Patent Application
20210309993
T cells are vital to the adaptive immune response, having roles in response to infection and cancer. T cells recognize proteins derived from foreign pathogens as well as self, such as in cases of autoimmunity. Fragments of these proteins (e.g., peptides) are presented by human leukocyte antigen (HLA) molecules and recognized by the T cell via the T cell receptor (TCR).
Major histocompatibility class (MEW) I HLA molecules display peptides generated largely from processing endogenous antigens produced by the cell, such as self-antigens, but also foreign intracellular antigens such as peptides derived from viral proteins, into smaller peptides. Once a peptide is bound into the HLA peptide binding cleft, MHC class I HLA molecules interact with and stimulate CD8+ cytotoxic T cells. MHC class I has 3 main loci A, B, and C, with each loci divided into many alleles. Alleles refer to the DNA sequence of a gene at the given locus and is usually denoted by at least a four-digit number (e.g., A*24:02) the first letter designating the locus, a first number defining an allele group (or type) and the second number defining a specific protein within the allele group. A second and third number can be appended indicating silent coding variants and non-coding variants respectively.
Upon recognition of a specific peptide-HLA complex (pHLA), the T cell becomes activated and can (1) become cytotoxic, (2) secrete cytokines, and/or (3) recruit other immune cells. This complex interaction between a foreign or self-peptide, HLA molecule, and TCR is central to identifying how the immune system responds to recognized pathogens at the molecular level. One of the greatest difficulties in this complex interaction during an immune response is understanding the specificities of TCRs in terms of the identity of the peptides that are recognized. New methods of identifying TCRs and the pHLAs that they recognize are needed.
Provided herein in some embodiments are antigen screening libraries comprising a plurality of Human Leukocyte Antigen (HLA)-antigen polypeptide complexes, the HLA-antigen polypeptide complexes comprising (a) an HLA polypeptide, the HLA polypeptide comprising a peptide binding cleft, (b) a randomized antigen polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 209, wherein the randomized antigen polypeptide specifically binds to the peptide binding cleft of the HLA polypeptide, and (c) a Beta-2 (β2) microglobulin polypeptide.
In some embodiments, the plurality of HLA-antigen complexes comprises an HLA polypeptide selected from the list consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises at least five, ten, fifteen, twenty, or twenty-five different HLA polypeptides selected from the list consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises all of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E HLA polypeptides.
In some embodiments, the plurality of HLA-antigen complexes comprises an HLA polypeptide comprising an amino acid sequence at least 87.5%, 90%, 95%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 427 to 455.
In some embodiments, the plurality of the HLA-antigen polypeptide complexes comprises at least about 105 different HLA-antigen polypeptide complexes comprising at least about 105 different randomized antigen polypeptides.
In some embodiments, the HLA polypeptide, the randomized antigen polypeptide, and the β2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, the single polypeptide further comprises a first flexible polypeptide linker and a second flexible polypeptide linker. In some embodiments, the randomized antigen polypeptide is N-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is N-terminal to the β2-microglobulin polypeptide on the single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the randomized antigen polypeptide, and a second flexible polypeptide linker separates the HLA polypeptide from the β2-microglobulin polypeptide. In some embodiments, the randomized antigen polypeptide is C-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is N-terminal to the β2-microglobulin polypeptide on the single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the randomized antigen polypeptide, and a second flexible polypeptide linker separates the HLA polypeptide from the β2-microglobulin polypeptide. In some embodiments, the randomized antigen polypeptide is N-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is C-terminal to the β2-microglobulin polypeptide on the single polypeptide. In these embodiments, the first flexible polypeptide linker separates the randomized antigen polypeptide from the β2-microglobulin polypeptide, and a second flexible polypeptide linker separates the β2-microglobulin polypeptide from the HLA polypeptide. In some embodiments, the randomized antigen polypeptide is C-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is C-terminal to the β2-microglobulin polypeptide on the single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the β2-microglobulin polypeptide, and a second flexible polypeptide linker separates the randomized antigen polypeptide from the HLA polypeptide. In some embodiments, the β2-microglobulin polypeptide is C-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is N-terminal to the randomized antigen polypeptide on the single polypeptide. In these embodiments, the first flexible polypeptide linker separates the HLA polypeptide from the randomized antigen polypeptide, and a second flexible polypeptide linker separates the randomized antigen polypeptide from the β2-microglobulin polypeptide. In some embodiments, the randomized antigen polypeptide is C-terminal to the β2-microglobulin on the single polypeptide, and the HLA polypeptide is C-terminal to the randomized antigen polypeptide on the single polypeptide. In these embodiments, the first flexible polypeptide linker separates the β2-microglobulin polypeptide from the randomized antigen polypeptide, and a second flexible polypeptide linker separates the randomized antigen polypeptide from the HLA polypeptide.
In some embodiments, each of the HLA-antigen complexes of the plurality of the HLA-antigen complexes do not comprise an epitope tag. In some embodiments, at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes comprise an epitope tag. In some embodiments, at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes does not comprise an epitope tag and at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes does comprise an epitope tag. In some embodiments, the epitope tag comprises a FLAG tag, a c-MYC tag, a HIS-tag, a hemagglutinin (HA) tag, a VSVg tag, or a V5 tag.
In some embodiments, the HLA-antigen complexes each comprise a membrane tethering domain. In some embodiments, the membrane tethering domain comprises Aga2. In some embodiments, the antigen screening library is expressed on a plurality of cells.
In some embodiments, the plurality of cells are a plurality of yeast cells. In some embodiments, the plurality of yeast cells are a plurality of yeast cells of the EBY100 strain of Saccharomyces cerevisiae.
In some embodiments, each cell of the plurality of cells expresses a specific HLA-antigen complex.
Provided herein in some embodiments are antigen screening libraries comprising a plurality of Human Leukocyte Antigen (HLA)-antigen polypeptide complexes, the HLA-antigen polypeptide complexes comprising an HLA polypeptide, the HLA polypeptide comprising a peptide binding cleft, and a randomized antigen polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 209, wherein the randomized antigen polypeptide specifically binds to the peptide binding cleft of the HLA polypeptide.
In some embodiments, the plurality of HLA-antigen complexes comprises an HLA polypeptide selected from the list consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises at least five, ten, fifteen, twenty, or twenty-five different HLA polypeptides selected from the list consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises all of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E HLA polypeptides.
In some embodiments, the plurality of HLA-antigen complexes comprises an HLA polypeptide comprising an amino acid sequence at least 87.5%, 90%, 95%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 427 to 455.
In some embodiments, the plurality of the HLA-antigen polypeptide complexes comprises at least about 105 different HLA-antigen polypeptide complexes comprising at least about 105 different randomized antigen polypeptides.
In some embodiments, the HLA polypeptide, the randomized antigen polypeptide, and the β2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, the single polypeptide further comprises a first flexible polypeptide linker separating the HLA polypeptide from the randomized antigen polypeptide. In certain of these embodiments, the randomized antigen polypeptide is N-terminal to the HLA polypeptide on the single polypeptide. In certain of these embodiments, the randomized antigen polypeptide is C-terminal to the HLA polypeptide on the single polypeptide.
In some embodiments, each of the HLA-antigen complexes of the plurality of the HLA-antigen complexes do not comprise an epitope tag. In some embodiments, at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes comprise an epitope tag. In some embodiments, at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes does not comprise an epitope tag and at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes does comprise an epitope tag. In some embodiments, the epitope tag comprises a FLAG tag, a c-MYC tag, a HIS-tag, a hemagglutinin (HA) tag, a VSVg tag, or a V5 tag.
In some embodiments, the HLA-antigen complexes each comprise a membrane tethering domain. In some embodiments, the membrane tethering domain comprises Aga2. In some embodiments, the antigen screening library is expressed on a plurality of cells.
In some embodiments, the plurality of cells are a plurality of yeast cells. In some embodiments, the plurality of yeast cells are a plurality of yeast cells of the EBY100 strain of Saccharomyces cerevisiae.
In some embodiments, each cell of the plurality of cells expresses a specific HLA-antigen complex.
Provided herein in some embodiments are antigen screening libraries comprising a plurality of antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes, the antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes. In these embodiments, the antigen screening libraries further comprise a randomized antigen polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 209, wherein the randomized antigen polypeptide specifically binds to the peptide binding cleft of the HLA polypeptide; and a Beta-2 (β2) microglobulin polypeptide. In these embodiments, the antigen screening libraries also further comprise a plurality of HLA polypeptides constitutively expressed by one or more yeast cells and comprising a peptide binding cleft.
In some embodiments, the plurality of HLA-antigen complexes comprises an HLA polypeptide selected from the list consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises at least five, ten, fifteen, twenty, or twenty-five different HLA polypeptides selected from the list consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises all of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E HLA polypeptides.
In some embodiments, the plurality of HLA-antigen complexes comprises an HLA polypeptide comprising an amino acid sequence at least 87.5%, 90%, 95%, 97%, 98%, 99%, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 427 to 455.
In some embodiments, the plurality of the antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes comprises at least about 105 different antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes comprising at least about 105 different randomized antigen polypeptides.
In some embodiments, the randomized antigen polypeptide and the β2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, the single polypeptide further comprises a first flexible polypeptide linker. In certain of these embodiments, the randomized antigen polypeptide is N-terminal to the β2-microglobulin polypeptide on the single polypeptide. In certain of these embodiments, the randomized antigen polypeptide is C-terminal to the β2-microglobulin polypeptide on the single polypeptide.
In some embodiments, each of the antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes of the plurality of the antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes do not comprise an epitope tag. In some embodiments, at least one of the antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes of the plurality of antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes comprise an epitope tag. In some embodiments, at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes does not comprise an epitope tag and at least one of the HLA-antigen complexes of the plurality of HLA-antigen complexes does comprise an epitope tag. In some embodiments, the epitope tag comprises a FLAG tag, a c-MYC tag, a HIS-tag, a hemagglutinin (HA) tag, a VSVg tag, or a V5 tag.
In some embodiments, the antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complexes each comprise a membrane tethering domain. In some embodiments, the membrane tethering domain comprises Aga2. In some embodiments, the antigen screening library is expressed on a plurality of cells.
In some embodiments, the plurality of cells are a plurality of yeast cells. In some embodiments, the plurality of yeast cells are a plurality of yeast cells of the EBY100 strain of Saccharomyces cerevisiae.
In some embodiments, each cell of the plurality of cells expresses a specific antigen polypeptide-Beta-2 (β2) microglobulin polypeptide complex.
Provided herein in some embodiments are a plurality of nucleic acids encoding the antigen screening libraries in accordance with the present technology.
In some embodiments, the HLA polypeptide of the HLA-antigen complex is encoded by a nucleic acid that is at least about 85%, 87.5%, 90%, 95%, 97%, 98%, or 99% homologous to any one of SEQ ID NOs: 456 to 484. In some embodiments, the randomized antigen polypeptide of the HLA-antigen complex is encoded by a nucleic acid set forth in any one of SEQ ID NOs: 210 to 426.
In some embodiments, the plurality of nucleic acids is expressed by a plurality of cells.
Provided herein in some embodiments are a plurality of cells expressing the antigen screening library in accordance with the present technology.
In some embodiments, the plurality of cells is a plurality of yeast cells. In some embodiments, the plurality of yeast cells is a plurality of cells of the EBY100 strain of Saccharomyces cerevisiae. In some embodiments, each cell of the plurality of cells comprises a nucleic acid of the plurality of nucleic acids encoding a specific of HLA-antigen complex.
Provided herein in some embodiments are methods of selecting an antigen comprising contacting the plurality of cells in accordance with the present technology with a T cell receptor (TCR).
In some embodiments, the TCR is immobilized on a substrate. In some embodiments, the TCR is expressed by a cell.
In some embodiments, the selection is repeated for 2, 3, 4, or 5 cycles.
In some embodiments, the antigen is a polypeptide antigen. In some embodiments, the antigen is a polypeptide antigen that does not naturally occur. In some embodiments, the antigen is a polypeptide antigen that does not naturally occur in a human.
Described herein are antigen screening libraries useful for selection and/or identification of polypeptide ligands for T cell receptors (TCRs). In many cases, the antigen screening libraries are useful to discover polypeptide antigens that are capable of interacting with and stimulating human T cells as TCR ligands, including both endogenous TCR antigens and non-endogenous TCR antigens which may be novel TCR antigens and/or novel epitopes. Such novel antigens and/or novel epitopes are useful, at least for example, to stimulate one or more TCRs on T cells that may have become exhausted or anergized, and revive immune responses against cancer, tumors, or chronic viral infections. Accordingly, the present disclosure includes peptide library display, such as randomized peptide antigen libraries, in the context of a given HLA to determine the specificities and general recognition properties of TCRs restricted to HLA-mediated peptide recognition.
Once expressed using the methodologies described herein, a randomized peptide antigen library may be displayed by HLA molecules that are expressed on the surface of cells. In general, the cells that display these HLA-antigen polypeptide complexes are not normal antigen presenting cells of a host's immune system but rather are cells that can easily be transformed, transfected, transduced, and/or electroporated with a nucleic acid encoding an HLA-antigen polypeptide, including without limitation, insect cells, yeast cells, and bacterial cells. In some embodiments, the randomized peptide antigen library is expressed by yeast cells. A mixture of plasmids that encode at least 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or 1015 distinct polypeptide antigens, and either one or a plurality of different HLA molecules, are transformed into yeast cells. Following transformation with the randomized peptide antigen library, the yeast cells that express the HLA-antigen polypeptide complex library are then contacted by a TCR, or other macromolecule having one or more antigen binding domains, serving as a bait. The TCRs are either (1) expressed by a cell or (2) recombinantly produced and, optionally, multimerized and/or immobilized, on a solid structure, such as a bead, or via a protein scaffold such as streptavidin or streptavidin conjugated dextran (referenced as the selection reagent). The cells expressing HLA-antigen polypeptide complexes that interact with the TCR selection reagent can be selected by an appropriate modality, and after 2, 3, 4, 5, 6, 7 or more rounds of enrichment (e.g., cycles) the nucleic acids encoding the HLA-antigen polypeptide complexes can be extracted from the enriched cells and sequencing can be performed to determine the polypeptide antigens that have been enriched. The enriched polypeptide antigens define the structural attributes that interact with a given TCR.
In some embodiments, the present disclosure includes an antigen screening library which comprises a plurality of HLA-antigen polypeptide complexes. In some embodiments, the HLA-antigen polypeptide complexes comprise (a) an HLA polypeptide, the HLA polypeptide comprising a peptide binding cleft; (b) a randomized antigen polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 194, wherein the randomized antigen polypeptide is selected to specifically bind to the peptide binding cleft of the HLA polypeptide; and (c) a beta-2 (β2) microglobulin polypeptide. Also provided herein are derivatives of randomized peptide antigens and libraries thereof, compositions thereof, pharmaceutical compositions thereof, and uses of the same. Also provided herein are nucleic acid sequences encoding one or more randomized peptide antigen libraries disclosed herein and derivatives thereof, and methods for expressing the one or more randomized peptide antigen libraries, peptides thereof, and derivatives thereof in one or more cells.
As set forth in the examples provided herein, a randomized peptide antigen library was designed (Example 1) and includes nucleic acid constructs (
The following description of the invention is merely intended to illustrate various embodiments of the present disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the present disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments are to be included herein.
All references listed herein are incorporated by reference, in their entirety. Methods and apparatuses are provided here by way of example and are not intended to be limiting to the present disclosure.
In the following description, some specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length, though a number of amino acid residues may be specified (e.g., 9mer is nine amino acid residues). Polypeptides may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E.
The term “amide residue” refers to amino acids in D- or L-form having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q.
The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.
The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, and R.
The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, and Q.
The term “nonfunctional residue” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle).
The term “neutral hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic, acidic, or polar groups. Exemplary neutral hydrophobic amino acid residues include A, V, L, I, P, W, M, and F.
The term “polar hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q, and N.
The term “hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic or acidic groups. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q, and N.
“Percent (%) sequence identity” with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that is identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software, or other software appropriate for nucleic acid sequences. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a some % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe to a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin & Altschul 1990, modified as in Karlin & Altschul 1993. Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul 1990. Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.
“T cell receptor” (TCR), refers to an antigen/MHC binding heterodimeric protein product of a vertebrate, e.g. mammalian, TCR gene complex, including the human TCR α, β, γ and δ chains. For example, the complete sequence of the human (3 TCR locus has been sequenced, as published by Rowen 1996; the human TCR locus has been sequenced and resequenced, for example see Mackelprang 2006; see a general analysis of the T-cell receptor variable gene segment families in Arden 1995; each of which is herein specifically incorporated by reference for the sequence information provided and referenced in the publication.
“Bait” refers to a TCR or “other macromolecule having one or more antigen binding domains” that binds to an antigen of the present technology. The other macromolecule having one or more antigen binding domains is an antibody, a DARPin, or a synthetic molecule, including aptamers. The antigen binding domain binds a peptide, such as one or more of the HLA-peptide complexes of the present technology, or a nucleic acid, such as DNA and RNA.
“Exogenous” with respect to a nucleic acid or polynucleotide indicates that the nucleic acid is part of a recombinant nucleic acid construct or is not in its natural environment. For example, an exogenous nucleic acid can be a sequence from one species introduced into another species, i.e., a heterologous nucleic acid. Typically, such an exogenous nucleic acid is introduced into the other species via a recombinant nucleic acid construct. An exogenous nucleic acid also can be a sequence that is native to an organism and that has been reintroduced into cells of that organism. An exogenous nucleic acid that includes a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., nonnative regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. The exogenous elements may be added to a construct, for example, using genetic recombination. Genetic recombination is the breaking and rejoining of DNA strands to form new molecules of DNA encoding a novel set of genetic information.
As used herein the term “about” refers to an amount that is near the stated amount by 10%.
Disclosed herein are antigen screening libraries, such as randomized peptide antigen libraries, which include a plurality of HLA-antigen polypeptide complexes. The HLA-antigen polypeptide complexes of the current disclosure minimally comprise at least three constituents: (a) a randomized antigen polypeptide, (b) a major histocompatibility class I (MHC I) HLA molecule, and (c) a β2-microglobulin. In some embodiments, the randomized antigen polypeptide of (a) is randomized having at least one or more residues conserved that serve as anchor residues to bind to an HLA molecule of a specific type. Exemplary, but not limiting, randomized antigen polypeptide antigens and the HLA type with which they associate are shown in Table 1 and given by SEQ ID NOs: 1 to 194 and Table 2 and given by SEQ ID NOs: 195 to 209. In some embodiments, the randomized polypeptide antigens comprises a sequence that is at least about 70%, 75%, 80%, 85%, 87%, 87.5%, 90%, 95%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of, but not limited to, the amino acid sequences set forth in any one of SEQ ID NOs: 1 to 194 and SEQ ID NOs: 195 to 209. In some embodiments, the randomized polypeptide antigens comprise a sequence identical to any one of those set forth in any one of SEQ ID NOs: 1 to 194 and SEQ ID NOs: 195 to 209. Also envisioned within the present disclosure are randomized polypeptide antigen truncations that have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acids truncated from the N-terminus or truncated from the C-terminus of any one of SEQ ID NOs: 1 to 194 and SEQ ID NOs: 195 to 209. In some embodiments, the HLA molecule of (b) is a HLA polypeptide and comprises a peptide binding cleft. Once expressed, in some embodiments, the randomized antigen polypeptide of (a) binds the HLA polypeptide of (b) at the peptide binding cleft.
In some embodiments, antigen screening libraries of the present disclosure include (b) randomized antigen polypeptides encoded at least by, but not limited to, nucleotide sequences SEQ ID NOs: 210 to 411 provided at least in Table 4. In some embodiments, antigen screening libraries of the present disclosure include (b) randomized antigen polypeptides encoded at least by, but not limited to, nucleotide sequences SEQ ID NOs: 412 to 426 provided at least in Table 5. Nucleic acids that encode the randomized antigen polypeptides of (b) are encoded by a degenerate base sequence, effectively allowing any amino acid to be encoded at a given position corresponding to the degenerate base sequence. Each randomized antigen polypeptide has at least one conserved anchor position that is encoded by a restricted degenerate code, or a specific sequence, which allows the randomized antigen polypeptide to more efficiently interact with a certain HLA type. Having at least one conserved anchor position per randomized antigen polypeptide increases efficiency of formation of a randomized antigen polypeptide and HLA complex compared to formation of an HLA complex with a fully randomized antigen polypeptide. In some embodiments, 1, 2, or 3 of the amino acid residues of a randomized antigen polypeptide are constant. In some embodiments, the randomized antigen polypeptide antigens comprises a sequence that is at least about 70%, 75%, 80%, 85%, 87%, 87.5%, 90%, 95%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of, but not limited to, the amino acid sequences set forth in any one of SEQ ID NOs: 210 to 411 and SEQ ID NOs: 412 to 426. In some embodiments, the randomized antigen polypeptide antigens comprise a sequence identical to any one of those set forth in any one of SEQ ID NOs: 210 to 411 and SEQ ID NOs: 412 to 426. Also envisioned within the present disclosure are randomized antigen polypeptide antigen polypeptide truncations that have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acids truncated from the N-terminus or truncated from the C-terminus of any one of SEQ ID NOs: 210 to 411 and SEQ ID NOs: 412 to 426.
In some embodiments, amino acid residues of a randomized antigen polypeptide vary by 2, 3, or 4 different amino acids. For example, referring to Table 1, the second and the last position of a randomized antigen polypeptide that binds to HLA-A2 will comprise leucine or methionine; and leucine, methionine, or valine, respectively.
The amino acid sequences in Tables 1 and 2 above include random amino acid residues (‘X’) and explicitly defined amino acids located at residues referred to collectively as anchor positions. The anchor positions specified in the library design can be altered, for example, based on amino acid substitutions set forth in Table 3. One of ordinary skill in the art would appreciate that possible substitutions for X residue in the amino acid sequences of Tables 1 and 2 are not limited and can include additional substitutions without departing from the scope of the disclosure. For example, amino acid substitutions can be used to identify important residues of the peptide sequence that contribute to binding of the HLA or to constrain of expand the members of the library described herein.
Conservative modifications will produce peptides having functional and chemical characteristics similar to those of the peptide from which such modifications are made. In contrast, substantial modifications in the functional and/or chemical characteristics of the peptides may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.
For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis” (see, for example, MacLennan 1998 and Sasaki & Sutoh 1998, which discuss alanine scanning mutagenesis).
Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. Exemplary amino acid substitutions are set forth in Table 3.
In certain embodiments, conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems.
As noted in the foregoing section “Certain Definitions,” naturally occurring residues may be divided into classes based on common sidechain properties that may be useful for modifications of sequence. For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the peptide that are homologous with non-human orthologs, or into the non-homologous regions of the molecule. In addition, one may also make modifications using P or G for the purpose of influencing chain orientation.
In making such modifications, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics; these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte & Doolittle 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. with a biological property of the protein.
The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”
A skilled artisan will be able to determine suitable variants of the polypeptide as set forth in the foregoing sequences using well known techniques. For identifying suitable areas of the molecule that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a peptide to similar peptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a peptide that are not conserved relative to such similar peptides would be less likely to adversely affect the biological activity and/or structure of the peptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (conservative amino acid residue substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the peptide structure.
Additionally, one skilled in the art can review structure-function studies identifying residues in similar peptides that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a peptide that correspond to amino acid residues that are important for activity or structure in similar peptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues of the peptides.
One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of that information, one skilled in the art may predict the alignment of amino acid residues of a peptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such data could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change would be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided, either alone or in combination with other mutations.
A number of scientific publications have been devoted to the prediction of secondary structure (see, e.g., Moult 1996; Chou & Fasman 1974a; Chou & Fasman 1974b; Chou & Fasman 1978a; Chou & Fasman 1978b; and Chou & Fasman 1979). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure (Holm & Sander 1999). It has been suggested that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will gain dramatically in accuracy (Brenner 1997).
Additional methods of predicting secondary structure include “threading” (Jones 1997; Sippl & Flockner 1996), “profile analysis” (Bowie 1991; Gribskov 1987; Gribskov 1990), and “evolutionary linkage” (Holm & Sander 1999; Brenner 1997).
One advantage of a randomized antigen polypeptide is that a single nucleic acid with a degenerate base code can potentially express a large amount of different randomized antigen polypeptides, which increases the chances that any one screening experiment will identify one or more randomized antigen polypeptides that interact with a certain TCR. In some embodiments, the nucleic acid that encodes the randomized antigen polypeptide can encode at least 1×104, at least 1×105, at least 1×106, at least 1×107, at least 1×108, at least 1×109, at least 1×1010, at least 1×1011, at least 1×1012, at least 1×1013, at least 1×1014, or at least 1×1015 different randomized polypeptide antigens.
Peptide antigens that bind in the binding cleft of an HLA molecule are generally of a restricted length range. The majority of polypeptides that bind to class I HLA molecules are 8, 9, 10, or 11 amino acids in length. In some embodiments, the randomized antigen polypeptide which binds to an HLA molecule and forming the HLA-antigen polypeptide complexes of the present disclosure is between 8 and 11 amino acids in length. In some embodiments, the randomized antigen polypeptide is between 8 and 10 amino acids in length. In some embodiments, the randomized antigen polypeptide is 8 amino acids in length. In some embodiments, the randomized antigen polypeptide is 9 amino acids in length. In some embodiments, the randomized antigen polypeptide is 10 amino acids in length. In some embodiments, the randomized antigen polypeptide is 11 amino acids in length.
Another constituent of the HLA-antigen polypeptide complexes described herein is an HLA molecule, such as an HLA polypeptide. For the purposes of the current disclosure, the HLA molecule is a class I major histocompatibility molecule. In some embodiments, the plurality of HLA polypeptides of the HLA-antigen polypeptide complexes of the current disclosure (HLA-antigen complexes) can comprise any of the following loci and alleles: A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, each of the HLA-antigen complexes in the plurality of HLA-antigen complexes comprise an HLA polypeptide selected from the group of HLA polypeptides consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises at least five, ten, fifteen, twenty, or twenty-five different HLA polypeptides selected from the group of HLA polypeptides consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E. In some embodiments, the plurality of HLA-antigen complexes comprises all of the HLA polypeptides in the group of HLA polypeptides consisting of A3, A11, A23, A24, A26, A30, A31, A33, A68, B7, B8, B15, B27, B40, B44, B51, B53, C1, C2, C3, C4, C5, C6, C7, C8, and E.
In some embodiments, the amino acid sequence of the HLA polypeptide of the HLA-antigen polypeptide complex can comprise any of the amino acid sequences set forth in Table 6. In some embodiments, the HLA polypeptide comprises an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 87%, 87.5%, 90%, 95%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% identical to any one of, but not limited to, the amino acid sequences set forth in any one of SEQ ID NOs: 427 to 455. In some embodiments, the HLA polypeptide comprises an amino acid sequence identical to any one of those set forth in any one of SEQ ID NOs: 427 to 455. In some embodiments, a portion of the HLA polypeptide that comprises the peptide binding cleft is identical to any one of SEQ ID Nos: 251 to 279, and the non-peptide binding cleft residues are at least about 70%, 75%, 80%, 85%, 87.5%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 427 to 455. Also envisioned within the present disclosure are HLA polypeptide truncations that have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acids truncated from the N-terminus or truncated from the C-terminus of any one of SEQ ID NOs: 427 to 455.
The HLA polypeptide of the HLA-antigen polypeptide complex can be encoded by a nucleic acid of any set forth in Table 7. In some embodiments, the HLA polypeptide is encoded by a nucleic acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% homologous to at least, but not limited to, any one of the nucleic acid sequences listed in Table 7, such as SEQ ID NOs: 456 to 484. In some embodiments, the HLA polypeptide is encoded by a nucleic acid sequence identical to that set forth in any one of SEQ ID NOs: 456 to 484.
In some embodiments, the plurality of the HLA-antigen polypeptide complexes of the randomized peptide antigen libraries comprise at least about 105 different HLA-antigen polypeptide complexes. Components of the 105 different HLA-antigen polypeptide complexes include, collectively, at least about 105 different randomized antigen polypeptides. In some embodiments, the plurality of the HLA-antigen polypeptide complexes of the randomized peptide antigen libraries comprise at least about 107 different HLA-antigen polypeptide complexes. Components of the 107 different HLA-antigen polypeptide complexes include, collectively, at least about 107 different randomized antigen polypeptides. In some embodiments, the plurality of the HLA-antigen polypeptide complexes of the randomized peptide antigen libraries comprise at least about 109 different HLA-antigen polypeptide complexes. Components of the 109 different HLA-antigen polypeptide complexes include, collectively, at least about 109 different randomized antigen polypeptides. In some embodiments, the plurality of the HLA-antigen polypeptide complexes of the randomized peptide antigen libraries comprise at least about 1011 different HLA-antigen polypeptide complexes. Components of the 1011 different HLA-antigen polypeptide complexes include, collectively, at least about 1011 different randomized antigen polypeptides.
In some embodiments, the plurality of the HLA-antigen polypeptide complexes of the randomized peptide antigen libraries further comprise a β2-microglobulin polypeptide, which interacts with and stabilizes the HLA-antigen polypeptide complexes on the surface of the cell. The amino acid sequence of human β2-microglobulin polypeptide is set forth in NCBI Seq. Ref. NP_004039. In some embodiments, the human β2-microglobulin polypeptide amino acid sequence of the present disclosure is a functional naturally occurring variant of the human β2-microglobulin polypeptide having an amino acid sequence at least about 90%, 95%, 97%, 98%, or 99% identical to the human β2-microglobulin polypeptide disclosed as NCBI Seq. Ref NP_004039.
The present disclosure also includes antigen screening libraries of a plurality HLA-antigen polypeptide where the β2-microglobulin is constitutively expressed by a cell. In some embodiments, the β2-microglobulin is encoded by a first nucleic acid, the randomized antigen polypeptide encoded by a second nucleic acid, and the HLA polypeptide is encoded by a third nucleic acid. In other embodiments, the β2-microglobulin is encoded by a first nucleic acid and the randomized antigen polypeptide and the HLA polypeptide is encoded by a second nucleic acid. When encoded by the first nucleic acid, the β2-microglobulin can be transduced, transfected, or transformed into a cell before or after the second nucleic acid or the third nucleic acid.
In some embodiments of the present disclosure, the β2-microglobulin is fused to at least one of the randomized antigen polypeptides of the antigen screening library using techniques known to those of ordinary skill in the art. In these embodiments, the HLA polypeptides may or may not be a component of the antigen screening library. In other embodiments of the present disclosure, at least one of the HLA polypeptides is fused to at least one of the randomized antigen polypeptides of the antigen screening library using techniques known to those of ordinary skill in the art. In these embodiments, the β2-microglobulin can be expressed by a cell that is transduced, transfected, or transformed to express other components of the antigen screening library, such as the randomized antigen polypeptides and the HLA polypeptides. Similar to other embodiments described herein, the β2-microglobulin is constitutively expressed by the cell. In certain of these embodiments, the cell is a yeast cell. In other embodiments, the β2-microglobulin is not expressed by the cell that is transduced, transfected, or transformed to express other components of the antigen screening library, such as the randomized antigen polypeptides and the HLA polypeptides. In certain of these embodiments, the cell is a mammalian cell.
In addition to the (a) randomized antigen polypeptide, (b) MHC I HLA molecule, and (c) β2-microglobulin features of the HLA-antigen polypeptide complex of the randomized peptide antigen libraries, the HLA-antigen polypeptide complexes of the present disclosure can further include (d) a signal sequence, (e) polypeptide linkers between any or all of (a), (b), or (c), (f) a membrane tethering domain, and, optionally, (g) an epitope tag, such as a FLAG tag, a c-Myc tag, a His-tag, a hemagglutinin (HA) tag, a VSVg tag, a V5 tag, an AU1 tag, an AU5 tag, a Glu-Glu tag, an OLLAS tag, a T7 tag, an S-TagHSV tag, a KT3 tag, a TK15 tag, an Fc tag, an Xpress tag, a Ty tag, a Strep tag, an NE tag, an E tag, a C-tag, and/or an AviTag. In some embodiments, the HLA-antigen complexes do not comprise an epitope tag. However, in some embodiments, at least one or more of each of the plurality of HLA-antigen complexes of the randomized peptide antigen libraries comprise the epitope tag which allows for confirmation of expression of at least one of the HLA-antigen complexes using an antibody specific for the epitope. In some embodiments, each of the plurality of HLA-antigen complexes of the randomized peptide antigen libraries comprise the epitope tag.
In some embodiments, the membrane tethering domain comprises a polypeptide linker separating the membrane tethering domain from one or more other features ((a)-(e) and (g)) of the HLA-antigen polypeptide complex. In some embodiments, the features ((a)-(g)) of the HLA-antigen polypeptide complex are expressed as a single polypeptide. In some embodiments, the (b) HLA molecule (e.g., HLA polypeptide), the (a) randomized antigen polypeptide, and the (c) β2-microglobulin polypeptide comprise a single polypeptide. In some embodiments, the (b) HLA polypeptide and the (a) randomized antigen polypeptide are expressed as a single polypeptide, while, the (c) β2-microglobulin is expressed separately. For example, the (c) β2-microglobulin can be supplied from a separate polypeptide encoded by the same nucleic acid that expresses the (a) randomized antigen polypeptide and the (b) HLA polypeptide, a separate nucleic acid, or endogenously produced by the cell. In some embodiments, the randomized antigen polypeptide is N-terminal to the HLA polypeptide, and the HLA polypeptide is N-terminal to the β2-microglobulin polypeptide. In some embodiments, the randomized antigen polypeptide is C-terminal to the HLA polypeptide, and the HLA polypeptide is N-terminal to the β2-microglobulin polypeptide. In some embodiments, the randomized antigen polypeptide is N-terminal to the HLA polypeptide, and the HLA polypeptide is C-terminal to the β2-microglobulin polypeptide. In some embodiments, the randomized antigen polypeptide is C-terminal to the HLA polypeptide, and the HLA polypeptide is C-terminal to the β2-microglobulin polypeptide.
The (a) a randomized antigen polypeptide, (b) a major histocompatibility class I (MHC I) HLA molecule, and (c) a β2-microglobulin, can be separated by at least one flexible polypeptide linker, such as a first flexible polypeptide linker, a second flexible polypeptide linker, a third flexible polypeptide linker, a fourth flexible polypeptide linker, a fifth flexible polypeptide linker, or more flexible polypeptide linkers. In some embodiments, the at least one flexible polypeptide linker can range between about 3 and about 100 amino acid residues in length, between about 5 and about 80 amino acid residues in length, between about 10 and about 70 amino acid residues in length, between about 3 and about 100 amino acid residues in length, between about 20 and about 60 amino acid residues in length. In some embodiments, the linker can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. In some embodiments, the linker can be a glycine linker, or a Gly-Ser linker of the formula (GGGGS)X, wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker can suitably comprise a protease cleavage site such as a thrombin cleavage site.
In some embodiments, the HLA-antigen polypeptide complexes of the randomized peptide antigen libraries comprise a signal polypeptide which directs the HLA-antigen polypeptide complex to the cell surface via the secretory pathway. This signal peptide is cleaved in the endoplasmic reticulum and is not expressed by the HLA-antigen polypeptide complex when located on the cell-surface. The signal sequence can be any suitable sequence such as an endogenous HLA leader sequence, or a heterologous leader sequence imported from a different secretory or transmembrane molecule, such as an immunoglobulin leader sequence.
The HLA-antigen polypeptide complexes further comprise a membrane tethering domain, such as an anchor domain from a glycosylphosphatidylinositol (GPI) protein and/or a domain from yeast proteins having internal repeats (PIR protein). This membrane tethering domain can comprise a transmembrane domain or a domain that interacts with a cell surface protein. In some embodiments, the membrane tethering domain comprise at least one anchor domain of a GPI protein selected from the group consisting of yeast Aga2, Cwp1p, Cwp2p, Aga1p, Tip1p, Flo1p, Sed1p, YCR89w, and Tir1p and/or a PIR protein selected from the group consisting of yeast Pir1p, Pir2p, Pir3p, Pir4p, and Pir5p. A non-limiting example of membrane domain tethering is provided in
In other embodiments, components of the antigen screening libraries of a plurality HLA-antigen polypeptide complexes are expressed as more than one polypeptide and include a cleavage sequence which separates components of the antigen screening libraries of a plurality HLA-antigen polypeptide complexes from one another. For example, the randomized peptide antigen is separated from the HLA polypeptide and/or from the Beta-2 (β2) microglobulin polypeptide by the cleavage sequence. As another example, the HLA peptide is separated from the Beta-2 (β2) microglobulin polypeptide by the nucleotide encoded cleavage sequence. In some embodiments, the components of the antigen screening libraries are separated by more than one cleavage sequence. Suitable cleavage sequences are known to those of ordinary skill in the art and include, but are not limited to, self-cleaving peptides (P2A, T2A, F2A, and E2A), proteolytic cleavage sites (a 3C site, a thrombin site, a TEV site, a Factor Xa site, and an EKT site) and an internal ribosome entry sequence (IRES).
In some embodiments, the antigen screening library and/or the HLA-antigen polypeptide complexes can be expressed by one or more cells that can easily be transfected, transduced, electroporated, or transformed with the nucleic acids described herein. In some embodiments, the antigen screening library and/or the HLA-antigen polypeptide complexes are expressed on a plurality of cells. In some embodiments, each cell of the plurality of cells expresses a specific HLA-antigen complex of the HLA-antigen polypeptide complexes and/or another component of the antigen screening library. In some embodiments, a nucleic acid or a plurality of nucleic acids encode the antigen screening library and/or the HLA-antigen polypeptide complexes. In some embodiments, the antigen screening library and/or the HLA-antigen polypeptide complexes comprise prokaryotic cells. In some embodiments, the cell expressing the HLA-antigen polypeptide complexes comprise eukaryotic cells. In some embodiments, the eukaryotic cells comprise yeast cells. In some embodiments, the yeast cells are a cell of Saccharomyces cerevisiae. In some embodiments, the Saccharomyces cerevisiae is of the strain EBY100. Transforming Saccharomyces cerevisiae with nucleic acids can be achieved by standard methods as long as the efficiency is sufficient to produce at least 107, 108, 109, or 1010 transformants.
In addition to the plurality of HLA-antigen polypeptide complexes of the antigen screening libraries described above, the present technology also includes at least two or more antigen screening libraries having HLA-antigen polypeptide complexes that differ from those described above. In some embodiments, the HLA-antigen polypeptide complexes have fewer components and/or at least one different component than the plurality of HLA-antigen polypeptide complexes described above. For example, in some embodiments, HLA-antigen polypeptide complexes can also comprise (a) an HLA polypeptide having a peptide binding cleft; and (b) a randomized antigen polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 209 that specifically binds to the peptide binding cleft of the HLA polypeptide. In these embodiments, the HLA polypeptide, and the randomized antigen polypeptide comprise a single polypeptide. Also in these embodiments, the single polypeptide further comprises a first flexible polypeptide linker separating the HLA polypeptide from the randomized antigen polypeptide. When expressed on a single polypeptide separated by the first flexible polypeptide linker, the randomized antigen polypeptide is N-terminal to the HLA polypeptide on the single polypeptide or the randomized antigen polypeptide is C-terminal to the HLA polypeptide on the single polypeptide.
As another example, in some embodiments, antigen screening libraries of the present technology comprise (a) an HLA polypeptide constitutively expressed by one or more yeast cells, the HLA polypeptide comprising a peptide binding cleft, and (b) a plurality of Beta-2 (β2) microglobulin polypeptide-antigen polypeptide complexes. In these embodiments, the plurality of Beta-2 (β2) microglobulin polypeptide complexes include a randomized antigen polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 209, wherein the randomized antigen polypeptide specifically binds to the peptide binding cleft of the HLA polypeptide; and (c) a Beta-2 (β2) microglobulin polypeptide. In these embodiments, the randomized antigen polypeptide and the β2-microglobulin polypeptide comprise a single polypeptide. Also, in these embodiments, the single polypeptide further comprises a first flexible polypeptide linker separating the Beta-2 (β2) microglobulin polypeptide from the randomized antigen polypeptide. When expressed on a single polypeptide separated by the first flexible polypeptide linker, the randomized antigen polypeptide is N-terminal to the Beta-2 (β2) microglobulin polypeptide on the single polypeptide or the randomized antigen polypeptide is C-terminal to the Beta-2 (β2) microglobulin polypeptide on the single polypeptide.
Also disclosed herein are nucleic acids that encode HLA-antigen polypeptide complexes of the antigen screening libraries. Nucleic acids that encode the HLA-antigen polypeptide complexes of the current disclosure minimally encode: (a) a randomized antigen polypeptide, (b) an MHC I HLA molecule, and a (c) β2-microglobulin. In addition to the (a) randomized antigen polypeptide, (b) MHC I HLA molecule, and (c) β2-microglobulin features of the HLA-antigen polypeptide complex of the randomized peptide antigen libraries encoded by one or more nucleic acids, the HLA-antigen polypeptide complexes of the present disclosure further include nucleic acids which encode (d) a signal sequence, (e) polypeptide linkers between any or all of (a), (b), or (c), (f) a membrane tethering domain, and, optionally, (g) an epitope tag, such as a FLAG tag, a c-MYC tag, a HIS-tag, a hemagglutinin tag, a VSVg tag, a V5 tag, an AU1 tag, an AU5 tag, a Glu-Glu tag, an OLLAS tag, a T7 tag, an S-Tag, an HSV tag, a KT3 tag, a TK15 tag, an Fc tag, an Xpress tag, a Ty tag, a Strep tag, an NE tag, an E tag, a C-tag, and/or an AviTag (
In some embodiments, the nucleic acid encoding the (f) membrane tethering domain may further encode (e) one or more polypeptide linkers separating the membrane tethering domain from other features of the HLA-antigen polypeptide complex. In some embodiments, the nucleic acid encodes one or more flexible polypeptide linkers which separate the (a) HLA polypeptide from the (b) randomized antigen polypeptide and the (c) β2-microglobulin polypeptide when all three features are encoded on the single nucleic acid.
In some embodiments, the nucleic acid encoding the single polypeptide further comprises nucleotides which encode a first flexible polypeptide linker and a second flexible polypeptide linker, wherein the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the randomized antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the randomized antigen polypeptide from the nucleotide sequence encoding the β2-microglobulin polypeptide. In some embodiments, once expressed, the randomized antigen polypeptide is N-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is N-terminal to the β2-microglobulin polypeptide on the single polypeptide.
In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the randomized antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the β2-microglobulin polypeptide. In some embodiments, once expressed, the randomized antigen polypeptide is C-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is N-terminal to the β2-microglobulin polypeptide on the single polypeptide.
In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the randomized antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the β2-microglobulin polypeptide. In some embodiments, once expressed, the randomized antigen polypeptide is N-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is C-terminal to the β2-microglobulin polypeptide on the single polypeptide.
In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the randomized antigen polypeptide from the nucleotide sequence encoding the β2-microglobulin polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the β2-microglobulin polypeptide from the nucleotide sequence encoding the HLA polypeptide. In some embodiments, once expressed the randomized antigen polypeptide is C-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is C-terminal to the β2-microglobulin polypeptide on the single polypeptide.
In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the β2-microglobulin polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the randomized antigen polypeptide from the nucleotide sequence encoding the HLA polypeptide. In some embodiments, once expressed, the β2-microglobulin polypeptide is C-terminal to the HLA polypeptide on the single polypeptide, and the HLA polypeptide is N-terminal to the randomized antigen polypeptide on the single polypeptide.
In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the HLA polypeptide from the nucleotide sequence encoding the randomized antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the randomized antigen polypeptide from the nucleotide sequence encoding the β2-microglobulin polypeptide. In some embodiments, once expressed, the randomized antigen polypeptide is C-terminal to the β2-microglobulin on the single polypeptide, and the HLA polypeptide is C-terminal to the randomized antigen polypeptide on the single polypeptide. In some embodiments, the nucleotide sequence encoding the first flexible polypeptide linker separates the nucleotide sequence encoding the β2-microglobulin polypeptide from the nucleotide sequence encoding the randomized antigen polypeptide, and the nucleotide sequence encoding the second flexible polypeptide linker separates the nucleotide sequence encoding the randomized antigen polypeptide from the nucleotide sequence encoding the HLA polypeptide.
In other embodiments, components of the antigen screening libraries of a plurality HLA-antigen polypeptide complexes are expressed as more than one polypeptide despite being encoded by a single nucleic acid. In these embodiments, a nucleotide encoded cleavage sequence separates components of the antigen screening libraries of a plurality HLA-antigen polypeptide complexes from one another. For example, once expressed, the randomized peptide antigen is separated from the HLA polypeptide and/or from the Beta-2 (β2) microglobulin polypeptide by the cleavage sequence. As another example, once expressed, the HLA peptide is separated from the Beta-2 (β2) microglobulin polypeptide by the nucleotide encoded cleavage sequence. In these embodiments, a portion of the HLA polypeptide is expressed separately from other components of the antigen screening libraries of a plurality HLA-antigen polypeptide complexes and, when expressed separately, pairs naturally with the other components of the HLA-antigen polypeptide complexes inside the cell.
In some embodiments, the randomized antigen polypeptide of the HLA-antigen complex is encoded by a nucleic acid set forth in any one of SEQ ID NOs: 210 to 411. In some embodiments, the HLA polypeptide of the HLA-antigen complex is encoded by a nucleic acid at least 70%, 75%, 80%, 85%, 87.5%, 90%, 95%, 97%, 98%, 99%, or 100% homologous to any one of SEQ ID NOs: 210 to 411. In some embodiments, the randomized antigen polypeptide of the HLA-antigen complex is encoded by a nucleic acid set forth in any one of SEQ ID NOs: 412 to 426. In some embodiments, the HLA polypeptide of the HLA-antigen complex is encoded by a nucleic acid at least 70%, 75%, 80%, 85%, 87.5%, 90%, 95%, 97%, 98%, 99%, or 100% homologous to any one of SEQ ID NOs: 280 to 308. In some embodiments, one or more of the nucleic acids such as one or more of the nucleic acids of SEQ ID NOs: 210 to 411 and 412 to 426 are expressed by a plurality of cells. In some embodiments, each cell of the plurality of cells comprises a nucleic acid encoding a HLA-antigen complex. In some embodiments, the plurality of cells are a plurality of yeast cells. In some embodiments, the plurality of yeast cells are a plurality of cells of the of the EBY100 strain of Saccharomyces cerevisiae.
Nucleic acids encoding one or more components of the HLA-antigen polypeptide complexes can be delivered to the plurality of cells with a nucleic acid or a vector, such as an exogenous nucleic acid or exogenous vector. Suitable exogenous nucleic acids and exogenous vectors include plasmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), transposons, and viral vectors. These exogenous nucleic acids and exogenous vectors can further comprise components that allow for replication of the nucleic acids encoding one or more components of the HLA-antigen polypeptide complexes, permit antibiotic selection to allow for section of cells or other organisms expressing the nucleic acids encoding one or more components of the HLA-antigen polypeptide complexes, genes that complement yeast autotrophies to select for yeast transformants expressing the nucleic acids encoding one or more components of the HLA-antigen polypeptide complexes, promoters or enhancers for prokaryotic or eukaryotic expression of the HLA-antigen polypeptide complexes, polyadenylation sites, or marker genes that allow for visualization of transformed cells. In some embodiments, the nucleic acids that comprise a nucleic acid encoding the HLA-antigen polypeptide complexes of the current disclosure comprise an inducible promoter.
Methods of using the HLA-antigen polypeptide complexes and nucleic acids encoding such complexes minimally comprise contacting one or more cells, such as a plurality of cells, expressing the HLA antigen polypeptide complexes with a TCR and selecting for one or more cells that interact with the TCR. Selection can be performed, for example, by using the TCR in a “panning step” to capture the one or more cells expressing HLA-antigen polypeptide complexes that interact with the TCR, and washing away any non-interacting cells, such as one or more cells that do not express the HLA-antigen polypeptide complexes that do not interact with the TCR. Nucleic acids from interacting cells can be harvested and sequenced to elucidate the amino acid sequences of the randomized antigen polypeptide that interacted with the TCR. These nucleic acids can be re-transfected, transformed, or transduced into one or more different cells for another round of selection. This method can be iterated for any number of rounds of selection, such as 1, 2, 3, 4, 5, or more times (e.g., in cycles) to enrich for HLA-antigen polypeptide complexes that strongly interact with the TCR.
Sequencing platforms that can be used in the present disclosure include, but are not limited to: pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, second-generation sequencing, nanopore sequencing, sequencing by ligation, or sequencing by hybridization. Preferred sequencing platforms are those commercially available from Illumina (RNA-Seq) and Helicos (Digital Gene Expression or “DGE”). “Next generation” sequencing methods include, but are not limited to those commercialized by: 1) 454/Roche Lifesciences including but not limited to the methods and apparatus described in Margulies 2005 and in U.S. Pat. Nos. 7,244,559; 7,335,762; 7,211,390; 7,244,567; 7,264,929; and 7,323,305; 2) Helicos Biosciences Corporation (Cambridge, Mass.) as described in U.S. Pat. Nos. 7,501,245; 7,491,498; and 7,276,720; and in U.S. Patent Publ. Nos. 2006/0024711; 2009/0061439; 2008/0087826; 2006/0286566; 2006/0024711; 2006/0024678; 2008/0213770; and 2008/0103058; 3) Applied Biosystems (e.g. SOLiD sequencing); 4) Dover Systems (e.g., Polonator G.007 sequencing); 5) Illumina as described U.S. Pat. Nos. 5,750,341; 6,306,597; and 5,969,119; and 6) Pacific Biosciences as described in U.S. Pat. Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050; 7,462,468; 7,476,503; 7,315,019; 7,302,146; and 7,313,308; and in US Patent Publ. Nos. 2009/0029385; 2009/0068655; 2009/0024331; and 2008/0206764.
Described herein are methods of using the HLA-antigen polypeptide complexes of the present disclosure to select or enrich for antigens that bind to a TCR, such as a specific TCR. In some embodiments, the method includes selecting an antigen comprising contacting one or more cells, such as a plurality of cells, expressing at the HLA antigen polypeptide complexes with a TCR using one or more transgenic HLA-antigen polypeptide cell libraries, such as a transgenic HLA-antigen polypeptide yeast cell libraries. The methods described herein include methods for constructing one or more transgenic HLA-antigen polypeptide yeast cell libraries.
After construction of the one or more transgenic HLA-antigen polypeptide yeast cell libraries, the methods further include validating one or more transgenic HLA-antigen polypeptide yeast cell libraries using limiting dilution methods which include limited dilution of one or more cultures of proliferating yeast cells that each express at least one of the HLA-antigen polypeptides with nutrient-deficient yeast media. In some embodiments, the methods further include counting yeast from diluted yeast cultures and estimating HLA-antigen polypeptide yeast cell libraries with diversities of at least about 106, 107, 108, or 109 unique HLA-antigen polypeptide sequences (e.g., clones). In some embodiments, expression of an epitope tag by a yeast cell is measured to determine if any of the 106, 107, 108, or 109 clones are displayed on a yeast cell surface. For example, expression of the epitope tag can be determined as a surrogate value for total HLA-antigen polypeptide expression in the plurality of yeast cells and percent expression can be calculated. In some embodiments, the percent expression is an estimate of a number of yeast cells expressing a certain HLA-antigen polypeptide relative to the HLA-antigen polypeptide sequence library.
Referring to
In some embodiments, greater than at least 1×104, at least 1×105, at least 1×106, at least 1×107, at least 1×108, at least 1×109, at least 1×1010, at least 1×1011, at least 1×1012, at least 1×1013, at least 1×1014, or at least 1×1015 different HLA-antigen polypeptide complexes are screened with methods of the present disclosure, such as those illustrated in
Expression of naive yeast libraries, such as the HLA-antigen polypeptide sequence libraries described herein, minimally express at about 15% of total antigen polypeptide sequences in an antigen polypeptide sequence library for a single length 9mer peptide presented by HLA-A1 (Gee 2018b) and less than about 5% of a single length peptide (e.g., 8mer) expression in an antigen polypeptide sequence library having mixed length peptides (e.g., 8mer, 9mer, 10 mer, 11 mer, 12mer) presented by HLA-A2 (Gee 2018a). Despite less than about 5% single length expression of the antigen polypeptide sequence library having 8mer length peptides, TCRs isolated target 8mer antigens from the antigen polypeptide sequence library that stimulated the TCR in an in vitro co-culture assay (Gee 2018a). These antigen polypeptide sequence libraries have been screened and isolate peptides against TCRs of known specificity (Gee 2018a). While a minimum level of expression necessary for a functional library has not yet been determined, data shows that less than 15% expression can result in an antigen polypeptide sequence library useful with the methods described herein.
In some embodiments, methods of the present disclosure further include identifying a polypeptide antigen that interact with a TCR. For example, a method for determining TCR interacting polypeptide antigens can comprise any of the following steps:
The transgenic HLA-antigen polypeptide cell libraries and antigens of the HLA-antigen polypeptide complexes described herein can be used in conjunction with a given TCR. For example, the TCR, or other macromolecule having one or more antigen binding domains, is a positive selector or bait and once bound to an antigen (e.g., HLA-antigen polypeptide complex), identifies its cognate antigen. The TCRs described herein can be native or exogenous (e.g., recombinant) and expressed by a cell, such as a primary T cell, an immortalized T cell, or a non-T cell. In some embodiments, the TCR is immobilized on a solid support such as a column, a polystyrene plate or well of a multi-well plate, or a bead. In some embodiments, the TCR is multimerized as a plurality of TCRs immobilized on a bead. For example, the TCR can be multimerized on but not limited to magnetic beads, streptavidin, or dextran.
In some embodiments, the TCR is a soluble protein comprising at least one or more binding domains of a TCR of interest, e.g. TCRα/β, TCRγ/δ. The soluble protein may be a single chain, or a heterodimer. In some embodiments, the soluble TCR is modified by the addition of a biotin acceptor peptide sequence at the C terminus of one polypeptide. After biotinylation at the acceptor peptide, the TCR can be multimerized or added to substrate by binding to biotin binding partner, e.g. avidin, streptavidin, traptavidin, neutravidin, etc. In some embodiments, the biotin binding partner can comprise a detectable label, e.g. a fluorophore, mass label, etc., or can be bound to a particle, e.g. a paramagnetic particle. Selection of ligands bound to the TCR can be performed by flow cytometry, magnetic selection, and the like as known in the art.
To the extent the foregoing materials and/or any other materials incorporated herein by reference conflict with the present disclosure, the present disclosure controls.
The following examples provide further representative embodiments of the presently disclosed technology.
The following examples are provided to further illustrate embodiments of the present technology and are not to be interpreted as limiting the scope of the present technology. To the extent that certain embodiments or features thereof are mentioned, it is merely for purposes of illustration and, unless otherwise specified, is not intended to limit the present technology. One skilled in the art may develop equivalent means without the exercise of inventive capacity and without departing from the scope of the present technology. It will be understood that many variations can be made in the procedures herein described while remaining within the bounds of the present technology. Such variations are intended to be included within the scope of the presently disclosed technology. As such, embodiments of the presently disclosed technology are described in the following representative examples.
This example describes design of the antigen libraries of the present disclosure for use with a polypeptide antigen HLA complex. An exemplary algorithm to design and select anchor residues for each HLA allele is as follows, using data of known HLA binding epitopes ligands from a website such as www.IEDB.org/:
Step 1: download list of polypeptides that bind to a given allele which may comprise several hundred peptides or several thousand peptides.
Step 2: construct a frequency matrix of residues per position of the peptide based upon the downloaded known peptides.
Step 3: select composition of “anchors” for library design by using a cutoff of the top 4 residues at each position.
This example describes electroporating yeast cells with nucleic acids encoding an exemplary antigen library of the present disclosure having all HLA allotypes and using peptides of 8-11 amino acids in length (8mer-11 mer). In this example, yeast cells were electroporated with nucleic acids encoding the antigen library of HLA-antigen polypeptide complexes (pHLA library).
The electroporation methods for expression of pHLA on yeast are as follows:
Day 0:
Day 1: Passage the two yeast cultures from Day 0 step 3 by adding 100 μl of each of the two yeast cultures to 5 ml of fresh YPD and shake at 30° C. overnight.
Day 2:
Day 2: Determine Titer
Day 3: Measure the OD of the passage from step 8 after 24 hours. The OD should be at least 5. Passage the culture to an OD of 1 in a total volume of 500 mL SDCAA.
Day 4: Passage cells to an OD of 1 in a total volume of 500 mL SDCAA.
Day 5: 72 hours after step 18 from Day 2 was performed, induce in SGCAA (galactose casamino acids, which also includes, yeast nitrogen base without amino acids and with ammonium sulfate, sodium citrate, and citric acid monohydrate, at a pH of 4.5).
Recipes:
This example describes characterizing expression of HLA-antigen polypeptide complexes on the electroporated yeast cells of Example 2. These expression measurements include FACS analysis to determine the levels of peptide-MHC displayed on the surface of yeast cells and indicate functionality of the random yeast display library. The characterization methods for expression of pHLA on yeast are as follows:
Materials
Optional:
Cell Preparation
Staining Cells
Washing Cells and Determining pHLA Expression
Results: Expression of the HLA-antigen polypeptide complexes (peptides of SEQ ID Nos: 8, 11, 14, 18, 21+24, 28, 32, 36, 40-44, 47, 50, 53, 56, 65, 75, 69, 77+80, 89, 95, 99, 102+106, 108, 111+114, 117+120, 124, and 125) was determined by flow cytometry and shown in
This example describes functionally validating expression of pHLAs on the electroporated yeast cells of Example 2 with a candidate TCR. Expected target antigens of the pHLAs can be identified from up to 6 libraries when the candidate TCR is allotype-matched. The functional validation methods for expression of pHLA on yeast are as follows:
HLA-antigen polypeptide sequence libraries, such as those disclosed herein, minimally express about 25% of total antigen polypeptide sequences for a single length 9mer peptide presented by HLA-A1 (Gee 2018b) and express less than about 5% of a single length peptide (e.g., 8mer) having mixed length peptides (e.g., 8mer, 9mer, 10 mer, 11 mer) presented by HLA-A2 (Gee 2018a). Despite less than about 5% single length peptide expression of the HLA-antigen polypeptide sequence having 8mer length peptides, isolated TCRs of interest target 8mer antigens from the HLA-antigen polypeptide complexes. These isolated TCR of interest were stimulated by one or more HLA-antigen polypeptide complexes in an in vitro co-culture assay (Gee 2018a; see
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
The present application claims the benefit of U.S. Provisional Application No. 62/726,060, filed Aug. 31, 2018, the contents of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/049205 | 8/30/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62726060 | Aug 2018 | US |
