e.g., HLA-A) includes multiple alleles. For example, HLA-A includes over 2,430 non-redundant known alleles. Exemplary HLA-A alleles used in the protein constructs and methods described herein include, but are not limited to: HLA-A*02:01, HLA-A*24:02, HLA-A*68:01 or HLA-A*68:02.
In some embodiments, the MHC-I constructs provided herein include a single-chain MHC-I. Such single-chain MHC-I constructs include a MHC-I heavy chain covalently attached to a β2-microglobulin. In some embodiments of the MHC-I constructs, the single-chain MHC-I includes, from N- to C-terminus, MHC-I heavy chain-linker-02 microglobulin. In another exemplary embodiment, the single-chain MHC includes, from N- to C-terminus, β2 microglobulin-linker-MHC-I heavy chain. In other embodiments, the MHC-I constructs include an MHC-I where the MHC-I heavy chain and β2 microglobulin are separate and not covalently attached by a linker.
Any suitable MHC-I heavy chain can be included in the MHC-I constructs provided herein. In some embodiments, the MHC-I heavy chain is an HLA-A heavy chain. In certain embodiments, the MHC-I heavy chain is an HLA-B heavy chain. In other embodiments, the MHC-I heavy chain is an HLA-C heavy chain. In an exemplary embodiment, the MEW heavy chain is an HLA-A*02:01, HLA-A*24:02, HLA-A*68:01 or HLA-A*68:02 allele heavy chain. In other embodiments, the MHC-I protein construct includes a mouse H-2. In certain embodiments, the H-2 is an H-2D, H-2K or H-2L. In exemplary embodiments, the H-2 is H-2DD or H-2LD. In some embodiments, the MHC construct include a variant of a wild-type MHC-I heavy chain. In particular embodiments, the variant MHC-I heavy chain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a wild-type MHC-I heavy chain. Any suitable linker can be used to attach the MHC-I heavy chain to the β2 microglobulin. In certain embodiments, the linker is (GGGS)x, wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In an exemplary embodiment, the linker is (GGGS)4.
In some embodiments, the MHC-I protein constructs provided herein include a heterodimerization domain that, upon binding to a heterodimerization domain of the chaperone protein construct, forms a stable “zippered” heterodimeric MHC-I/chaperone complex. Such stable “zippered” heterodimeric MHC-I/chaperone complexes are subsequently purified using any technique in the art. Any suitable heterodimerization domain that facilitate the heterodimerization of a MHC protein construct and chaperone protein construct can be used. In some embodiments, the heterodimerization domains favor the formation of the heterodimeric MHC-I/chaperone complex over homodimers that include two MHC-I protein constructs or two chaperone protein constructs. In some embodiments, the heterodimerization domains include coiled-coil heterodimerization domains. In certain embodiments, the heterodimerization domains are leucine zipper domains. In an exemplary embodiment, the leucine zipper domain is a Fos or Jun leucine zipper domain. In particular embodiments, the MHC-I protein construct includes a Fos domain and the chaperone protein construct includes a Jun domain. In other embodiments, the MHC-I protein construct includes a Jun domain and the chaperone protein construct includes a Fos domain.
The MHC-I protein constructs provided herein can include a protease cleavage site that facilitates the cleavage of the heterodimerization domain from the MHC-I protein construct after co-purification of the “zippered” heterodimeric MHC-I/chaperone complex. Any suitable protease cleavage site can be incorporated into the MHC-I protein construct. The protease that recognizes the protease cleavage site does not cleave the MHC-I protein construct at any site or in any domain other than the protease cleavage site. In an exemplary embodiment, the same protease cleavage site included in the MHC protein construct is also include in the chaperone protein construct. In such an embodiment, one protease is used to remove the heterodimerization domains on each of construct of the “zippered” heterodimeric MHC-I/chaperone complex to produce a mature “unzippered” peptide receptive MHC-I complex. Suitable cleavage sites include, but are not limited to enterokinase (DDDK), Factor Xa (IEGR/IDGR), Tobacco Etch Virus (ENLYFQS), thrombin (LVPRGS) and PreScission (LEVLFQGP), furin (Arg-X-X-ArgV) and genenase (Arg-X-(Lys/Arg)-Arg) protease cleavage sites. In an exemplary embodiment, the protease cleavage site is a Tobacco Etch Virus (TEV) protease cleavage site.
In some embodiments, the MHC-I protein construct can further include e) one or more purification tags at its carboxyl terminal that facilitate purification of the MHC-I protein construct and/or a “zippered” heterodimeric MHC-I/chaperone complex. In such embodiments, the parts of the MHC protein construct are covalently linked from N- to C-terminus according to the following order: a) single-chain MHC-I, d) protein tag, c) protease cleavage site, b) heterodimerization domain and e) purification tag(s). In some embodiments, the purification tag allows for affinity purification of the “zippered” heterodimeric MHC-I/chaperone complexes from cell culture medium. Suitable purification tags that can be included in the chaperone protein construct include, but are not limited to, histidine tags, Strep-Tags®, MYC-tags and HA-tags. In an exemplary embodiment, the purification tag is a Strep-Tags®.
Following purification and “unzippering” of the glycosylated heterodimeric MHC-I/chaperone complexes by protease cleavage the resulting peptide receptive MHC-I complexes can be used to form multimers (e.g., tetramers or Dextramers®). To facilitate multimerization, the MHC-I protein constructs provided herein optionally include a protein tag that is capable of being biotinylated (see
In an exemplary embodiment, the MHC-I protein construct includes from N- to C-terminus orientation: a) a signal peptide; b) β2-microglobulin; c) an MHC-I heavy chain; d) a protein tag for multimerization; e) a protease cleavage site; and f) a leucine zipper heterodimerization domain (e.g., Fos or Jun domain) (see, e.g.,
C. Chaperone Protein Constructs
In another aspect, provided herein are chaperone protein constructs that include: a) a chaperone; b) a heterodimerization domain; and c) a protease cleavage site. Such chaperone protein constructs (e.g., TAPBPR protein constructs), together with the MHC protein constructs provided herein, are useful in making the subject glycosylated MHC-I/chaperone complexes.
The a) a chaperone; b) a heterodimerization domain; and c) a protease cleavage site of the chaperone protein constructs provided herein are covalently linked from N- to C-terminus according to the following order: a) chaperone, c) protease cleavage site, and b) heterodimerization domain. Any suitable linkers can be used to link the various parts of the chaperone construct together, including those provided herein. Subject MEW protein constructs provided herein are made using any suitable technique including standard molecule biology and cloning techniques as described by Maniatis et al., “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory, 1982, CSH, New York
In some aspects, the chaperone protein construct lacks a heterodimerization domain, including aspects where both the chaperone protein construct and the MHC-I protein construct lack heterodimerization domains.
In some embodiments, the chaperone included in the chaperone protein construct is a Tapasin Binding Protein Related (TAPBPR). TAPBPR protein includes a signal sequence, three extracellular domains comprising a unique membrane distal domain, an IgSF (immunoglobulin superfamily) V domain and an IgC1 domain, a transmembrane domain, and a cytoplasmic region. (Boyle et al., PNAS 110 (9) 3465-3470 (2013); incorporated by reference herein).
When co-expressed in mammalian cells, the chaperone protein constructs and MHC-I protein constructs provided are capable of forming “zippered” glycosylated heterodimeric MHC-I/chaperone complexes via the heterodimerization domains included in each construct. As discussed above, any suitable heterodimerization domain that facilitates the formation of the “zippered” glycosylated heterodimeric MHC-I/chaperone complexes over homodimeric species can be used. In some embodiments, the heterodimerization domains include coiled-coil heterodimerization domains. In certain embodiments, the heterodimerization domains are leucine zipper domains. In an exemplary embodiment, the leucine zipper domain is a Fos or Jun leucine zipper domain. In particular embodiments, the MHC-I protein construct includes a Fos domain and the chaperone protein construct includes a Jun domain. In other embodiments, the MHC-I protein construct includes a Jun domain and the chaperone protein construct includes a Fos domain.
The chaperone protein constructs provided herein include a protease cleavage site that facilitates the cleavage of the heterodimerization domain from the chaperone protein construct after co-purification of the zippered MHC protein construct/chaperone protein construct heterodimer. Any suitable protease cleavage site can be incorporated into the chaperone protein construct. The protease that recognizes the protease cleavage site does not cleave the chaperone protein construct at any site or in any domain other than the protease cleavage site. In an exemplary embodiment, the same protease cleavage site included in the chaperone protein construct is also include in the MHC-I protein construct. In such an embodiment, one protease is used to remove the heterodimerization domains on each of construct of the “zippered” glycosylated heterodimeric MHC-I/chaperone complex, thereby “unzippering” the complex and resulting in a peptide receptive MHC-I. Suitable cleavage sites include, but are not limited to enterokinase (DDDK), Factor Xa (IEGR/IDGR), Tobacco Etch Virus (ENLYFQS), thrombin (LVPRGS) and PreScission (LEVLFQGP), furin (Arg-X-X-ArgV) and genenase (Arg-X-(Lys/Arg)-Arg) protease cleavage sites. In an exemplary embodiment, the protease cleavage site is a Tobacco Etch Virus (TEV) protease cleavage site.
In some embodiments, the chaperone protein construct further includes: d) one or more purification tags that facilitate the co-purification of the zippered MHC protein construct/chaperone protein construct heterodimers (also referred to herein as glycosylated MHC-I/chaperone complexes). In such embodiments, the parts of the chaperone protein construct are covalently linked from N- to C-terminus according to the following order: a) chaperone, c) protease cleavage site, b) heterodimerization domain, and d) purification tag(s). Any tag that allows for co-purification of the zippered MHC-I protein construct/chaperone protein construct heterodimer can be included in the chaperone protein construct. In some embodiments, the purification tag allows for affinity purification of the “zippered” glycosylated heterodimeric MHC-I/chaperone complexes from cell culture supernatant. Suitable purification tags that can be included in the chaperone protein construct include, but are not limited to, histidine tags, Strep-Tags®, MYC-tags and HA-tags. In an exemplary embodiment, the purification tag is a Strep-Tags®.
Following purification and protease treatment of the “zippered” glycosylated heterodimeric MHC-I/chaperone complexes to yield peptide receptive MHC-I complexes, the peptide receptive MHC-I complexes can be multimerized, as discussed above. Such multimers can be stored or directly loaded with high-affinity peptides (pMHC-I multimers). Once loaded with high-affinity peptides, the glycosylated pMHC-I multimers can be used in applications wherein such multimers are useful, as disclosed herein (e.g., T cell repertoire analysis, receptor ligand characterization studies and T cell stimulation). In other embodiments, the mature “unzippered” glycosylated heterodimeric MHC-I/chaperone complexes are loaded with high-affinity peptides before the formation of multimers to form glycosylated pMHC-I complexes. In an exemplary embodiment, the chaperone protein construct includes from N- to C-terminus orientation: a) a TAPBPR chaperone; b) a protease cleavage site; c) a leucine zipper heterodimerization domain (e.g., Fos or Jun domain), and d) one or more purification tags (see, e.g.,
D. Glycosylated MHC/Chaperone Mammalian Expression Systems
In another aspect, provided herein are expression vectors that include a polynucleotides encoding one or more of the MHC protein constructs and/or chaperone protein constructs provided herein. Expression vector compositions that include a) a first polynucleotide encoding a MHC-I protein construct described herein; and b) a second polynucleotide encoding a chaperone protein construct described herein are also provided. In preferred embodiments, the expression vector is a mammalian expression vector. In exemplary embodiments, each of the first polynucleotide and second polynucleotide are included in the same expression vector (see
As discussed herein, the MHC-I protein constructs and chaperone protein constructs are expressed using a mammalian expression system and/or cell line that advantageously allows for post-translational glycosylation of the MHC-I protein at one or more native positions (e.g., N86). Such glycosylated MHC-I proteins, when multimerized, allow for the identification of high-affinity T cell and natural killer (NK) cell receptors previously unidentified using traditional unglycosylated pMHC-I tetramers produced in non-mammalian expression systems (e.g., Drosophila S2 or E. coli expression systems).
Cultured mammalian cell lines that are useful for making the glycosylated peptide receptive MHC-I complexes and tetramers described herein include, but are not limited to, Chinese hamster ovary (CHO), COS, HEK and HeLa cell lines. In certain embodiments, the protein constructs provided herein are expressed using a CHO-K1 cell line.
E. Methods of Making Glycosylated Peptide Receptive MHC-I Complexes
The MHC-I protein constructs, chaperone protein constructs and mammalian expression systems provided herein can be used to make peptide receptive MHC-I complexes, wherein the MHC-I molecule is glycosylated at one or more native glycosylation position (e.g., conserved N86 of MHC-I).
In such a method, a mammalian host cell (e.g., CHO or HEK cell) is first provided that includes an expression vector having a first nucleic acid encoding a MHC-I protein construct described herein. In some embodiments, wherein an MHC-I/chaperone construct is desired, the host cell further includes an expression vector having a second nucleic acid encoding a chaperone protein construct described herein. In other embodiments, each of the first nucleic acid and second nucleic acid are included in separate expression vectors in the mammalian host cell. The mammalian host cell is cultured under suitable conditions where the MHC-I protein construct and chaperone protein construct are co-expressed and the constructs undergo post-translation glycosylation at one or more native glycosylation positions (e.g., conserved N86 of a MHC-I molecule). As described herein, the MHC-I protein construct and chaperone protein construct each include a heterodimerization domain (e.g., leucine zipper domains) that facilitate the heterodimerization of the MHC-I protein construct and the chaperone protein construct to form “zippered” heterodimeric MHC-I/chaperone complexes. In some embodiments, the heterodimerization domains include coiled-coil heterodimerization domains. In certain embodiments, the heterodimerization domains are leucine zipper domains. In an exemplary embodiment, the leucine zipper domain is a Fos or Jun leucine zipper domain. In particular embodiments, the MHC-I protein construct includes a Fos domain and the chaperone protein construct includes a Jun domain. In other embodiments, the MHC-I protein construct includes a Jun domain and the chaperone protein construct includes a Fos domain.
The “zippered” heterodimeric MHC-I/chaperone complexes are purified from the cellular supernatant using any suitable methods. In some embodiments, a purification tag is included in the C-terminal of one or both of the MHC-I protein construct and chaperone protein construct. Suitable purification tags that can be included in the chaperone protein construct and/or MHC-I protein construct include, but are not limited to, histidine tags, Strep-Tags®, MYC-tags and HA-tags. In an exemplary embodiment, the chaperone protein construct includes a Strep-Tag®. The culture medium containing the purification tagged “zippered” heterodimeric MHC-I/chaperone complexes is applied to an affinity column that binds the complexes via the purification tags. In some embodiments, the affinity column includes streptavidin or Strep-Tactin®, which allows for the capture of zippered” heterodimeric MHC-I/chaperone complexes that include a Strep-Tag®. Following capture of the zippered” heterodimeric MHC-I/chaperone complexes, the complexes are eluted from the column and subsequently contacted with a protease (e.g., TEV protease) that cuts each of the MHC protein construct and chaperone protein construct at a protease cleavage site, thereby removing the heterodimerization domains and “unzippering” the mature glycosylated MHC-I/chaperone complexes. The “unzippered” glycosylated MHC-I/chaperone complexes can be purified away from the cleaved heterodimerization domains, for example, by size exclusion chromatography. Such constructs are capable of receiving a high affinity peptide of interest and are can therefore be termed glycosylated peptide receptive MHC-I complexes can be used to form MHC tetramers contacted with high-affinity peptides to form pMHC multimers (e.g., tetramers). The peptide receptive MHC-I chaperone constructs can be complexed with a chaperone protein, but need not be.
In still other embodiments, MHC-I protein constructs and chaperone protein constructs are engineered without a heterodimerization domain and coexpressed in mammalian cells. The chaperone constructs act upon the MHC-I protein constructs catalytically (e.g. where the MHC-I protein construct and the chaperone protein construct do not form a stable complex) to transform the MHC-I protein constructs into peptide receptive MHC-I complexes that can be purified and loaded with peptide as described herein. No protease treatment is necessary in this particular embodiment.
F. Glycosylated MHC-I/TAPBPR Multimers
The glycosylated peptide receptive MHC-I complexes provided herein can be used to form glycosylated peptide receptive MHC-I multimers and/or glycosylated multimers of an complexed with a peptide of interest (called a pMHC-I herein). Such multimers (e.g., tetramers or Dextramers®) can allow for the identification of high-affinity T cell and natural killer (NK) cell receptors previously unidentified using traditional unglycosylated MHC-I tetramers produced in non-mammalian expression systems (e.g., Drosophila S2 or E. coli expression systems).
In some embodiments, glycosylated peptide receptive MHC-I multimers can be produced by attaching biotinylated glycosylated peptide receptive MHC-I complexes to a backbone. Biotinylation of the glycosylated peptide receptive MHC-I complexes can be performed by contacting the complexes with biotin in the presence of a biotin ligase enzyme.
In some embodiments, the backbone is a streptavidin backbone. In certain embodiments, the backbone is an avidin backbone. In other embodiments, the backbone is a dextran backbone.
In some embodiments, contacting the glycosylated peptide receptive MHC-I complex with high-affinity peptide to form pMHC-I complexes occurs prior to multimerization. The pMHC-I complexes can then be biotinylated and attached to backbones to form pMHC-I multimers. In exemplary embodiments, peptide deficient MHC class I/chaperone complexes are biotinylated first and then attached to a backbone (e.g., a streptavidin, avidin or dextran backbone), thereby forming peptide deficient MEW class I/chaperone multimers (e.g., tetramers). Such peptide receptive MEW class I multimers can be used for the large scale production of multimers comprising one or more peptides of interest by contacting the peptide receptiveMHC class I multimers with the one or more peptides of interest. For example, in one embodiment, aliquots of the peptide receptive MHC-I multimers are contacted with different peptides of interest, thereby forming a library of pMHC-I multimers. After loading of the pMHC-I multimers with peptides of interest, the resulting loaded pMHC-I multimers can be washed to remove any free chaperones, labels (e.g., nucleic acid barcodes) or excess peptides of interest not bound in the pMHC-I complexes. Following such a washing step, the exchanged pMHC-I multimers can be stored (e.g., 4° C. for several weeks) or used immediately. In some embodiments, the free chaperones, labels and/or peptides of interest are removed by spin column dialysis.
In some embodiments, the MHC-I protein construct includes a protein tag that facilitates multimerization. Such protein tags are capable of being biotinylated, thereby allowing the attachment of the MHC-I protein constructs to backbones to form multimers. In some embodiments, the protein tag included in the MHC-I protein construct includes one or more amino acid residues that can be biotinylated. in an exemplary embodiment, the protein tag includes exactly one amino acid residue that can be biotinylated. In certain embodiments, the amino acid residue is a lysine residue. In particular embodiments, the protein tag is an AviTag (GLNDIFEAQKIEWHE) that includes one lysine residue.
In some embodiments, the glycosylated pMHC-I multimer is a dimer. In some embodiments, the pMHC-I multimer is a trimer. In preferred embodiments, the glycosylated pMHC-I multimer is a tetramer. In one embodiment, the multimer is a dextramer. Dextramers include ten glycosylated MHC-I complexes attached to a dextran backbone. Dextramers allow for the detection, isolation, and quantification of antigen specific T cell populations due to an improved signal-to-noise ratio not present in prior generations of multimers. See, e.g., Bakker and Schumacher, Current Opinion in Immunology 17(4): 428-433 (2005); and Davis et al., Nature Reviews Immunology 11:551-558 (2011).
In some embodiments, the glycosylated pMHC-I multimer is a glycosylated pMHC-I tetramer that includes four glycosylated pMHC-I molecules, wherein the four glycosylated pMHC-I molecules are each attached to a streptavidin backbone. In some embodiments, each of the four glycosylated pMHC-I molecules are biotinylated and attached to one of the four biotin binding subunits of the streptavidin backbone. In one embodiment each of the four glycosylated pMHC-I molecules is glycosylated at least one native glycosylation site. In an exemplary embodiments, each of the four glycosylated pMHC-I molecules is glycosylated at N86.
In some embodiments, the four glycosylated pMHC-I complexes each include a glycosylated single-chain MHC-I protein construct that includes an MHC-I heavy chain covalently linked to a β2 microglobulin. The single chain MHC-I protein construct is complexed with a peptide of interest. Any suitable MHC-I heavy chain allele can be included in the single-chain MHC-I protein construct. In some embodiments, the single-chain MHC-I protein construct includes an HLA-A heavy chain. In certain embodiments, the single-chain MHC-I protein construct includes an HLA-B heavy chain. In other embodiments, the single-chain MHC-I protein construct includes an HLA-C heavy chain. In an exemplary embodiment, the single-chain MHC-I protein construct includes an HLA-A01 or HLA-A02 allele heavy chain. In an exemplary embodiment, the MHC heavy chain is an HLA-A*02:01, HLA-A*24:02, HLA-A*68:01 or HLA-A*68:02 allele heavy chain. In other embodiments, the single chain MHC-I protein construct includes a mouse H-2. In certain embodiments, the H-2 is an H-2D, H-2K or H-2L. In exemplary embodiments, the H-2 is H-2DD or H-2LD. In some embodiments, the single-chain MHC-I protein construct include a variant of a wild-type MHC-I heavy chain. In particular embodiments, the variant MHC-I heavy chain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a wild-type MHC-I heavy chain. Any suitable linker can be used to attach the MHC-I heavy chain to the β2 microglobulin. In certain embodiments, the linker is (GGGS)x, wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In an exemplary embodiment, the linker is (GGGS)4.
In certain embodiments, at least one of the glycosylated MHC-I molecules of the multimer is complexed with a chaperone molecule. In some embodiments, one, two, three, four or more of the glycosylated MHC-I molecules of the multimer are each complexed with a chaperone molecule. In some embodiments, the chaperone molecule is TAPBPR. In exemplary embodiments, the glycosylated MHC-I molecules of the tetramer are each loaded with a peptide of interest (i.e., glycosylated tetramers).
In some embodiments, the backbone of the pMHC-I multimer is conjugated with a detectable label (e.g., a fluorophore or a radiolabel) that allow the multimer to be detected in various applications. In certain embodiments, the detectable label is as fluorophore. See, e.g., Nepom et al., J Immunol 188 (6) 2477-2482 (2012). In one embodiment, the detectable label is a radiolabel. In certain embodiments, the backbone includes a barcode (e.g., a nucleic acid barcode) that allows the glycosylated multimer to be used in large scale high throughput processes. See, e.g., Bentzen et al., Nature Biotechnology 34(1): 1037-1045 (2016). In an exemplary embodiment, unique barcodes are used for each of the different peptides of interest included in the glycosylated pMHC-I multimers, thereby allowing for the tracking, sorting and identification of particular glycosylated pMHC-I multimers in high throughput applications. In particular embodiments, each barcode includes a unique nucleotide sequence.
In some embodiments, the glycosylated pMHC-I multimer is coupled to a toxin (e.g., saporin). Such pMHC-I multimer conjugates can be used to modulate or deplete specific T cell populations. See, e.g., Maile et al., J. Immunol. 167: 3708-3714 (2001); and Yuan et al., Blood 104: 2397-2402 (2004).
G. Methods of Using Tetrameric Glycosylated MHC-I/TAPBPR Complexes
In certain embodiments, the pMHC-I multimer produced from the glycosylated peptide receptive MHC-I complex provided herein is a pMHC-I tetramer. pMHC-I tetramers provided herein can be used to study pathogen immunity, for the development of vaccines, in the evaluation of antitumor response, in allergy monitoring and desensitization studies, and in autoimmunity. See, e.g., Nepom et al., J. Immunol 188 (6) 2477-2482 (2012); and Davis et al., Nature Reviews Immunology 11:551-558 (2011).
In some embodiments, the pMHC-I multimers are used to characterize T cell (e.g., CD8 T cell) responses to a vaccine, including, but not limited to influenza, yellow fever, tuberculosis, coronavirus, (e.g. SARS-CoV-2), and HIV/SIV vaccines. In an exemplary embodiment, the vaccine is a cancer vaccine. In particular embodiments, the cancer vaccine is melanoma or chronic myeloid leukemia. In such embodiments, a sample (e.g., a blood sample) of a vaccinated patient is contacted with one or more of the subject pMHC-I multimers that include one or more peptide of interests derived from the vaccine to identify and monitor antigen specific T cells that are produced in response to the vaccine.
Peptide-MHC-I multimers provided herein can also be used to isolate and enrich particular antigen specific T cells for therapeutic use. See, e.g., Cobbold et al., J. Exp. Med. 202: 379-386 (2006); and Davis et al., Nature Reviews Immunology 11:551-558 (2011). In this particular application, patient samples are contacted with sortable pMHC-I multimers that include a peptide antigen of interest and a label that allows for sorting (e.g., a fluorophore or nucleic acid label). Antigen specific T cells that bind the pMHC-I multimer are subsequently isolated and purified, for example, using flow cytometry or similar cell sorting and identification techniques.
In certain embodiments, the pMHC-I multimers provided herein are used for epitope mapping. In this method, a plurality of pMHC-I multimers that include different peptides derived from an antigen of interest (e.g., a tumor antigen) are contacted with a sample from a subject. Antigen specific T cells are detected and the corresponding epitope peptide sequences are identified any technique known in the art, include, for example, flow cytometry and cell sorting techniques. See, e.g., Bentzen et al., Nat Biotechnol. 34(10):1037-1045 (2016).
In some embodiments, the pMHC-I multimers provided herein are used to determine a T cell profile of one or more subjects. In such an embodiment, a sample from a subject is contacted with a library of pMHC-I multimers that include a library of peptides of interest and a detectable label. Identification of antigen specific T cells that bind particular peptides of interest presented in the context of the pMHC-I multimers is achieved using the detectable label. The methods described herein allow for the large scale production of pMHC-I multimer libraries that can in turn be used for high throughput T cell profiling.
In another aspect, the pMHC-I multimers are used therapeutically for the targeted elimination of particular antigen specific T cells in a subject. In one embodiment, the pMHC-I multimers are conjugated to a cytotoxic agent or a toxin. When administered to a subject, the pMHC-I multimer conjugates attach to and facilitate the elimination of particular antigen specific T cells.
Peptide-WIC class I multimers used in the methods described herein can be tracked and detected using any suitable techniques including, but not limited to, techniques utilizing detectable labels and nucleic acid barcodes that allow identification of particular pMHC class I multimers. In addition, T cells of interest isolated in such methods can also be identified using similar techniques.
T cells of interest that interact with pMHC-I multimers can be isolated using any suitable technique including, for example, flow cytometry techniques. Isolated T cells and corresponding peptide-MHC class I multimers can then be characterized using any suitable method, for example, the ECCITE-seq method as explained below ((https://www.nature.com/articles/s41592-019-0392-0) in conjunction with 10× Genomics CHROMIUM SINGLE CELL IMMUNE PROFILING SOLUTION™ with FEATURE BARCODING™ technology (https://support.10xgenomics.com/single-cell-vdj/software/vdj-and-gene-expression/latest/overview). This method incorporates a cellular barcode into cDNA generated from both tetramer oligos and TCR mRNA, thus the pairing of cellular barcodes can connect TCR sequences and other mRNAs with pMHC-I multimers specificities.
Peptide exchange technologies are central to the generation of high throughput pMHC-multimer libraries currently used for probing polyclonal TCR repertoires. To date, only non-glycosylated MHC molecules produced in E. coli and refolded in vitro have been available for library construction. Although glycosylation is not essential for peptide loading, the biological significance of a single highly conserved glycan on MHC Class I molecules, remains to be determined.
Expression of MHC-I complexes with TAPBPR in mammalian cells provides native, peptide-receptive MHC-I/TAPBPR complexes that are glycosylated. For example, such complexes would include the critical glycosylation at the conserved N86 in HLA-A, HLA-B, and HLA-C. Upon multimerization and loading with high-affinity peptides, as described in Overall et al., BioRxiv doi: https://doi.org/10.1101/653477 (2019)), incorporated by reference herein, these complexes allow stable antigen presentation in a physiologically relevant form of the molecule that can in turn be used to identify high-affinity T cell and natural killer (NK) cell receptors. The disclosed method is modular, and is applicable to a range of applications, including immune repertoire characterization on patient samples, as outlined in detail below. Furthermore, the high level expression of peptide-receptive MHC-I molecules in mammalian cells can result in therapeutic and applications such as vaccines.
Previous work (Overall et al supra) made use of MHC refolding protocols from inclusion bodies expressed in E. coli and TAPBPR expressed in S2 Drosophila cells. Such methods of producing pMHC-I complexes require refolding and purification. In addition, MHC-I molecules produced in E. coli lack a glycan post-translational modification at the conserved N86 residue. The glycan modification is known to provide stability to the MHC-I. The glycan is at the face of the MHC which is known to interact with T cell and natural killer (NK) cell receptors, and could therefore play important roles in physiologically relevant immune recognition.
So disclosed herein is a mammalian expression system, including engineered protein constructs for the preparation of peptide-receptive MHC molecules in complex with the molecular chaperone TAPBPR which can be used directly for the preparation of MHC multimer libraries, and other applications.
The class I molecules of the Major Histocompatibility Complex (MHC) play a pivotal role in orchestrating an adaptive immune response by alerting the immune system to the presence of developing infections and tumors in the body. Immune surveillance is achieved through the display of short (8-11 residue long) peptides derived from viral proteins (or mutated oncogenes) via a tight interaction with the MHC-I peptide-binding groove. Such peptide/MHC-I protein complexes are assembled inside the cell and displayed on the surface of all antigen-presenting cells where they can interact with specialized receptors on T cells and natural killer (NK) cells. The MHC-I proteins are extremely polymorphic (more than 13,000 different alleles have been identified in the human population to date), and each allele can display an estimated 1,000-10,000 different peptides, which makes the characterization of specific T cell responses against a panel of known peptide epitopes a daunting task, further challenged by the fact that typical T cell receptor affinities for their cross-reactive pMHC ligands are low (e.g., in the micromolar range).
The use of multivalent, fluorescent pMHC-I multimers was pioneered by Altman and Davis in 1996 to stain T cells (Altman J D et al., Science 274, 94-96 (1996); Altman J D & Davis M M, Curr Prot Immunol Ch. 17, Unit 17.3 (2003); both of which are incorporated by reference). Cells that recognize a specific peptide/MHC multimer can be identified and sorted using flow cytometry, and their receptors can be sequenced in subsequent steps. Peptide-MHC-I (pMHC-I) tetramers have revolutionized experimental immunology and the development of new therapies, leading to a breadth of discoveries (Doherty P C, J Immunol 187, 5-6 (2011); incorporated by reference herein).
However, the preparation of properly conformed pMHC molecules via in vitro refolding of inclusion bodies expressed in E. coli, requires a laborious, multi-step process that is highly inefficient (typical refolding yields are <5% by weight). Moreover, all MHC molecules expressed in E. coli lack the functionally relevant post-translational glycosylations that are required for proper immune surveillance function (Garboczi D N et al., PNAS 89, 3429-3433 (1992); Barber et al., J Immunol 156, 3275-3284 (1996); both of which are incorporated by reference herein). Specifically, a conserved glycan at residue N86 of the MHC-I protein, which is located at a site near the TCR recognition surface, is not present in E. coli expressed MHC-I. This limits the application of refolded tetramers to identify high-affinity T cell receptors and natural killer cell receptors and results in a more limited TCR repertoire than would be present in vivo. The missing TCRs can include important targets for a number of applications including the study of antigen recognition processes, and the development of immunotherapies to combat bacterial and viral infections and cancer.
The use of MHC-I molecules expressed in mammalian cells as a high-affinity complex with the molecular chaperone TAPBPR (McShan A C et al., Nat Chem Biol 14, 811-820 (2018); incorporated by reference herein) results in a number of advantages. It provides native, peptide-receptive MHC-I complexes containing the critical glycosylation at the conserved N86. Upon multimerization and loading with high-affinity peptides as described in Overall et al supra, peptide receptive MHC-I complexes allow stable antigen presentation in a physiologically relevant form of the molecule towards the identification of high-affinity T cell and natural killer cell receptors. The use of affinity tags attached to the recombinant proteins results in specific binding for MHC-I molecules results in fewer non-specific peptides contaminating the library.
Efficient expression of a peptide receptive MHC-I complex in mammalian cells has the potential to simply the workflow to prepare peptide loaded tetramers for T cell analysis. Previously disclosed methods involve the refolding of E. coli inclusion-body expressed protein. In addition, MHC molecules produced in prokaryotic systems lack glycosylation. Human MHC-I (HLA A, B and C) are highly polymorphic but the N86 PNGS shows considerable conservation across phylogeny (Grossberger D and Parham P, Immunogenetics 36, 166 (1992); incorporated by reference herein). Glucose trimming of ER associated core N-glycan Glc3Man9GlcNAc2 facilitates proper interactions with the lectin chaperones calnexin and calreticulin, but the function of glycans subsequently added by the N-linked glycosylation pathway is yet to be determined (Barber L D et al., J Immunol 156, 3275-3284 (1996) and Ryan S O and Cobb B A, Semin Immunopathol 34, 424-441 (2012); both of which are incorporated by reference herein). A simplified version of the mammalian N-linked glycan pathway is shown in
Based on an early report of proteolysis of MHC-I heavy chain by complement C1S enzyme between the α2 and α3 heavy chains (Erikson H and Nissen M H, Biochem Biophys Res Commun 187, 832-838 (1989)); incorporated by reference herein), initial small-scale expression trials for the production of MHC-I/TAPBPR were performed in a C1s−/− CHOK1s knockout (Li S et al., Biotechnol Bioeng, doi: 10.1002/bit.27016 (2019); incorporated by reference herein). Constructs used herein are shown in
Small scale (C400/15 mL) 5 day transfections yielded between 10-30 mg/L of purified complex, prior to any optimization. Typical cell growth is shown in
The glycosylated peptide receptive MHC-I complexes produced in CHO cells can be tetramerized and loaded with peptide, in the same manner as bacterially expressed and refolded (and therefore glycan free) peptide receptive MHC-I complexes.
As further shown in
Molecular cloning. Vectors designated (Z1 and Z2) are suitable for transient and stable high level expression of secreted proteins (particularly proteins that are engineered to include leucine zipper domains) in mammalian cells. The recombinant proteins comprising the leucine zippers can be purified directly from tissue culture supernatant using a StrepTrap HP affinity column (GE Healthcare, Chicago Ill.). The leucine zipper domain can be removed by TEV protease digestion, and peptide receptive MHC-I molecules purified by size exclusion chromatography Construct Z1 expresses a single MHC-I single-chain gene that expresses a recombinant protein comprising the human B2M sequence (UniProtKB P61769) a flexible linker sequence (Hansen et al., 2009), and the ecto-domain of MHC-I HLA* 0201 exons 1-4. Construct Z2 expresses TAPBPR (UniProtKB Q9BX59-1) and a Strep-Tactin® tag. Standard molecular protocols were used to construct expression vectors. Briefly, synthetic, codon optimized B2M MHC-I and TAPBPR genes were purchased from IDT (Coralville, Iowa) and cloned independently into a CMV driven expression cassette within a plasmid vector. Plasmids were propagated in the DH5 alpha strain of E. coli, and purified using an endotoxin free PureLink extraction kit (Life Technologies, Thermo Fisher, Carlsbad, Calif.). DNA sequencing was carried out at the University of California at Berkeley Core Sequencing facility using Sanger chain termination sequencing. The complete mature protein sequences are provided herein.
Cells and antibodies. CHO-K1 cells were obtained from ATCC (ATCC, Manassas, Va.) and adapted to suspension culture by serial passage in suspension (CHO-K1s). A CHOK1s variant (CHO-K1 s C1S−/−) was provided by Dr. Phil Berman (Li S et al., Biotechnol Bioeng, doi: 10.1002/bit.27016 (2019); incorporated by reference herein). HEK293F cells were obtained from Life Technologies (Thermo Fisher Carlsbad, Calif.). Anti-TAPBPR antibodies were purchased from Life Technologies (Thermo Fisher Carlsbad, Calif.) or raised by immunization of rabbits immunized using a Complete Freund's Adjuvant/Incomplete Freund's Adjuvant (CFA/IFA) protocol (Pocono Rabbit Farms, AAALAC #926, Canadensis, Pa.) with TAPBPR produced in CHO-K1s cells as an antigen. Anti-B2M macroglobulin antibodies were purchased from R & D Systems (Minneapolis, Minn.). Flow cytometry antibodies were purchased from BD Biosciences (San Jose, Calif.).
Cell culture conditions. Stocks of suspension adapted CHO-K1s, 293 HEKF, and CHO-K1s C1s−/− cells were maintained in shake flasks (Corning, Corning N.Y.) using a Kuhner ISF1-X shaker incubator (Kuhner, Birsfelden, Switzerland). For normal cell propagation shake flasks cultures were maintained at 37° C., 8% CO2, and 135 rpm. TCR β-chain deficient Jurkat-MA T cells expressing the DMF5 TCR recognizes Melan-A epitope MART-1 bound to HLA-A*02:01, were grown in DMEM supplemented with 10% heat inactivated FBS, 25 mM HEPES pH 7, 2 μM β-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin/streptomycin and 1×non-essential amino acids. All supplements were obtained from Life Technologies (Carlabad Calif.) unless stated otherwise. Static cultures were maintained in 96 or 24 well cell culture dishes and grown in a Sanyo incubator (Sanyo, Moriguchi, Osaka, Japan) at 37° C. and 5% CO2.
Cell culture media. For normal CHO-K1s cell growth, cells were maintained in BalanCD CHO Growth A (Irvine Scientific, Santa Ana, Calif.) supplemented with 0.1% pluronic acid, 8 mM GlutaMax and 1× Hypoxanthine/Thymidine (Thermo Fisher, Life Technologies, Carlsbad, Calif.). 293 HEK (Freestyle) cells were maintained in Freestyle 293 cell culture media (Life Technologies, Carlsbad, Calif.). For CHO cell protein production, the cells were maintained at 32° C. (24 hours after transfection) in in BalanCD CHO Growth A medium supplemented with 0.1% pluronic acid, 2 mM GlutaMax and 1×H/T (Thermo Fisher, Life Technologies, Carlsbad, Calif.), and fed daily with MaxCyte CHO A Feed which is comprised of 0.5% Yeastolate, B D, Franklin Lakes, N.J.; 2.5% CHO-CD Efficient Feed A, 2 g/L Glucose (Sigma-Aldrich, St. Louis, Mo.) and 0.25 mM GlutaMax).
Cell counts and growth calculation. All cell counts were performed using a TC20™ automated cell counter (BioRad, Hercules, Calif.) with viability determined by trypan blue (Thermo Fisher, Life Technologies, Carlsbad, Calif.) exclusion. Cell-doubling time in hours was calculated using the formula: (((time2−time1)×24)×ln (2)/(ln (density2)−ln (density1)).
Electroporation. Electroporation was performed using a MaxCyte STX scalable transfection system (MaxCyte Inc., Gaithersburg, Md.) according to the manufacturer's instructions, using aseptic technique. Cells were maintained at >95% viability prior to transfection, and sub-cultured one day prior to transfection. The day of transfection, cells were pelleted at 250 g for 10 minutes, and then re-suspended in MaxCyte EP buffer (MaxCyte Inc., Gaithersburg, Md.) at a density of 2×108 cells/mL. Transfections were carried out in the OC-400 processing assembly (MaxCyte Inc., Gaithersburg, Md.) with a total volume of 400 μL and 8×107 total cells. Plasmid DNA in endotoxin-free water was added for a final concentration of 300 μg of DNA/ml. The processing assemblies were then transferred to the MaxCyte STX electroporation device and appropriate conditions (CHO protocol) were selected using the MaxCyte STX software. Following completion of electroporation, the cells in Electroporation buffer were removed from the processing assembly and placed in 125 mL Erlenmeyer cell culture shake flasks (Corning, Corning N.Y.). The flasks were placed into 37° C. incubators with no agitation for 40 minutes. Following the rest period, pre-warmed OPTI-CHO media (Thermo Fisher, Invitrogen, Carlsbad, Calif.) supplemented with 0.1% pluronic acid, 2 mM GlutaMax and 1×H/T, was added to the flasks for a final cell density of 4×106 cells/mL. Flasks were then moved the Kuhner shaker and agitated at 135 rpm.
ImmunoBlot. Proteins (from cell supernatant and cytoplasmic lysate) were electrophorized on 12% SDS gels in MOPS gel running buffer (Thermo Scientific, Waltham, Mass.). For Immunoblot, proteins were electrophoresed, transferred to a PDVF membrane, then probed with a polyclonal rabbit anti-TAPBPR antibody or a murine anti-B2M followed by an affinity purified secondary HRP conjugated anti-species antibody (Jackson ImmunoResearch, West Grove, Pa.) and visualized using an Innotech FluoChem2 system (Genetic Technologies Grover, Mo.).
Peptide Receptive MHC-I Complex Purification. Culture media was harvested and pre-cleared by centrifugation at 250 g for 10 minutes. The media was adjusted to contain 25 mM Tris pH 8, 1 mM EDTA and 27 mg/L of avidin and filtered (0.22 micron) before affinity purification on a StrepTrap HP affinity column (GE Healthcare, Chicago Ill.). Bound protein was washed with 10 column volumes of wash buffer (25 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA) and eluted with 2.5 mM desthiobiotin/wash buffer. The complex was concentrated from approximately 6 mL to 0.5 mL on a 30 kD cutoff MicrosepAdvance filter (Pall, New York, N.Y.), and digested overnight with TEV (Tobacco Etch Protein) in TEV cleavage buffer (25 mM Tris pH 8, 100 mM NaCl 1 mM EDTA, 3 mM/0.3 M glutathione redox buffer at 4° C. Complex was recovered by gel filtration (SEC) on a Superdex 200 10/300 increase column (GE Healthcare, Chicago Ill.) at a flow rate of 0.5 mL/min in 50 mM Tris pH 7.5 buffer containing 100 mM NaCl at room temperature. MHC-I/TAPBPR complexes eluted at 26.5-27 minutes GM did peptide was (10 mM) was added to the running buffer during chromatography.
LC-MS. The molecular mass of TEV digested TAPBPR was determined by HPLC separation on a Higgins PROTO300 C4 column (5 μm, 100 mm×21 mm) followed by electrospray ionisation performed on a Thermo Finnigan LC/MS/MS (LQT) instrument. Peptides were identified by extracting expected m/z ions from the chromatogram and deconvoluting the resulting spectrum in MagTran.
Native gel shift assay of peptide binding to empty complex. Peptide-receptive MHC-I complexes were incubated with the indicated molar ratio of relevant (TAX or MART1) or irrelevant (P18410 or NIH) peptide for 1 h at room temperature at pH 7.5 in Tris buffer with 50 mM NaCl. Samples were electrophoresed at 90 V on a 12% polyacrylamide gel in 25 mM T IS pH 8.8, 192 mM glycine, at 4° C. for 4.0 hours and developed using InstantBlue (Expedeon San Diego, Calif.).
Tetramer formation. The procedure for production of peptide loaded tetramers using TAPBPR mediated exchange is described in Overall et. al., (2019) (referenced above). Briefly, SEC purified (unzipped) peptide receptive MHC-I complex molecules were biotinylated via an AviTag (GLNDIFEAQKIEWHE) on the MHC-I molecule using biotin ligase (BirA) (Avidity.com Co), according to the manufacturer's instructions. Biotinylated MHC-I/TAPBPR complex was buffer exchanged into PBS pH 7.4 using a PD-10 desalting column. Biotinylation was confirmed by SDS-PAGE in the presence of excess streptavidin. Tetramerization of empty-MHC-I/TAPBPR was performed by adding a 2:1 molar ratio of biotinylated MHC-I/TAPBPR to streptavidin-PE or streptavidin-APC (Prozyme Hayward, Calif.) in five additions over 1 h on ice. Peptide-receptive MHC-I tetramers were then contacted with peptides of interest by adding a 20-molar excess of peptide to each well and incubating for 1 hour. A solution of 8M biotin (to block any free streptavidin sites) was added and incubated for a further 1 h at room temperature. After exchange, tetramers were transferred to 100 kDa spin columns (Amicon, Millipore, Burlington, Ma) and washed with 1000 volumes of PBS to remove TAPBPR and excess peptide. After washing, exchanged tetramers were pooled and stored at 4° C. for up to 3 weeks.
Flow cytometry. Tetramer analysis was carried out as described in Overall et al. (2019) supra by staining 2×105 Jurkat/MA cells transduced with the DMF5 receptor specific to the MART-1, with an anti-CD8a mAb (BD Biosciences) and 1 μg/mL HLA-A02:01/MART-1 tetramer or HLA-A02:01/TAX tetramer for 1 h on ice, followed by two washes with 30 volumes of FACS buffer (PBS, 1% BSA, 2 mM EDTA). All flow cytometric analysis was performed using a BD LSR II instrument equipped with FACSDiva software (BD Biosciences).
For EC50 determination. Tetramer concentrations were calculated based on total amount of pMHC-I at the time of exchange. Titrations were performed on the appropriate cell line in duplicate in two independent experiments. The percentage of tetramer+ T cells was measured relative to the staining achieved at the highest concentration tested within each experiment. EC50 values were calculated by fitting a Boltzmann sigmoidal function to the data with the lower constraint set to 0 and upper constraints set to 95 for B4.2.3 and 28 for DMF5 in GraphPad Prism 7.
Differential scanning Fluorimetry (DSF). To measure thermal stability of pMHC-I complexes, 2.5 μM of protein was mixed with 10×Sypro Orange dye in matched buffers (20 mM sodium phosphate pH 7.2, 100 mM NaCl) in MicroAmp Fast 96 well plates (Applied Biosystems) at a final volume of 50 DSF was performed using an Applied Biosystems ViiA qPCR machine with excitation and emission wavelengths at 470 nm and 569 nm respectively. Thermal stability was measured by increasing the temperature from 25° C. to 95° C. at a scan rate of 1° C./min. Melting temperatures (Tm) were calculated in GraphPad Prism 7 by plotting the first derivative of each melt curve and taking the peak as the Tm Determination of Tm values of TAPBPR exchanged molecules additionally required subtraction of the TAPBPR melt curve from the curve obtained for the complex, then calculating the first derivative. This procedure, on average, enhanced the Tm values calculated for TAPBPR exchanged pMHC-I complexes by 1.5° C., compared to refolded and photo-exchanged pMHC-I complexes. All samples were analyzed in duplicate and the error is represented as the standard deviation of the duplicates analyzed independently.
Current approaches for generating MHC Class-I proteins with peptides of interest (pMHC-I) for diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP Binding Protein Related (TAPBPR), a robust method has been developed to produce properly conformed, peptide-receptive molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E. coli. Purified peptide receptive MHC-I/complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of principle, both HLA allele-specific peptide binding, and MHC-restricted antigen recognition by T cells for a panel of HLA-A*02:01 epitopic peptides predicted from the SARS-CoV-2 genome were demonstrated. The system disclosed herein provides a facile, high-throughput approach to probe polyclonal TCR repertoires against their cognate pMHC-I antigens.
Introduction
The Class-I proteins of the Major Histocompatibility Complex (MHC-I) play a pivotal role in orchestrating immune responses through their interactions with specialized receptors on T cells and Natural Killer (NK) cells (Germain and Margulies, Annu. Rev. Immunol., 11, 403-450 (1993); Jiang et al., Adv. Exp. Med. Biol., 1172, 21-62 (2019)). Immune surveillance by αβ T cell receptors (TCRs) is achieved through the display of short (8-11 residue long) peptides derived from viral proteins (or mutated oncogenes) via tight capture within the MHC-I peptide-binding groove as an obligate protein complex (Rossjohn et al., Annu. Rev. Immunol., 33, 169-200 (2015)). MHC-I molecules are assembled on the endoplasmic reticulum (ER) from component heavy and β2 microglobulin (β2m) light chains and loaded with peptides in the context of a multi-subunit membrane complex (Cresswell et al., Immunol. Rev., 172, 21-28 (1999)). Interactions of nascent MHC-I with molecular chaperones (tapasin and TAPBPR) select for high-affinity peptides to ensure the prolonged stability and immunogenicity of resulting peptide/MHC-I (pMHC-I) complexes (Blum et al., Annu. Rev. Immunol., 31, 443-473 (2013)). As an additional quality control step, glucose trimming of the ER-associated Glc3Man9GlcNAc2 moiety found at the conserved Asn 86 (N86) residue ensures that only correctly folded molecules are trafficked further along the antigen processing pathway towards the cell surface (Wearsch et al., roc. Natl. Acad. Sci. U.S.A, 108, 4950-4955 (2011)). Although tapasin is an integral part of the ER-anchored peptide loading complex, TAPBPR is found throughout the ER and cis-Golgi network and has independent, auxiliary functions in MHC-I quality control (Neerincx et al., eLife, 6 (2017)) and in shaping the displayed peptide repertoire (Boyle et al., Proc. Natl. Acad. Sci. U.S.A, 110, 3465-3470 (2013); Hermann et al., eLife, 4 (2015); Hermann et al., Tissue Antigens, 85, 155-166 (2015)).
Detecting and quantifying antigen-specific TCRs during the course of disease, treatment or immunization was revolutionized by the use of multivalent, fluorescent, pMHC-I complexes (Altman et al., Science, 274, 94-96 (1996); Hadrup and Schumacher, Cancer Immunol. Immunother., 59, 1425-1433 (2010)). Empty MHC-I molecules are unstable and highly prone to aggregation, so pMHC-I proteins are commonly produced by in vitro refolding of light and heavy chain components, derived from E. coli inclusion bodies, in the presence of large molar excess of a synthetic peptide which involves a laborious multi-step process with typical yields of <5% by weight (Garboczi et al., Proc. Natl. Acad. Sci., 89, 3429-3433 (1992)). There have been considerable efforts to develop peptide-exchange methods, including photolabile peptides (Bakker et al., Proc. Natl. Acad. Sci. U.S.A, 105, 3825-3830 (2008)), dipeptide catalysts (Saini et al., Proc. Natl. Acad. Sci. U.S.A, 112, 202-207 (2015)), thermal exchange (Luimstra et al., J. Exp. Med., 215, 1493-1504 (2018)) or disulfide-linked MHC-I molecules (Moritz et al., Sci. Immunol., 4(37):eaav0860 (2019)). All these methods make use of refolded MHC-I molecules, lacking important post-translational modifications which are likely to influence peptide repertoire selection, and T cell and NK cell recognition. Methods for producing pMHC-I complexes in mammalian cells using covalently linked peptides as single-chain (Jurewicz et al., Anal. Biochem., 584, 113328 (2019)) or fused antibody-pMHC-I constructs (Schmittnaegel et al., Mol. Cancer Ther., 15, 2130-2142 (2016)) have been described, however both approaches necessitate cleavage of the bound peptide for exchange to occur, which results in low protein yields.
The use of molecular chaperones for peptide exchange applications was first explored in the context of Tapasin (Chen and Bouvier, EMBO J., 26, 1681-1690 (2007)). TAPBPR, known to stabilize the empty MHC-I peptide-binding groove in a widened conformation (Jiang et al., Adv. Exp. Med. Biol., 1172, 21-62 (2017); Thomas and Tampé, Science, eaao6001 (2017)) and to promote peptide exchange in vitro (Morozov et al., Proc. Natl. Acad. Sci. U.S.A, 113, E1006-E1015 (2016)), offers an attractive alternative to Tapasin from a biochemical perspective and has been exploited to load MHC-I molecules with peptides directly on the cell surface, independently of the peptide-loading complex (Ilca et al., Proc Natl Acad Sci U.S.A. 115(40), E9353-E9361 (2018); Ilca et al., eLife:7 (2019)). A detailed characterization of the TAPBPR catalytic cycle (McShan et al., Nat Biochem 14(8), 811-820 (2018)) has recently been leveraged to develop a high-throughput exchange methodology for multiple murine and human MHC-I allotypes expressed in E. coli and refolded with an exchangeable peptide (Overall et al., Nat. Commun., 11, 1-13 (2020)). Here, the chaperone function of TAPBPR was explored to develop a similar approach for producing soluble pMHC-I complexes, using a mammalian protein expression system. This approach was to engineer suitable MHC-I and TAPBPR constructs with a cleavable heterodimeric leucine-zipper, a system which enables the production of pMHC-I complexes of desired peptide specificities at mg quantities. Recombinant MHC-I/TAPBPR complexes produced in mammalian cells bypass the requirements and restrictions of the peptide loading complex, are subject to standard eukaryotic post-translational modification, and can be readily loaded with peptides towards functional, biochemical and structural characterization of interactions with their cognate immune receptors.
Results
Generating Properly Conformed MHC-I Molecules in CHO Cells
The full arrangement of the MHC-I and TAPBPR transgenes engineered to co-express a leucine zippered MHC-I/TAPBPR complex is shown in
An electrophoretic mobility shift assay on a non-denaturing (native) gel was used to further confirm that the chaperoned MHC-I protein was peptide-receptive (Morozov et al., Proc. Natl. Acad. Sci. U.S.A, 113, E1006-E1015 (2016)). In this assay, incubation of the cleaved complex with 10-fold molar excess of two high-affinity peptides (TAX9 or MART-1), led to the formation of discrete bands corresponding to properly conformed pMHC species of slightly different electrophoretic mobilities due to the charge of bound peptides (
Complex-Type Glycosylation Patterns of MHC-I Molecules
Glycans play important roles in the immune response by affecting folding, multimerization, trafficking, cell surface stability and half-life of both antigens and their receptors (Baum and Cobb, Glycobiology, 27, 619-624 (2017)). Despite the highly polymorphic nature of HLA-A, HLA-B, and HLA-C alleles, all class-I molecules share a conserved glycan at Asn 86, and the oligosaccharide structures that predominate appear to be highly processed, biantennary N-linked-oligosaccharides (Parham et al., J. Biol. Chem., 252, 7555-7567 (1977)). Mass-spectroscopy confirmed that proteolysis of CHO-derived recombinant MHC-I resulted in isolation of a single N-glycosylation site at N86 within a unique 15-residue fragment. Peaks corresponding to this peptide revealed high intensities in both the MS1 and MS2 dimensions following analysis by ESI-MS/MS (
A biantennary glycan bearing two non-reducing terminal sialic acid residues on the HLA-A*02:01/MART-1 X-ray structure (
Screening Peptide Binding on CHO-Derived MHC-I/TAPBPR Complexes
CHO-derived HLA-A*02:01/TAPBPR complexes can be loaded with high-affinity TAX9 and MART-1 peptides (
Peptide/MHC-I complex assembly for a panel of HLA-A*02:01-restricted epitopic peptides predicted from the SARS-CoV-2 genome was demonstrated using a developed structure-guided method developed (Nerli and Sgourakis, bioRxiv, 2020.03.23.004176 (2020)) (Table 1). Peptides were incubated with MHC-I/TAPBPR complex, then analyzed for binding using the described electrophoretic mobility shift assay. Of 13 predicted peptides, 11 produced strong pMHC-I bands. The two peptides that were predicted to be weak binders (TLACFVLAAV and WLMWLIINL) had little or no binding, as evidenced by no pMHC-I band formation. The specificity of peptide binding is reflected by the observed electrophoretic mobilities of the resulting pMHC-I species, which correlate with the overall charges and hydrodynamic radii of the resulting protein complexes (
Antigen-Specific T Cell Recognition of Native Peptide/HLA Antigens
CHO-derived MHC-I/TAPBPR complexes may be readily multimerized via a streptavidin fluorophore conjugate (Altman et al., Science, 274, 94-96 (1996)). TAPBPR-promoted peptide loading can be then utilized to generate pMHC-I tetramers of desired peptide specificities. The efficiency of antigen-specific staining of a human lymphocyte line (DMF5) transduced with a T cell receptor (Melan-A) specific for the melanoma-associated MART-1 peptide (Johnson et al., J. Immunol. Baltim. Md. 1950, 177, 6548-6559 (2006)) was demonstrated. DMF5 cells were incubated with phycoerythrin (PE) labelled MART-1/MHC-I tetramers prepared using either i) in vitro refolded pMHC-I (used as a positive control) vs ii) empty MHC/TAPBPR complexes or iii) TAPBPR-exchanged pMHC-I complexes loaded with the heteroclitic MART-1 peptide or iv) a different antigenic peptide, NY-ESO-1, corresponding to the cancer-testis antigen 1B (Gnjatic et al., Adv. Cancer Res., 95, 1-30 (2006)). To demonstrate antigen/receptor-specific tetramer staining, a complementary set of flow cytometry experiments was performed using a NY-ESO-1 specific T cell line (Bethune et al., Proc. Natl. Acad. Sci. U.S.A, 115, E10702-E10711 (2018)). Flow cytometry (
Discussion
Murine MHC-I molecules engineered as single-chain constructs with a covalently linked peptide were first expressed in mammalian cells (Mage et al., Proc. Natl. Acad. Sci. U. S. A., 89, 10658-10662 (1992)) and are reported to stimulate both antigen-specific B and T cells (Yu et al., 2002). Mammalian expression systems for human HLA antigens with the potential for immune stimulation include both single-chain constructs (Jurewicz et al., Anal. Biochem., 584, 113328 (2019)), and pMHC-IgG fusions (Wooster et al., J. Immunol. Methods, 464, 22-30 (2019)), but significant challenges remain with respect to loading such molecules with high-affinity peptides of choice. Here, systems and methods are provided to produce soluble, peptide-receptive human MHC-I proteins in the biopharmaceutical standard Chinse Hamster Ovary line, suitable for generating natively-folded and glycosylated pMHC-I with controlled peptide specificities. The above results demonstrate reconstitution of three commonly occurring human HLA allotypes of the A02 supertype (HLA-A*02:01, HLA-A*68:02 and HLA-A*24:02) (Sidney et al., BMC Immunol., 9, 1 (2008)) as fully functional, properly conformed pMHC-I complexes. The antigen specific staining of T cells with CHO-derived pMHC-I tetramers encompassing known tumor antigens is comparable to that of refolded pMHC-I complexes, while allowing for a significantly more convenient process which includes functionally important post translational modifications. Finally, an application of this platform was demonstrated to assay the specificity of several predicted epitopic peptides from the SARS-CoV-2 genome against the common allotype HLA-A*02:01, which provides a convenient approach to validate peptide/HLA binding. Combined with previously described multiplexed tetramer (Bentzen and Hadrup, Annu. Rev. Immunol., 31, 443-473 (2017); Overall et al., Nat. Commun., 11, 1-13 (2020)) and nanoparticle methods (Ichikawa et al., Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res., doi: 10.1158/1078-0432.CCR-19-3487 (2020)), the disclosed system can be leveraged for the development of antigen libraries to monitor and expand polyclonal T cell specificities in various research and clinical settings.
Materials and Methods
Purification of peptide receptive MHC-I Complexes. Culture media was harvested and pre-cleared by centrifugation at 250×g for 10 minutes before adjusting to 25 mM Tris pH 8, 1 mM EDTA and adding 27 mg/L of avidin. The media was filtered (0.22 μm) and affinity purified on a StrepTrap HP affinity column (GE Healthcare, Chicago Ill.). Bound protein was washed with 10 column volumes of wash buffer (25 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA) and eluted with 5 mM desthiobiotin/wash buffer. Leucine zippers were removed by a 2-hour digestion at room temperature with Tobacco Etch Protein (TEV) in 25 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA, 3 mM/0.3 mM glutathione redox buffer. Complex was polished by size exclusion gel filtration (SEC) at room temperature on a Superdex 200 10/300 increase column (GE Healthcare, Chicago Ill.) at a flow rate of 0.5 mL/min in 50 mM Tris pH 7.5 buffer containing 100 mM NaCl. Protein concentrations were determined using A280 measurements on a NanoDrop spectrophotometer.
Native gel-shift assay of peptide binding peptide receptive MHC-I. Peptide-receptive MHC-I complexes were incubated with a 10-molar excess of high affinity or non-binding peptide overnight at 4° C. temperature at pH 7.2 in phosphate buffer with 150 mM NaCl. Samples were electrophoresed at 90V on a 12% polyacrylamide gel in 25 mM Tris pH 8.8, 192 mM glycine, at 4° C. for 5 hours, and developed using InstantBlue (Expedeon San Diego, Calif.). Gels were imaged using an Innotech FluoChem2 system (Genetic Technologies Grover, Mo.).
PNGase F digestion assay. Five μg aliquots of purified HLA-A*02:01 and HLA-A*02:01 S88A (ΔN86 glycan) TAPBPR complex were denatured then either treated with PNGase F (NEB, Ipswich, Mass.) or incubated in glycosidase buffer alone at 37° C. for 1 hour, then reduced with DTT and electrophoresed.
Glycan Mapping by LC-MS/MS N-Glycan analysis. All materials were purchased from Millipore-Sigma unless otherwise noted. Purified MHC-I (20 μg) was buffer exchanged into 50 mM ammonium bicarbonate (pH 8.0) and incubated at 90° C. for 5 min. Following trypsin digestion and reduction, the sample was iodoacetamide treated. Glycopeptides were then enriched using the ProteoExtract Glycopeptide Enrichment Kit according to the manufacturing guidelines, and lyophilized before resuspension in 204, of 5% Acetonitrile and 0.1% Trifluoroacetic acid in ddH2O. Glycopeptides (5 μL) were injected on to a 75 mm×20 cm column packed with C18 Zorbax resin equipped using a Thermo Scientific EASY-nLC 1200 nanopump. Analytes were eluted with a linear gradient of increasing acetonitrile and injected into a Q Exactive hybrid quadrupole mass spectrometer (Thermo Scientific). MS2 spectra for intense ions were collected with stepped NCE energies of 15, 25 and 35 eV. The 10 most abundant N-glycopeptides, based on spectra counts, were annotated using GlycoWorkbench Version 2.1
E. coli protein expression, refolding and purification of conventional refolded pMHC-I. Luminal domain HLA-A*02:01 and human (32m expression plasmids were provided by the NIH Tetramer Core Facility. Proteins were expressed previously described and in vitro refolded in the presence of 10-fold molar excess of synthetic peptides.
Tetramer formation. MHC-I molecules were biotinylated using biotin ligase (BirA) (Avidity.com, Co.). Tetramerization of peptide receptive-MHC-I complexes was performed by adding a 2:1 molar ratio of biotinylated peptide receptive MHC-I to streptavidin-PE or streptavidin-APC (Prozyme Hayward, Calif.) in five additions over 1 hr on ice. Peptide-receptive MHC-Itetramers were then exchanged with peptides of interest by adding a 20-molar excess of peptide and incubating for 1 hour. A solution of 8M biotin (to block any free streptavidin sites) was added and incubated for a further 1 hr at room temperature. After exchange, tetramers were transferred to 100 kDa spin columns (Amicon, Millipore, Burlington, Ma) and washed with 1000 volumes of PBS to remove TAPBPR and excess peptide. For T-cell receptor staining comparison, conventionally produced MHC-I molecules were biotinylated and assembled onto streptavidin-PE.
Flow cytometry. Tetramer analysis of cell lines was carried out by staining 2×105 DMF5 cells, with an anti-CD8a FITC conjugated mAb (BD Biosciences) and 1 μg/mL of either conventionally refolded PE-HLA-A*02:01/MART-1 tetramer, CHO-derived PE-HLA-A*02:01 tetramer loaded with MART-1 peptide, CHO-derived PE-HLA-A*02:01 tetramer loaded with the neo-antigen NY-ESO-1, or left peptide-free and incubated for 1 hr on ice. Cells were washed twice with 30 volumes of FACS buffer (PBS, 1% BSA, 2 mM EDTA) before analysis, and gated by forward and side scattering properties. All experiments were performed using an LSRII (BD) and data analysis performed using FACSDiva (BD) and FlowJo (Tree Star, Ashland Oreg.). Cells tested negative for mycoplasma using the universal mycoplasma test kit (ATCC).
All cited references are herein expressly incorporated by reference in their entirety.
Whereas particular embodiments of the invention have been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.
This application claims priority to U.S. Provisional Patent Application No. 62/900,260, filed Sep. 13, 2019, U.S. Provisional Patent Application No. 63/047,812, filed Jul. 2, 2020, U.S. Provisional Patent Application No. 63/011,221, filed on Apr. 16, 2020, U.S. Provisional Patent Application No. 63/076,601, filed on Sep. 10, 2020, and U.S. Provisional Patent Application No. 62/975,040, filed on Jan. 3, 2020, which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62900260 | Sep 2019 | US | |
| 63047812 | Jul 2020 | US | |
| 63011221 | Apr 2020 | US | |
| 63076601 | Sep 2020 | US | |
| 62957040 | Jan 2020 | US |
