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NL2021034B1 - TEIPP neoantigens and uses thereof - Google Patents

TEIPP neoantigens and uses thereof Download PDF

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NL2021034B1
NL2021034B1 NL2021034A NL2021034A NL2021034B1 NL 2021034 B1 NL2021034 B1 NL 2021034B1 NL 2021034 A NL2021034 A NL 2021034A NL 2021034 A NL2021034 A NL 2021034A NL 2021034 B1 NL2021034 B1 NL 2021034B1
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acid sequence
peptide
seq
cell
nucleic acid
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NL2021034A
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Van Hall Thorbald
Henricus Van Der Burg Sjoerd
Marijt Koen
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Academisch Ziekenhuis Leiden
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Priority to PCT/NL2019/050316 priority patent/WO2019231326A1/en
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract

Novel nucleic acid sequences, vectors, modified cells, binding agents, peptides and pharmaceutical compositions are provided that are useful as a medicament, for example in the prevention or treatment of cancer or viral infections associated with impaired HLA class I antigen presentation. Corresponding methods and uses are also provided.

Description

Background
Many T-cell based immunotherapies used for treatment of cancer in humans are based on recognition of tumor antigens presented in HLA class I (HLA-I) molecules by tumor cells1’2. Point mutated peptides constitute formidable tumor antigens clue to their non-self nature for which a non-curtailed T cell repertoire is available. An absolute requirement for such T cells to exert their action against cancer is the display of HLA-I at the surface of tumor cells. However, HLA-I down modulation on cancer cells is observed in many immune-escaped cancers, often caused by epigenetic silencing of antigen processing components, like the peptide transporter TAP5’7.
A novel category of tumor antigens, referred to as TEIPP (T cell epitopes associated with peptide processing), are presented at the surface of tumor cells carrying defects in antigen processing13·14. TEIPPs are derived from ubiquitously expressed non-mutated self’ proteins, however, their processed peptides fail to be loaded into HLA-I in healthy cells. Their surface presentation is highly promoted by defects in the antigen processing machinery, especially in the absence of the peptide transporter TAP. By this virtue, TEIPP peptides constitute tumorspecific antigens. In mouse tumor models in which MHC-I presentation is clown modulated by defects in the peptide transporter TAP, selective presentation of TEIPP peptides and successful targeting of immune-escaped tumor variants by TEIPP specific T cells has been shown 15J5. Thus, targeting TEIPP neoantigens is a potent strategy to induce anti-tumor responses for tumors with low MHC-I expression.
HLA-I down modulation has been observed upon infection with certain viruses (for example HIV), resulting in surface presentation of TEIPP antigens on virally infected cells. Accordingly, TEIPP peptides may also be considered as target antigens for treating or preventing a viral infection associated with impaired antigen presentation on HLA class I molecules.
US2009/0220534 and Weinzierl et al., Eur. J. Immunol. 2008. 38: 1503-1510 describe screening methods for identifying TEIPP epitopes that are presented on the surface of cells via a TAP independent mechanism. They identify over fifty potential immunogenic peptides that make it to the surface of antigen processing deficient cells. However, Weinzierl and colleagues note that, on average, TAP-independently presented peptides had a 15-fold decrease in their BIMAS score, indicating a reduced binding affinity to their respective HLA molecules. They also raise the possibility that some of these HLA:peptide complexes resulted from the association of cytosolic peptides with HLA post-lysis.
Despite their potential as neoantigen targets for T-cell based immunotherapies, to date only one human TEIPP neoantigen has been identified at the molecular level17·18.
Brief summary of the disclosure
The inventors have developed a hybrid forward-reverse immunologic screen to identify novel TAP-independent non-mutated neoantigens that are selectively presented by immuneescaped cancers. Their approach encompassed an in silico prediction of TEIPP neoantigencandidates from the whole human proteome, matching of candidates to the cancer-specific peptidome, and an ex vivo screen to confirm the presence of a TEIPP T cell repertoire in healthy donors.
This strategy yielded a short-list of 65 TEIPP neoantigen-candidates of which 40 had a predicted binding to the HLA-A*02:01 allele. Analysis of the 40 HLA-A*02:01 binding TEIPP candidates revealed a consistent CD8+ T cell repertoire to only 16 of these 40 peptides. Although three of the sixteen peptides have previously been described to bind HLA-A*02:01 (namely, FLGPWPAAS (SEQ ID NO:1); LLLDVPTAAV (SEQ ID NO: 12) and LLSAEPVPA (SEQ ID NO:13); see US2009/0220534 and Weinzierl et al., Eur. J. Immunol. 2008. 38: 1503-1510) the existence of cognate CD8+ T cells to these specific peptides (and not other peptides) in the repertoire of healthy donors was not reported and could not have been predicted from the previously published data.
Advantageously, the CD8+ T cells identified in the repertoire of healthy donors were predominantly in the naive state. The sixteen peptides presented herein are therefore ideal candidates for inducing a T-cell based immune response in vivo without autoimmune reactivity. The data presented herein therefore provides evidence, for the first time, that these peptides (and corresponding nucleic acid sequences or vectors encoding them) could be used as a novel therapy to induce a tumor-specific T cell based immune response in vivo (by activating the naive cognate T cells present within the natural T cell repertoire of the patient) without risking autoimmunity. In addition, binding agents such as antibodies, TCRs or CARs (or modified cells expressing the same) that specifically bind to these peptides may advantageously be used as a novel immunotherapy for the prevention or treatment of cancer or viral infections associated with impaired HLA class I presentation.
Advantageously, the peptides described herein are not derived from the mutanome of cancers, but are of ‘self’ origin and therefore constitute universal neoantigens that may be presented on the cell surface of any cell with impaired antigen presentation on HLA class I molecules. The inventors have shown that CD8+ T cell clones specific for one of the TEIPP peptides (p14; the leader peptide of the ubiquitously expressed LRPAP protein) recognized TAP-deficient, HLA class llow lymphomas, melanomas, colon carcinomas and renal cell carcinomas, but not healthy TAP-proficient melanocytes and kidney epithelial cells. These data demonstrate the universal nature of the TEIPP neoantigens described herein across cancers of different histological origins.
In one aspect, the invention provides an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence FLGPWPAAS (SEQ ID NO:1); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence FLGPWPAAS (SEQ ID NO: 1).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide FLGPWPAAS (SEQ ID NO: 1).
Suitably, the CDR3 of (a) has an amino acid sequence having at least 90% sequence identity to VVMGYGQNFV (SEQ ID NO:24).
Suitably, the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO:23.
Suitably, the CDR3 of (b) has an amino acid sequence having at least 90% sequence identity to SAMGRQSTDTQY (SEQ ID NO: 32).
Suitably, the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 31.
Suitably, the CDR3 of (a) is within a TCR a chain variable region that specifically binds to FLGPWPAAS (SEQ ID NO: 1), optionally wherein (a) further comprises a TCR a chain constant region.
Suitably, the TCR a chain variable region has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18.
Suitably, the TCR a chain variable region of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 17.
Suitably, the CDR3 of (b) is within a TCR β chain variable region that specifically binds to FLGPWPAAS (SEQ ID NO: 1), optionally wherein (b) further comprises a TCR β chain constant region.
Suitably, the TCR β chain variable region has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:26.
Suitably, the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 25.
Suitably, the CDR3 of (a) is within a TCR a chain variable region having at least 90% sequence identity to SEQ ID NO: 18, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 24; and optionally wherein (a) comprises a TCR a chain constant region.
Suitably, the TCR a chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 20 and the TCR a chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 22.
Suitably, the CDR3 of (b) is within a TCR β chain variable region having at least 90% sequence identity to SEQ ID NO: 26, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 32; and optionally wherein (b) comprises a TCR β chain constant region.
Suitably, the TCR β chain variable region CDR1 has an amino acid sequence of SEQ ID NO: 28 and the TCR β chain variable region CDR2 has an amino acid sequence of SEQ ID NO: 30.
Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is also provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1).
Suitably, the target binding moiety is encoded by a nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence FLGPWPAAS (SEQ ID NO: 1); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence FLGPWPAAS (SEQ ID NO: 1) as described elsewhere herein.
The isolated nucleic acid sequence provided herein may be part of a vector. A vector is therefore provided comprising a nucleic acid sequence described herein. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence or vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising:
a) an isolated nucleic acid sequence described herein;
b) a vector described herein;
c) a modified cell described herein;
d) an isolated peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1);
e) an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1);
f) a vector comprising an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1); or
g) a binding agent that specifically binds to a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1);
and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), d) e), f) and g) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of a), b), e) or f) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of d) or g) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition comprises an isolated peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence FLGPWPAAS (SEQ ID NO: 1).
Suitably, the composition comprises an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). Suitably, the nucleic acid sequence is mRNA or DNA.
Suitably, the composition comprises a binding agent that specifically binds to a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). Suitably, the binding agent is an antibody.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is also provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence FLGPWPAAS (SEQ ID NO:1). Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, wherein the peptide comprises the amino acid sequence FLGPWPAAS (SEQ ID NO: 1).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided comprising:
determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLGPWPAAS (SEQ ID NO:1), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLGPWPAAS (SEQ ID NO:1); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLGPWPAAS (SEQ ID NO: 1).
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
Various aspects of the invention are described in further detail below.
Brief description of the drawings
Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:
Figure 1: Shows the identification approach of TEIPP neoantigens associated with immune-edited cancers.
(A) A schematic overview of the applied screening approach. Human peptide sequences were selected via algorithms-based predictions on the whole human proteome for presentation by HLA class I molecules independent of the peptide transporter TAP. Two main unraveled pathways have been described for which algorithms are available: the processing of signal peptides and of C-terminal ‘tail’ peptides. The resulting peptide selection was matched with a large database of naturally presented peptides from human cancers and healthy tissue. The subsequent shortlist of candidates with a predicted binding to HLAA*02:01 was subjected to functional testing for T cell immunogenicity and recognition of tumor cells deficient for the peptide transporter. (B, top panel) Graphical display of the two molecularly described TAP-independent processing mechanisms. Signal peptide containing proteins are processed in the ER membrane by the proteolytic enzymes signal peptidase (SP) and signal peptide peptidase (SPP), resulting in the liberation of small peptides in the lumen of the ER where they can bind to HLA class I molecules19·51. (B, bottom panel) Proteins with their C-terminal tail protruding in the ER can be cleaved by proteases like furins or SPP21. (C) Venn diagram of in silico predicted TEIPP neoantigens matched with databases comprising naturally presented peptides from tumor tissues or healthy tissues.
Figure 2: Prediction results of HLA-I binding of TEIPP neoantigen candidates.
TEIPP neoantigen candidate peptides were tested for HLA specificity using a web-based algorithm. (A-B) Absolute number of TEIPP candidates with predicted binding in HLA superclasses for “leader” peptides and “tail” peptides. (C-D) Percentage of total TEIPP candidates with predicted binding in HLA superclasses for “leader” peptides and “tail” peptides.
Figure 3: Detection of CD8+ T cells against TEIPP neoantigens in PBMC of healthy donors.
PBMCs of healthy donors were screened with HLA-A*02:01 tetramers to identify whether a T cell repertoire is present for TEIPP neoantigens. HLA-A*02:01 tetramers folded with the selected 40 TEIPP neoantigen candidates were arranged in eight pools based on predicted peptide binding. (A) TEIPP enriched T cell pools were analyzed by flow cytometry. Collective data of representative dot plots of TEIPP neoantigens specific CD8 T cells. (B) PBMCs of additional healthy donors were tested to validate the presence of a TEIPP T cell repertoire. All 40 TEIPP neoantigen-candidates were tested in at least three different donors. Shown are data of all tested donors with fraction of T cell-responsive donors indicated above each bar. Each bar represents one TEIPP neoantigen-candidate. The darker bar represents the percentage of T cell positive donors based on flow cytometry analysis.
Figure 4: Characterization of TEIPP T cells and cognate antigen.
(A) Flow cytometry gating strategy for flow sorting PBMCs into experienced T cells and naive T cells. (B) Overview of bacterial peptide sequences with TEIPP peptide homology. (C) Gene name, peptide sequence and predicted peptidase cleavage sites (SP (right triangle) and SPP (left triangle)) of p14, p29, p34, p35, and p55. (D) pH LA peptide affinity was measured by a cellular based assay. Percentage of maximum HLA-I expression was determined by flow cytometry to calculate EC50 values. (E) pH LA peptide stability was measured by calculating EC50 values of maximum HLA-I expression over time. (F) Multiple T cell clones with different VP’s for the different specificities were isolated. Shown is T cell clone name and corresponding T cell receptor beta variable gene name, determined by using a TCR \/β Screening kit analyzed by flow cytometry. (G) Comparison of GM-CSF production of various T cell clones upon 518A2 TAP KO and 518A2 WT recognition.
Figure 5: Most TEIPP-specific CD8+ T cells reside in the naïve repertoire. To study the T cell status of TEIPP specific T cells, clones were generated for peptide-candidates p14, p29, p34, p35, and p55, of which T cells were most abundantly detected in PBMCs of healthy donors by single cell sorting HLA-A*02:01 tetramer-positive cells and subsequent expansion. (A) Representative dot plots of pHLA-A*02:01 tetramer stained CD8+ T cells cultured from either the naive subset or the experienced subset. Percentage is pHLA-A*02:01 tetramer positive cells out of total CD8+ T cells. (B) Pie-chart for each of the TEIPP neoantigencandidates. Five donors were tested and each slice represents one donor. Relative percentage of naive cells (dark grey) and percentage of experienced cells (black) is calculated out of total pHLA-A*02:01 tetramer positive CD8+ cells. (C) Functional T cell avidity was examined by measuring cytokine production of activated T cells recognizing TEIPP peptide pulsed HLA-A*02:01 positive T2 cells with different concentrations of peptide. EC50 was determined by calculating the peptide concentration (nM) corresponding to the 50% cytokine production of the maximum response.
Figure 6: mRNA and protein expression analysis on tumor panel.
(A) Peptide specificity of the LRPAP21-30 specific-T cell clone was assessed by testing different length variants of the p14 peptide at different locations of the signal peptide. Peptides with a predicted HI_A-A*02:01 binding (ic50<500nM) were tested. GM-CSF cytokine production was measured by ELISA. (B) LRPAP1 mRNA levels of the tumor panel were quantified by PCR. Delta CT values were calculated. (n=3) (C) TAP1 protein levels of the 518A2 skin melanoma cell line variants were measured using western-blot. No TAP1 expression was observed in the 518A2 TAP KO variant. (D) mRNA expression of TAP1gene of the 518A2 skin melanoma cell line variants were determined by quantitative PCR. (E) TAP1 protein levels of the T1 and T2 lymphoma were measured using western-blot. No TAP1 expression was observed in the T2 lymphoma. (F) LRPAP1 mRNA expression levels of the T1 and T2 lymphoma were determined by quantitative PCR. Both cell lines had equal LRPAP1 mRNA expression. (n=3) (G) LRPAP1 mRNA expression levels of the tumor panel was examined by quantitative PCR. Relative mRNA gene expression of the TAP KO variant compared to WT variant was calculated. (n=3) (H) Relative TAP1 mRNA levels of the tumor panel were determined by quantitative PCR. 518A2 skin melanoma was set to 1 (n=3).
Figure 7: CD8+ T cells against p14 recognize TAP-deficient tumors of different histological origin.
T cell clones were tested for their specificity to selectively recognize the LRPAP21.30 TEIPP epitope presented on APM-impaired tumors. To examine this the inventors established immune-escaped variants of human primary melanomas tumor panel and human renal cell carcinoma panel. (A) LRPAP1 Gene expression data collected from TCGA database. (B) To make immune-escaped variants the inventors knocked out TAP1 in tumors (TAP KO). Additionally the inventors knocked-out the LRPAP21-30 epitope (ag-KO). mRNA expression of LRPAP1 in 518A2 melanoma (WT vs. TAP-KO: p= n.s, TAP-KO vs. TAP-KO/Ag-KO: p=0.001). (C) HLA-A*02:01 surface expression on 518A2 melanoma variants, measured by flow cytometry. (D) Relative GM-CSF production of T cell clone upon 518A2 TAP KO recognition. When LRPAP21.30 epitope was knocked out (ag-KO), recognition by the T cell was abolished. Representative experiment is shown (n=3). (E) To assess recognition was pHLA-TCR mediated, HLA-ABC or HLA-A*02:01 was blocked by specific blocking antibodies. (F) TEIPP T cell specificity was determined by co-culturing T cells together with
TAP- proficient (T1) cells and TAP deficient (T2) tumor cells. Cytokine responses were measured to detect T cell recognition. Each symbol represents one unique T cell clone. (G) HLA-A*02:01 surface expression of the APM-impaired tumor panel measured by flow cytometry.
Figure 8: IFN-y production of T cell clones upon antigen recognition of four melanomas and two RCCs. Each dot represents one isolated T cell clone specific for the LRPAP21-30 epitope. Representative data is shown (n=3). Student T-test is used to calculate statistical difference, where KIRC represents kidney renal clear cell carcinoma, and SKCM represents skin cutaneous melanoma.
Figure 9: Targeting TEIPP antigens is safe despite ubiquitous expression of their proteins in healthy cells.
To assess the safety of targeting nonmutated TEIPP neoantigens the inventors examined the T cell recognition of healthy cells. (A) Histological tissue stainings for LRPAP1 were collected from the online accessible database protein atlas. Positive protein detection was observed in various healthy organs. (B) LRPAP1 mRNA levels were determined of two primary melanocytes and two non-transformed kidney epithelial cells by quantitative PCR.
(C) Surface HLA-A*02:01 expression of the healthy cells was determined by flow cytometry.
(D) No cytokine responses were seen when T cell specific for LRPAP21.30 specific T cells were co-cultured with healthy cells. (E) To assess the ability of the healthy cells to present the epitope in HLA-A*02:01, exogenous peptide was presented. T cells were able to recognize peptide pulsed healthy cells.
Figure 10: Database analysis of TEIPP candidates.
(A) LRPAP1 protein expression in 44 different healthy tissues. Data collected from proteinatlas.org. (B) mRNA expression of 16 identified TEIPP antigens in the thymus, expressed in Tag per Million (TPM). Data collected from FANTOM5 datasets. (C) mRNA expression data of the TEIPP antigens were of different tumor types were extracted from the TCGA database. Our list with 16 TEIPP antigens are categorized in two groups. Ubiquitous TEIPP; all examined tumor types have mRNA expression of the respective TEIPP antigen (TPM>5). Tissue restricted TEIPP; mRNA expression was only detected in some tumor types (TPM>5).
Figure 11: CD8 T cell recognition of two TAP-deficient colon carcinoma cell lines (A) LRPAP1 mRNA expression levels of two colon carcinoma cell lines were determined by quantitative PCR. Equal levels of LRPAP1 mRNA expression was observed. (n=2). (B) HLA
A*02:01 surface expression of the APM-impaired colon carcinoma lines were measured by flow cytometry. (C) GM-CSF production of T cell clone 1A8 upon antigen recognition of two colon carcinomas (WT vs. TAP KO).
Detailed Description
The inventors have identified sixteen peptide antigens that are selectively presented by immune-escaped TAP-deficient cells. Naive state, cognate CD8+ T cells to these specific peptides have also been identified in the repertoire of healthy donors. The sixteen peptides presented herein are therefore ideal candidates for inducing a T-cell based immune response in vivo without autoimmunity. The universal nature of these TEIPP neoantigens across cancers of different histological origins is also described.
Advantageously, these peptides (or corresponding nucleic acid sequences that encode said peptides) can be used as a medicament e.g. for preventing or treating viral infections or cancers associated with impaired HLA class I antigen presentation. In addition, virus infected or cancerous cells displaying impaired HLA class I antigen presentation can be specifically targeted using binding agent therapies e.g. antibody, adoptive T cell or CAR-T cell therapies that are specific to one or more of these peptides without causing autoimmunity. Advantageously, these peptides are not derived from the mutanome of cancers but are of ‘self’ origin and therefore constitute universal neoantigens.
The peptides have been identified as being HLA-A*02:01 restricted. The invention described herein is therefore particularly useful in the treatment or prevention of viral infection or cancer associated with impaired HLA class I antigen presentation in a HLA-A*02:01 positive human subject.
Accordingly, preferably, the subject is a HLA-A*02:01 positive human subject.
Each of the peptides and their uses are described below.
FLGPWPAAS
Aspects relating to FLGPWPAAS (also known as p14 herein; or SEQ ID NO:1) are described above under “brief summary of the disclosure”.
LLLDVPTAAV
Aspects relating to LLLDVPTAAV (also known as p30 herein; or SEQ ID NO: 12) are described below.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLDVPTAAV (SEQ ID NO:12); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide LLLDVPTAAV (SEQ ID NO: 12). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLDVPTAAV (SEQ ID NO:12); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12) as described elsewhere herein.
The isolated nucleic acid sequence provided herein may be part of a vector. A vector is therefore provided comprising a nucleic acid sequence described herein. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence or vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising:
a) an isolated nucleic acid sequence described herein;
b) a vector described herein;
c) a modified cell described herein;
d) an isolated peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12);
e) an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12);
f) a vector comprising an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12); or
g) a binding agent that specifically binds to a peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12);
and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), d) e), f) and g) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of
a), b), e) or f) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of d) or g) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition comprises an isolated peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12).
Suitably, the composition comprises an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12). Suitably, the nucleic acid sequence is mRNA or DNA.
Suitably, the composition comprises a binding agent that specifically binds to a peptide comprising the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12). Suitably, the binding agent is an antibody.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is also provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12). Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence LLLDVPTAAV (SEQ ID NO: 12).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided comprising:
determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLLDVPTAAV (SEQ ID NO: 12), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLLDVPTAAV (SEQ ID NO:12); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLLDVPTAAV (SEQ ID NO: 12).
LLLSAEPVPA
Aspects relating to LLLSAEPVPA (also known as p32 herein; or SEQ ID NO: 13) are described below.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TOR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13); and/or (b) a polypeptide comprising a CDR3 of a TOR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide LLLSAEPVPA (SEQ ID NO: 13). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13).
Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TOR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13); and/or (b) a polypeptide comprising a CDR3 of a TOR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13) as described elsewhere herein.
The isolated nucleic acid sequence provided herein may be part of a vector. A vector is therefore provided comprising a nucleic acid sequence described herein. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence or vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising:
a) an isolated nucleic acid sequence described herein;
b) a vector described herein;
c) a modified cell described herein;
d) an isolated peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13);
e) an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13);
f) a vector comprising an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13); or
g) a binding agent that specifically binds to a peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13);
and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), d) e), f) and g) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of a), b), e) or f) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of d) or g) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition comprises an isolated peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13), Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13).
Suitably, the composition comprises an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13). Suitably, the nucleic acid sequence is mRNA or DNA.
Suitably, the composition comprises a binding agent that specifically binds to a peptide comprising the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13). Suitably, the binding agent is an antibody.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is also provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence LLLSAEPVPA (SEQ ID NO:13).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence LLLSAEPVPA (SEQ ID NO: 13).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLLSAEPVPA (SEQ ID NO: 13), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLLSAEPVPA (SEQ ID NO: 13); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLLSAEPVPA (SEQ ID NO: 13).
LTLLGTLWGA
Aspects relating to LTLLGTLWGA (also known as p34 herein; or SEQ ID NO:2) are described below.
An isolated peptide is provided comprising the amino acid sequence LTLLGTLWGA (SEQ ID NO: 2). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence LTLLGTLWGA (SEQ ID NO: 2).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence LTLLGTLWGA (SEQ ID NO: 2). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LTLLGTLWGA (SEQ ID NO:2); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LTLLGTLWGA (SEQ ID NO: 2).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide LTLLGTLWGA (SEQ ID NO: 2). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence LTLLGTLWGA (SEQ ID NO: 2). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LTLLGTLWGA (SEQ ID NO:2); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LTLLGTLWGA (SEQ ID NO: 2) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence LTLLGTLWGA (SEQ ID NO:2).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence LTLLGTLWGA (SEQ ID NO: 2).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LTLLGTLWGA (SEQ ID NO:2), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LTLLGTLWGA (SEQ ID NO:2); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is LTLLGTLWGA (SEQ ID NO:2).
SVLWLGALGL
Aspects relating to SVLWLGALGL (also known as p35 herein; or SEQ ID NO:3) are described below.
An isolated peptide is provided comprising the amino acid sequence SVLWLGALGL (SEQ ID NO: 3). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence SVLWLGALGL (SEQ ID NO: 3).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence SVLWLGALGL (SEQ ID NO: 3). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence SVLWLGALGL (SEQ ID NO:3); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence SVLWLGALGL (SEQ ID NO: 3).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide SVLWLGALGL (SEQ ID NO: 3). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence SVLWLGALGL (SEQ ID NO: 3). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence SVLWLGALGL (SEQ ID NO:3); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence SVLWLGALGL (SEQ ID NO: 3) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence SVLWLGALGL (SEQ ID NO:3).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence SVLWLGALGL (SEQ ID NO: 3).
A method of diagnosing a cancer or viral infection associated with impaired HI_A class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is SVLWLGALGL (SEQ ID NO:3), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HI_A class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is SVLWLGALGL (SEQ ID NO:3); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is SVLWLGALGL (SEQ ID NO:3).
VIIKPLVWV
Aspects relating to VIIKPLVWV (also known as p55 herein; or SEQ ID NO:4) are described below.
An isolated peptide is provided comprising the amino acid sequence VIIKPLVWV (SEQ ID NO: 4). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence VIIKPLVWV (SEQ ID NO: 4).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence VIIKPLVWV (SEQ ID NO: 4). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence VIIKPLVWV (SEQ ID NO:4); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence VIIKPLVWV (SEQ ID NO: 4).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide VIIKPLVWV (SEQ ID NO: 4). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence VIIKPLVWV (SEQ ID NO: 4). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence VIIKPLVWV (SEQ ID NO:4); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence VIIKPLVWV (SEQ ID NO: 4) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence VIIKPLVWV (SEQ ID NO:4).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence VIIKPLVWV (SEQ ID NO: 4).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is VIIKPLVWV (SEQ ID NO:4), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is VIIKPLVWV (SEQ ID NO:4); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is VIIKPLVWV (SEQ ID NO:4).
LLALAAGLAV
Aspects relating to LLALAAGLAV (also known as p29 herein; or SEQ ID NO:5) are described below.
An isolated peptide is provided comprising the amino acid sequence LLALAAGLAV (SEQ ID NO: 5). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence LLALAAGLAV (SEQ ID NO: 5).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence LLALAAGLAV (SEQ ID NO: 5). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLAI_AAGI_AV (SEQ ID NO:5); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLAI_AAGLAV (SEQ ID NO: 5).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide LLAI_AAGI_AV (SEQ ID NO: 5). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence LLAI_AAGI_AV (SEQ ID NO: 5). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLAI_AAGI_AV (SEQ ID NO:5); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLAI_AAGI_AV (SEQ ID NO: 5) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence LLALAAGLAV (SEQ ID NO:5).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence LLALAAGLAV (SEQ ID NO: 5).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLALAAGLAV (SEQ ID NO:5), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLALAAGLAV (SEQ ID NO:5); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLALAAGLAV (SEQ ID NO:5).
FLSELQYYL
Aspects relating to FLSELQYYL (also known as p1 herein; or SEQ ID NO:6) are described below.
An isolated peptide is provided comprising the amino acid sequence FLSELQYYL (SEQ ID NO: 6). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence FLSELQYYL (SEQ ID NO: 6).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence FLSELQYYL (SEQ ID NO: 6). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence FLSELQYYL (SEQ ID NO:6); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence FLSELQYYL (SEQ ID NO: 6).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide FLSELQYYL (SEQ ID NO: 6). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence FLSELQYYL (SEQ ID NO: 6). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence FLSELQYYL (SEQ ID NO:6); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence FLSELQYYL (SEQ ID NO: 6) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence FLSELQYYL (SEQ ID NO:6).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence FLSELQYYL (SEQ ID NO: 6).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLSELQYYL (SEQ ID NO:6), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLSELQYYL (SEQ ID NO:6); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLSELQYYL (SEQ ID NO:6).
VLLDHLSLA
Aspects relating to VLLDHLSLA (also known as p2 herein; or SEQ ID NO:7) are described below.
An isolated peptide is provided comprising the amino acid sequence VLLDHLSLA (SEQ ID NO: 7). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence VLLDHLSLA (SEQ ID NO: 7).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence VLLDHLSLA (SEQ ID NO: 7). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence VLLDHLSI_A (SEQ ID NO:7); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence VLLDHLSLA (SEQ ID NO: 7).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide VLLDHLSLA (SEQ ID NO: 7). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence VLLDHLSLA (SEQ ID NO: 7). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence VLLDHLSI_A (SEQ ID NO:7); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence VLLDHLSLA (SEQ ID NO: 7) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence VLLDHLSLA (SEQ ID NO:7).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence VLLDHLSLA (SEQ ID NO: 7).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is VLLDHLSLA (SEQ ID NO:7), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is VLLDHLSLA (SEQ ID NO:7); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is VLLDHLSLA (SEQ ID NO:7).
ALFSFVTAL
Aspects relating to ALFSFVTAL (also known as p4 herein; or SEQ ID NO:8) are described below.
An isolated peptide is provided comprising the amino acid sequence ALFSFVTAL (SEQ ID NO: 8). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence ALFSFVTAL (SEQ ID NO: 8).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence ALFSFVTAL (SEQ ID NO: 8). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence ALFSFVTAL (SEQ ID NO:8); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence ALFSFVTAL (SEQ ID NO: 8).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide ALFSFVTAL (SEQ ID NO: 8). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence ALFSFVTAL (SEQ ID NO: 8). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence ALFSFVTAL (SEQ ID NO:8); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence ALFSFVTAL (SEQ ID NO: 8) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence ALFSFVTAL (SEQ ID NO:8).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence ALFSFVTAL (SEQ ID NO: 8).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is ALFSFVTAL (SEQ ID NO:8), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is ALFSFVTAL (SEQ ID NO:8); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is ALFSFVTAL (SEQ ID NO:8).
VLAVFIKAV
Aspects relating to VLAVFIKAV (also known as p9 herein; or SEQ ID NO:9) are described below.
An isolated peptide is provided comprising the amino acid sequence VLAVFIKAV (SEQ ID NO: 9). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence VLAVFIKAV (SEQ ID NO: 9).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence VLAVFIKAV (SEQ ID NO: 9). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence VLAVFIKAV (SEQ ID NO:9); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence VLAVFIKAV (SEQ ID NO: 9).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide VLAVFIKAV (SEQ ID NO: 9). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence VLAVFIKAV (SEQ ID NO: 9). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence VLAVFIKAV (SEQ ID NO:9); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence VLAVFIKAV (SEQ ID NO: 9) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence VLAVFIKAV (SEQ ID NO:9).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence VLAVFIKAV (SEQ ID NO: 9).
A method of diagnosing a cancer or viral infection associated with impaired HI_A class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is VLAVFIKAV (SEQ ID NO:9), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HI_A class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HI_A class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is VLAVFIKAV (SEQ ID NO:9); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HI_A class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is VLAVFIKAV (SEQ ID NO:9).
LLWGRQLFA
Aspects relating to LLWGRQLFA (also known as p17 herein; or SEQ ID NO: 10) are described below.
An isolated peptide is provided comprising the amino acid sequence LLWGRQLFA (SEQ ID NO: 10). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence LLWGRQLFA (SEQ ID NO: 10).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence LLWGRQLFA (SEQ ID NO: 10). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLWGRQLFA (SEQ ID NO: 10); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLWGRQLFA (SEQ ID NO: 10).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide LLWGRQLFA (SEQ ID NO: 10). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence LLWGRQLFA (SEQ ID NO: 10). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LLWGRQLFA (SEQ ID NO: 10); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LLWGRQLFA (SEQ ID NO: 10) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence LLWGRQLFA (SEQ ID NO: 10).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence LLWGRQLFA (SEQ ID NO: 10).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLWGRQLFA (SEQ ID NO: 10), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLWGRQLFA (SEQ ID NO: 10); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is LLWGRQLFA (SEQ ID NO: 10).
TLLGASLPA
Aspects relating to TLLGASLPA (also known as p18 herein; or SEQ ID NO: 11) are described below.
An isolated peptide is provided comprising the amino acid sequence TLLGASLPA (SEQ ID NO: 11). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence TLLGASLPA (SEQ ID NO: 11).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence TLLGASLPA (SEQ ID NO: 11). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence TLLGASLPA (SEQ ID NO: 11); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence TLLGASLPA (SEQ ID NO: 11).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide TLLGASLPA (SEQ ID NO: 11). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence TLLGASLPA (SEQ ID NO: 11). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence TLLGASLPA (SEQ ID NO: 11); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence TLLGASLPA (SEQ ID NO: 11) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence TLLGASLPA (SEQ ID NO: 11).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence TLLGASLPA (SEQ ID NO: 11).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is TLLGASLPA (SEQ ID NO: 11), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is TLLGASLPA (SEQ ID NO: 11); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is TLLGASLPA (SEQ ID NO: 11).
FLYPFLSHL
Aspects relating to FLYPFLSHL (also known as p44 herein; or SEQ ID NO: 14) are described below.
An isolated peptide is provided comprising the amino acid sequence FLYPFLSHL (SEQ ID NO: 14). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence FLYPFLSHL (SEQ ID NO: 14).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence FLYPFLSHL (SEQ ID NO: 14). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence FLYPFLSHL (SEQ ID NO: 14); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence FLYPFLSHL (SEQ ID NO: 14).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide FLYPFLSHL (SEQ ID NO: 14). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence FLYPFLSHL (SEQ ID NO: 14). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence FLYPFLSHL (SEQ ID NO: 14); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence FLYPFLSHL (SEQ ID NO: 14) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence FLYPFLSHL (SEQ ID NO: 14).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence FLYPFLSHL (SEQ ID NO: 14).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLYPFLSHL (SEQ ID NO: 14), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLYPFLSHL (SEQ ID NO: 14); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is FLYPFLSHL (SEQ ID NO: 14).
ILEYLTAEV
Aspects relating to ILEYLTAEV (also known as p49 herein; or SEQ ID NO: 15) are described below.
An isolated peptide is provided comprising the amino acid sequence ILEYLTAEV (SEQ ID NO: 15). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence ILEYLTAEV (SEQ ID NO: 15).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence ILEYLTAEV (SEQ ID NO: 15). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence ILEYLTAEV (SEQ ID NO: 15); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence ILEYLTAEV (SEQ ID NO: 15).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide ILEYLTAEV (SEQ ID NO: 15). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence ILEYLTAEV (SEQ ID NO: 15). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence ILEYLTAEV (SEQ ID NO: 15); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence ILEYLTAEV (SEQ ID NO: 15) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence ILEYLTAEV (SEQ ID NO: 15).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence ILEYLTAEV (SEQ ID NO: 15).
A method of diagnosing a cancer or viral infection associated with impaired HI_A class I antigen presentation in a human subject is also provided comprising: determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is ILEYLTAEV (SEQ ID NO: 15), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HI_A class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is ILEYLTAEV (SEQ ID NO: 15); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is ILEYLTAEV (SEQ ID NO: 15).
LSEKLERI
Aspects relating to LSEKLERI (also known as p67 herein; or SEQ ID NO: 16) are described below.
An isolated peptide is provided comprising the amino acid sequence LSEKLERI (SEQ ID NO: 16). Suitably, the peptide has no more than 20 amino acids. Suitably, the peptide consists of the amino acid sequence LSEKLERI (SEQ ID NO: 16).
An isolated nucleic acid sequence encoding the peptide described herein is also provided. Suitably, the nucleic acid sequence is mRNA or DNA.
A binding agent is provided that specifically binds to a peptide comprising the amino acid sequence LSEKLERI (SEQ ID NO: 16). Suitably, the binding agent is a binding protein such as an antibody.
An isolated nucleic acid sequence is provided encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LSEKLERI (SEQ ID NO: 16); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LSEKLERI (SEQ ID NO: 16).
Suitably, the nucleic acid sequence encodes both (a) and (b), wherein (a) and (b) together specifically bind to the peptide LSEKLERI (SEQ ID NO: 16). Suitably, the nucleic acid sequence encodes a T cell receptor.
An isolated nucleic acid sequence is provided encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence LSEKLERI (SEQ ID NO: 16). Suitably, the target binding moiety is encoded by an isolated nucleic acid sequence encoding:
(a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide having the amino acid sequence LSEKLERI (SEQ ID NO: 16); and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide having the amino acid sequence LSEKLERI (SEQ ID NO: 16) as described elsewhere herein.
The isolated nucleic acid sequences provided herein may be part of a vector. A vector comprising a nucleic acid sequence described herein is therefore also provided. Suitably, the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
A modified cell transfected or transduced with a nucleic acid sequence described herein or a vector described herein is also provided. Suitably, the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line or a NK-92 cell line. Suitably, the modified cell is a human cell.
A pharmaceutical composition is provided comprising an a) isolated peptide, b) nucleic acid sequence, c) vector, d) binding agent or e) modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, any one of a), b), c) or d) may be present in the pharmaceutical composition by virtue of them being encoded or expressed (as appropriate) by a cell that is present within the pharmaceutical composition. As an example, any one of b) or c) may be encoded by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition; or any of a) or d) may be expressed by a cell that is combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate the pharmaceutical composition. More details on this are provided below under “detailed description”.
Suitably, the composition is formulated as a vaccine.
A pharmaceutical composition described herein is provided for use as a medicament. Suitably, the pharmaceutical composition is for use in the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation in a human subject.
A method of treating a condition in a human subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Suitably, the method is for the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
A method of generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or vector described herein with a cell under conditions in which the nucleic acid sequence or vector is incorporated and expressed by the cell to generate the T cell receptor that specifically binds to a peptide having the amino acid sequence LSEKLERI (SEQ ID NO: 16).
Suitably, the method is ex vivo.
Use of a peptide as a biomarker for a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided, wherein the peptide comprises the amino acid sequence LSEKLERI (SEQ ID NO: 16).
A method of diagnosing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is also provided comprising:
determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LSEKLERI (SEQ ID NO: 16), wherein the presence of the peptide in the sample identifies the subject as having a cancer or viral infection associated with impaired HLA class I antigen presentation and wherein the absence of the peptide in the sample identifies the subject as not having a cancer or viral infection associated with impaired HLA class I antigen presentation.
A method of treating a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject is provided, the method comprising:
(i) determining the presence of a peptide in a sample isolated from the subject, wherein the peptide is LSEKLERI (SEQ ID NO: 16); and (ii) administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
A pharmaceutical composition described herein is provided for use in treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject, wherein the subject has been identified as having a cancer or viral infection associated with impaired HLA class I antigen presentation by the presence of a peptide in a sample isolated from the subject, wherein the peptide is LSEKLERI (SEQ ID NO: 16).
Definitions and embodiments
The following definitions and embodiments apply to each aspect of the invention described herein.
Immunogenic peptides
The inventors have identified sixteen peptides that are present in the HLA class I ligandome of tumor cells displaying impaired HLA class I antigen presentation. T cells that recognise each of these peptides have been identified within the T cell repertoire of healthy donors. The data presented herein verifies that these peptides are presented on the cell surface of cells with impaired HLA class I presentation and that cognate T cell receptors that specifically recognise these HLA class I restricted peptides on the cell surface are present in vivo. Advantageously, these peptides may be used as an immunogenic peptide vaccine which can be administered to a human subject in order to treat or prevent a cancer or viral infection associated with impaired HLA class I antigen presentation. Nucleic acid sequences and vectors encoding these peptides may also be useful for this purpose.
The peptides described herein represent bonafide immunogenic tumor-specific antigens (or antigens that are presented in viral infections associated with impaired HLA class I antigen presentation) that may be further exploited in the development of personalized vaccines, which may be particularly useful as an adjunct to other therapies (e.g. ACT as described herein). These immunogenic peptides can therefore be used as an immunotherapy in the form of e.g. peptide, RNA, DNA, dendritic cell based therapies, and adoptive TCR transgenic T cell-based therapies.
As used herein, an “isolated peptide” refers to a peptide that is not in its natural environment. The peptide may therefore be of synthetic origin (or alternatively, of natural original, but isolated from its natural environment). In the context of this disclosure, the natural environment of these peptides is within the human body. Accordingly, when the peptides are present e.g. in a pharmaceutical composition (comprising adjuvants etc) they are considered to be in isolated form, as they are not in their natural environment.
The isolated peptide may be relatively short (i.e. no more than 20 amino acids; e.g. no more than 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids). The peptide may consist of the amino acid sequence of any one of SEQ ID NO:1 to 16 only.
The isolated peptide may be administered to a human subject in order to treat or prevent a cancer or viral infection associated with impaired HLA class I antigen presentation. For example, the isolated peptide may be administered to the subject in order to induce or enhance their immune response. The peptide may therefore be administered to the subject to induce T cell activation (e.g. in vivo T cell activation) in the subject, wherein the activated T cells are specific for the peptide (and thus will specifically target the cancerous or virally infected cells).
The isolated peptide may be administered as a peptide vaccine for treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation. The isolated peptide may be administered to induce or enhance activation of T cells specific for cancerous or virally infected cells.
Similarly, nucleic acid sequences and vectors encoding the peptides described herein may be administered as a nucleic acid vaccine for treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation. The isolated nucleic acid sequences and vectors may be administered to induce or enhance activation of T cells specific for cancerous or virally infected cells.
The peptides described herein (and corresponding nucleic acid sequences or vectors encoding the same) may be particularly useful as an immunotherapy for human subjects that are positive for HLA-A*02:01.
Isolated peptides described herein (or their corresponding nucleic acid sequences or vectors) may also be provided in compositions that comprise more than one of the peptides (or nucleic acid sequences or vectors) discussed above. The composition may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more etc of these peptides. Byway of example, an isolated peptide may be provided (and/or administered) as a composition that comprises a mixture of (a) an isolated peptide comprising the amino acid sequence of SEQ ID NO:1; and (b) an isolated peptide comprising an amino acid sequence selected from SEQ ID NO:2 to SEQ ID NO:16. The composition may comprise at least a peptide comprising the amino acid sequence of SEQ ID NO:1 (with additional peptide(s) comprising the amino acid sequence of any of SEQ ID NO:2 to 16); or at least a peptide comprising the amino acid sequence of SEQ ID NO:2 (with additional peptide(s) comprising the amino acid sequence of any of SEQ ID NO:1 or 3 to 16); or at least a peptide comprising the amino acid sequence of SEQ ID NO:3 (with additional peptide(s) comprising the amino acid sequence of any of SEQ ID NO:1, 2 or 4 to 16); or at least a peptide comprising the amino acid sequence of SEQ ID NO:4 (with additional peptide(s) comprising the amino acid sequence of any of SEQ ID NO:1, 2, 3 or 5 to 16); or at least a peptide comprising the amino acid sequence of SEQ ID NO:5 (with additional peptide(s) comprising the amino acid sequence of any of SEQ ID NO:1 to 4 or 6 to 16) etc. Any combination of isolated peptides may be provided within one composition. The combinations and permutation recited above for peptide compositions apply equally to compositions comprising two or more nucleic acid sequences or vectors that encode such peptides.
Binding agents
Binding agents are described herein that specifically bind to a peptide comprising (or consisting of) the amino acid sequence of one of SEQ ID NO: 1 to 16. The binding agent is useful in the prevention or treatment of a cancer or viral infection associated with impaired HLA class I antigen presentation in a human subject.
The binding agent may specifically bind to an epitope within the recited peptide (i.e. within the amino acid sequence provided by SEQ ID NO: 1 to 16). As used herein the term “epitope” refers to a site on a target molecule (in this case the recited peptide) to which a binding agent binds. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single peptide (antigen) may have more than one epitope. Epitopes can be formed both from contiguous or adjacent noncontiguous residues (e.g., amino acid residues) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acid residues) typically are also called linear epitopes. An epitope typically includes at least 5 and up to about 12 residues, mostly between 6 and 10 residues (e.g. amino acid residues). Epitopes may also be conformational (i.e. non-linear).
In one example, the binding agent (e.g. antibody) specifically binds to an epitope generated by the peptide itself. In another example, the binding agent (e.g. antibody) binds to an epitope generated by the combination of the peptide and the HLA molecule that presents it (i.e. an epitope that is generated when the peptide is presented on the cell surface by HLA class I, e.g. HLA*0201).
Examples of binding agents include, but are not limited to antibodies, chimeric antigen receptors (CAR), T cell receptors (TCR), aptamers (e.g. nucleic acid aptamers peptide aptamers, aptabodies, affimers) etc. Other appropriate binding agents are also well known and readily identifiable to a person of skill in the art using routine experimental procedures.
In one example, the binding agent is a binding protein. Examples of appropriate binding proteins include chimeric antigen receptors (CAR), T cell receptors (TCR), antibodies and antibody mimetics. Examples of appropriate antibody mimetics include affibody molecules (including affimabs) affilins, peptide aptamers (including affimers), affitins, alphabodies, anticalins, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies and nanoCLAMPs, all of which are well known in the art.
In one example, the binding agent is an isolated binding agent. As used herein, an “isolated binding agent” refers to a binding agent that is not in its natural environment. The binding agent may therefore be a recombinant binding agent, or the binding agent may be of synthetic origin (or alternatively, of natural original, but isolated from its natural environment). In the context of this disclosure, the natural environment of binding agents such as antibodies is within the human body. Accordingly, when the binding agent (e.g. antibody) are present e.g. in a pharmaceutical composition (comprising adjuvants etc) they are considered to be in isolated form, as they are not in their natural environment.
Antibodies
As stated above, the binding agent may be a binding protein such as an antibody. The antibody may be an isolated antibody (as defined above).
As used herein, “antibody” refers to antibodies, antibody mimetics and antibody fragments unless the context specifically requires otherwise.
The terms “antibody” or “antibodies” as used herein refer to molecules or active fragments of molecules that bind to known antigens, and particularly refers to immunoglobulin molecules and to immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that immunospecifically binds an antigen. The immunoglobulin can be of any class (IgG, IgM, IgD, IgE, IgA and IgY) or subclass (e.g. IgG 1, lgG2, IgG3, lgG4, IgA1 and lgA2) or subclasses (isotypes) of immunoglobulin molecule (e.g. IgG in lgG1, lgG2, lgG3, and lgG4, or IgA in lgA1 and lgA2).
Each antibody typically comprises a variable region. Each variable region as a whole is considered to constitute one antigen binding site (or epitope binding site).
The term variable region refers to the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK, νλ, and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively. More specifically, the term refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).). A single VH or VL domain may be sufficient to confer antigen-binding specificity.
The term hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a complementarity determining region or CDR (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain according to Kabat et al, Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a hypervariable loop (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain according to Chothia and LeskJ. Mol. Biol. 196:901-917(1987)).
The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, generally referred to in the art and herein as the Fv domain or Fv region. In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigenbinding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a CDR), in which the variation in the amino acid sequence is most significant. Variable refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. Variability within the variable region is not evenly distributed. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called hypervariable regions that are each 9-15 amino acids long or longer. Each VH and VL is composed of three hypervariable regions (complementary determining regions, CDRs) and four FRs, arranged from amino- terminus to carboxy-terminus in the following order: FR1 -CDR1 -FR2-CDR2-FR3- CDR3-FR4.
The term “antibody” or “antibodies” include monoclonal, polyclonal, chimeric, single chain, bispecific, human and humanized antibodies as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens include F(abj2, F(abjs, diabodies, triabodies, scFv-Fc, di-scFv, minibodies, Fab, bispecific Fab2, trispecific Fabs, scFv, bispecific di-scFv, bispecific scFv-Fc, bispecific diabodies, a trispecific triabodies and bispecific minibodies, including the products of an Fab immunoglobulin expression library and epitope-binding fragments of any of the antibodies and fragments mentioned above.
In a particular example, the antibody may be a monoclonal antibody. As used herein, the term “monoclonal antibody” refers to an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically made by fusing a normally short-lived, antibody-producing B cell to a fast-growing cell, such as a cancer cell (sometimes referred to as an “immortal” cell). The resulting hybrid cell, or hybridoma, multiplies rapidly, creating a clone that produces large quantities of the antibody. As used herein, “monoclonal antibody” is also to be understood to comprise antibodies that are produced by a mother clone which has not yet reached full monoclonality.
As used herein, the term “chimeric antibody” refers to a monoclonal antibody comprising a variable region, i.e., binding region, from mouse and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are exemplary embodiments. Such mouse/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding mouse immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of “chimeric antibodies” encompassed by the present disclosure are those in which the class or subclass has been modified or changed from that of the original antibody. Such “chimeric” antibodies are also referred to as “class-switched antibodies.” Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S. L, et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244.
In a particular example, the antibody may be a human antibody or a humanized antibody.
As used herein the term “humanized antibody” or “humanized version of an antibody” refers to antibodies in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In some exemplary embodiments, the CDRs of the VH and VL are grafted into the framework region of human antibody to prepare the “humanized antibody.” See e.g. Riechmann, L, et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be the sequences of naturally occurring human antibodies. Human heavy and light chain variable framework regions are listed e.g. in Lefranc, M.-P., Current Protocols in Immunology (2000)—Appendix 1P A.1P.1-A.1P.37 and are accessible via IMGT, the international ImMunoGeneTics information System® (http://imgt.cines.fr) or via http://vbase.mrc-cpe.cam.ac.uk, for example. Optionally the framework region can be modified by further mutations. Exemplary CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. In some embodiments, such humanized version is chimerized with a human constant region.
As used herein the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G.,
Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., etal., Nature 362 (1993) 255-258; Brueggemann, M. D., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., etal., J. Mol. Biol. 222 (1991) 581597). The techniques of Cole, A., et al. and Boerner, P., et al. are also available for the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A. R. (1985) p. 77; and Boerner, P., etal., J. Immunol. 147 (1991) 8695).
As used herein “single chain antibody” refers to single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird etal., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 or a bispecific single chain Fv (WO 03/11161). Typical scFv linkers are well known in the art, are generally 10 to 25 amino acids in length and include glycines and serines.
As used herein, a “di-ScFv” refers to a dimerised scFV.
As used herein, “minibodies” are minimized antibody-like proteins comprising a scFv joined to a CH3 domain. See Hu etal., 1996, Cancer Res. 56:3055-3061. In some cases, the scFv can be joined to the Fc region, and may include some or the entire hinge region.
By Fab or Fab region as used herein is meant the polypeptides that comprise the VH, CH1, VH, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody or antibody fragment or fab fusion protein.
The terms “Fab”, “Fab region”, “Fab portion” or “Fab fragment” are understood to define a polypeptide that includes a VH, a CH1, a VL, and a CL immunoglobulin domain. Fab may refer to this region in isolation, or this region in the context of an antibody molecule according to the invention, as well as a full length immunoglobulin or immunoglobulin fragment. Typically a Fab region contains an entire light chain of an antibody. A Fab region can be taken to define “an arm” of an immunoglobulin molecule. It contains the epitope binding portion of that Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by a papain-digestion. A “F(ab')2 portion” is the proteolytic fragment of a pepsin-digested immunoglobulin. A “Fab' portion” is the product resulting from reducing the disulfide bonds of an F(ab')2 portion. As used herein the terms “Fab”, “Fab region”, “Fab portion” or “Fab fragment” may further include a hinge region that defines the C-terminal end of the antibody arm (cf. above). This hinge region corresponds to the hinge region found C-terminally of the CH1 domain within a full length immunoglobulin at which the arms of the antibody molecule can be taken to define a Y. The term hinge region is used in the art because an immunoglobulin has some flexibility at this region.
By Fc fusion as used herein is meant a protein wherein one or more polypeptides is operably linked to Fc. Fc fusion is herein meant to be synonymous with the terms immunoadhesin, Ig fusion, Ig chimera, and receptor globulin (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide or small molecule. The role of the non-Fc part of an Fc fusion, i.e., the fusion partner, is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody. Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion. Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such targets may be any molecule, e.g., an extracellular receptor that is implicated in disease.
As used herein the term “antibody fragments” refers to a portion of a full length antibody, for example possibly a variable domain thereof, or at least an antigen binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g., described in Huston, J. S., Methods in Enzymol. 203 (1991) 46-88. Antibody fragments can be derived from an antibody of the present invention by a number of art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al.
Methods Enzymology, 121:663-69, Academic Press, 1986. Antibody fragments useful to the invention are multivalent antibody fragments.
In a particular example, the binding agent described herein may be an IgG. The IgG may be selected from lgG1, lgG2, lgG3 and lgG4.
As used herein the terms “specific binding” and “binding specifically” (or other equivalent terms) are used interchangeably to indicate that other biomolecules do not significantly bind to the region (binding region e.g. variable region) that is specifically binding to the peptide of interest (i.e. the recited peptide comprising the amino acid sequence of one of SEQ ID NO:1 to 16). In some embodiments, the level of binding to a biomolecule other than the specified peptide results in a negligible (e.g., not determinable) binding affinity by means of ELISA or an affinity determination. 'By “together specifically bind” is meant that in the context of a single polypeptide chain that comprises multiple TCR binding chains, e.g. a TCR a chain and a TCR b chain, the single polypeptide specifically binds to the peptide of interest through the collective binding of each of the TCR binding chains within the polypeptide.
By “negligible binding” a binding is meant, which is at least about 85%, particularly at least about 90%, more particularly at least about 95%, even more particularly at least about 98%, but especially at least about 99% and up to 100% less than the binding to the peptide of interest (i.e. the recited peptide comprising the amino acid sequence of one of SEQ ID NO:1 to 16).
The binding affinity of the binding agent to the peptide of interest (i.e. the recited peptide comprising the amino acid sequence of one of SEQ ID NO:1 to 16) may be determined using a standard binding assay, such as surface plasmon resonance technique (BIAcore®, GEHealthcare Uppsala, Sweden). The term surface plasmon resonance, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., etal. (1993) Ann. Biol. Clin. 51 : 19-26; Jonsson, U., etal. (1991) Biotechniques 11 :620-627; Johnsson, B., etal. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., etal. (1991) Anal. Biochem. 198:268-277.
For example, “specifically binding” in the context of the binding of an antibody to a predetermined antigen/epitope means binding with an affinity corresponding to a KD of about 10~7 M or less, such as about 10~8 M or less, such as about 10-9 M or less, about 10-1°
M or less, or about 10’11 M or even less when determined for instance by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antibody as the analyte. This term also means that the antibody binds to the predetermined antigen/epitope with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g. bovine serum albumin, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is very specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term “KD” as used herein, means the dissociation rate constant of a particular antibody-antigen interaction.
T cell receptors
As stated above, the binding agent may be a binding protein such as a T cell receptor. The binding agent may be an isolated T cell receptor (i.e. a T cell receptor that that is not in its natural environment). The T cell receptor may therefore be a recombinant TCR, or the binding agent may be of synthetic origin (or alternatively, of natural original, but isolated from its natural environment).
A T cell receptor (TCR) is a molecule found on the surface of T cells (T lymphocytes) that is responsible for recognising a peptide that is bound to (presented by) a major histocompatibility complex (MHC) molecule on a target cell. TCRs interact with a particular peptide in the context of the appropriate serotype of MHC.
The human leukocyte antigen (HLA) system or complex is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. HLAs corresponding to MHC class I (A, B, and C) present peptides from inside the cell. HI_A-A*02:01 is a globally common human leukocyte antigen serotype within the HLA-A serotype group. Peptides that are presented by HLA-A*02:01 to TCRs are described as being “HLA-A*02:01 restricted”.
In a particular example, the TCRs described herein interact with the peptide of interest (i.e. the recited peptide comprising the amino acid sequence of one of SEQ ID NO:1 to 16) in the context of HLA-A*02:01. In other words, the peptides of interest are HLA-A*02:01 restricted peptides.
The TCR is composed of two different polypeptide chains. In humans, 95% of TCRs consist of an alpha (a) chain and a beta (β) chain (encoded by TRA and TRB respectively). When the TCR engages with peptide in the context of HLA (e.g. in the context of HLA-A*02:01, as appropriate), the T cell is activated through signal transduction.
The alpha and beta chains of the TCR are highly variable in sequence. Each chain is composed of two extracellular domains, a variable region (V) and a constant region (C). The constant region is proximal to the T cell membrane followed by a transmembrane region and a short cytoplasmic tail while the variable region binds to the peptide/HLA-A complex.
The variable region of each chain has three hypervariable regions (also called complementarity determining regions (CDRs)). Accordingly, the TCR alpha chain comprises a CDR1, a CDR2 and a CDR3 and the TCR beta chain also comprises a (different) CDR1, CDR2, and a CDR3. In each of the alpha and beta chains, it is CDR3 that is mainly responsible for recognizing the peptide being presented by HLA-A.
Chimeric antigen receptor proteins
As stated above, the binding agent may also be a binding protein such as a chimeric antigen receptor protein (CAR). CARs are typically expressed once they have been introduced into a T cell (i.e. they are expressed on the surface of a modified T cell, called a CAR-T cell). CART cells create a link between an extracellular antigen recognition domain (referred to as a target binding moiety herein) and an intracellular signalling molecule which in turn activates the T-cells. The specificity and safety of CAR T-Cells are determined by the choice of molecule that is targeted. CAR-T cells may be autologous or allogenic.
CARs typically comprise at least the following three domains: (i) an (extracellular) target binding moiety (forming part of the ectodomain of the CAR); (ii) a transmembrane domain, and (iii) an (intracellular) signalling region (forming part of the endodomain of the CAR).
The ectodomain is the region of the receptor that is exposed to the extracellular fluid and typically comprises: a signalling peptide, a target binding moiety and a spacer. A signal peptide directs the nascent protein into the endoplasmic reticulum. A target binding moiety interacts with specifically binds to) the target peptide (i.e. the recited peptide comprising the amino acid sequence of one of SEQ ID NO: 1 to 16). The spacer separates the target binding moiety from the transmembrane domain. The transmembrane domain is typically a hydrophobic alpha helix that spans the membrane. In one example the CD28 transmembrane domain or CD3-zeta transmembrane domain may be used. The endodomain comprises the intracellular signalling region that activates the T cell. In one example, a CD3-zeta endodomain component may be used which contains three ITAMs. It transmits an activation signal to the T cell after the antigen is bound. In some examples, costimulatory signalling may also be used (e.g. by using chimeric CD28 and 0X40 together with with CD3-Zeta).
CARs are introduced into autologous or allogeneic T cells using standard techniques e.g. transfection of a nucleic acid sequence or vector encoding the CAR into the T cell (e.g. using an integrating gammaretrovirus (RV) or a lentivirus (LV) vector). The CARs are then expressed by the modified T cell, which can be administered to a subject as an immunotherapy.
Chimeric antigen receptor proteins (CARs) are typically introduced into T cells to specifically recognise surface antigens on e.g. tumor cells. CARs are fusion target-binding proteins that can utilise antibodies, antibody fragments (e.g. scFvs), or TCR sequences to confer specificity of binding (i.e. act as a target binding moiety), and intracellular signalling regions to determine the specific biological activity required. Various different generations of CARs are known, and each of these different generations represents a suitable example of a chimeric antigen receptor described herein, unless the context of the present disclosure requires otherwise.
For the avoidance of doubt, chimeric antigen receptor proteins referred to herein may also be taken to encompass T cell receptor (TCRs) modified to comprise an intracellular signalling region from a different source. In these examples, the binding specificity to the target peptide (i.e. the recited peptide comprising an amino acid sequence of one of SEQ ID NO:1 to 16) may be provided by the TCR alpha and TCR beta chains of the receptor, which act as the target binding moiety of the chimeric antigen receptor proteins. As the target binding moiety and the signalling region are from different sources, such modified TCRs are chimeric for the purposes of this disclosure.
As described herein, the chimeric antigen receptor proteins described herein comprise a target binding moiety that specifically binds to a (target) peptide comprising the amino acid sequence of one of SEQ ID NO: 1 to 16. The target binding moiety that specifically binds to the recited peptide may comprise an antibody, an antibody fragment (e.g. scFvs), a TCR, or a TCR fragment.
Nucleic acid sequences and vectors
Nucleic acid sequences and vectors that encode TCR polypeptide components or chimeric antigen receptor proteins are described herein that specifically bind to a target peptide (i.e. a recited peptide comprising an amino acid sequence of one of SEQ ID NO: 1 to 16).
Nucleic acid sequences and vectors that encode peptides comprising an amino acid sequence of any one of SEQ ID NO:1 to 16 are also described herein, as well as nucleic acid sequences and vectors encoding binding agents described herein.
Details of appropriate chimeric antigen receptor proteins that may be encoded by the nucleic acid sequences or vectors described herein are provided above. As stated elsewhere herein, the nucleic acid sequences or vectors may encode a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence of one of SEQ ID NO: 1 to 16. In one example, the target binding moiety is encoded by an isolated nucleic acid sequence encoding one or more TCR polypeptide components described in more detail below.
Nucleic acid sequences that encode a TCR polypeptide component may form part of a larger nucleic acid sequence that encodes a larger component part of a T cell receptor (e.g. TCR a chain variable region, a TCR β chain variable region, a TCR a chain, a TCR β chain etc). The nucleic acid sequences may also form part of a larger nucleic acid sequence that encodes a functioning T cell receptor (i.e. encodes a functional TCR a chain and a functional TCR β chain, optionally separated by a linker sequence that enables coordinate expression of two proteins or polypeptides by the same vector). More details on this are provided below.
The nucleic acid sequences may encode a small component of a T cell receptor e.g. a CDR3 domain of a TCR a chain polypeptide, or a CDR3 domain of a TCR β chain polypeptide only. The nucleic acid sequence may therefore be considered as a “building block” that provides an essential component for peptide specificity. The nucleic acid sequence may be incorporated into a distinct nucleic acid sequence (e.g. a vector) that encodes the other elements of a TCR variable chain, such that when the nucleic acid sequence is incorporated, a new nucleic acid sequence is generated that encodes a TCR a chain variable region and/or a TCR β chain variable region that specifically binds to a target peptide (i.e. a recited peptide comprising an amino acid sequence of one of SEQ ID NO: 1 to 16). A nucleic acid sequence described herein therefore has utility as an essential component of TCR specificity for the selected peptide, and thus can be used to generate a nucleic acid sequence encoding a TCR variable region with the required antigen binding activity and specificity to target cancerous cells or virally infected cells associated with impaired HLA class I antigen presentation.
An isolated nucleic acid sequence encoding: (a) a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to the target peptide; and/or (b) a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to the target peptide (i.e. specifically binds to one of the following peptides: SEQ ID NO: 1 to SEQ ID NO: 16). As will be clear throughout the application the “target peptide” refers to the peptide that is being targeted i.e. it refers a peptide selected from those comprising an amino acid sequence of SEQ ID NO: 1 to 16.
The nucleic acid sequence may encode (a), (b), or (a) and (b). The nucleic acid sequence therefore encodes at least one polypeptide comprising a CDR3 of a T cell receptor polypeptide, wherein the CDR3 specifically binds to one of the following peptides: SEQ ID NO: 1 to SEQ ID NO: 16.
The nucleic acid sequence may include an alpha chain CDR3 and a beta chain CDR3, wherein the alpha chain CDR3 and the beta chain CDR3 together specifically bind to the selected peptide.
The nucleic acid sequence therefore encodes a “CDR3 of a TCR a chain polypeptide” (also referred to herein as an alpha chain CDR3, or an a chain CDR3) and/or a “CDR3 of a TCR β chain polypeptide” (also referred to herein as a beta chain CDR3, or an β chain CDR3).
The alpha chain CDR3 may be that of SEQ ID NO:24 or one of the variants described below. Similarly, the beta chain CDR3 may be that of SEQ ID NO:32 or one of the variants described below. It is noted that these specific CDR3 have been found by the inventors to specifically bind to the peptide of SEQ ID NO:1.
Any of the permutations described below for (a) may be combined with the permutations described below for (b) (e.g. to form an appropriate nucleic acid sequence that encodes a functioning T cell receptor (i.e. encodes a functional TCR a chain and TCR β chain, optionally separated by a linker sequence that enables coordinate expression of two proteins or polypeptides by the same vector).
Polypeptide (a) - components of the TCR alpha chain
In one embodiment, the CDR3 of (a) may have an amino acid sequence of SEQ ID NO:24, or be functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:24. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:24, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO: 24 that do not specifically bind to SEQ ID NO:1. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:24 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
In one embodiment, the CDR3 of (a) may have an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:24, whilst retaining the ability to specifically bind to the peptide of SEQ ID NO:1. In other words, a functional CDR3 with one amino acid substitution compared to the sequence of SEQ ID NO:24 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:24).
In examples where the CDR3 of (a) has the amino acid sequence of SEQ ID NO:24, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO:23 or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO:23, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
In one embodiment, the polypeptide of (a) comprises the CDR3 (e.g. the CDR3 of SEQ ID NO:24 or a variant thereof as defined above) within a TCR a chain variable region that specifically binds to the peptide of SEQ ID NO:1. In other words, the polypeptide of (a) may comprise a TCR a chain variable region which includes the specified CDR3, wherein the TCR a chain variable region (and the CDR3 within it) specifically binds to the peptide of SEQ ID NO:1. As will be clear to a person of skill in the art, the phrase “TCR a chain variable region” refers to the variable (V) region (extracellular domain) of a TCR alpha chain, and thus includes three hypervariable regions (CDR1, CDR2 and the specified CDR3), as well as the intervening sequences, but does not include the constant (C) region of the alpha chain, which does not form part of the variable chain.
The encoded TCR a chain variable region may comprise, in addition to the specified CDR3, a CDR1 having an amino acid sequence of SEQ ID NQ:20, or functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the N-terminus of the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NQ:20. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NQ:20, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO: 20 that do not specifically bind to the N-terminus of the peptide of SEQ ID NO:1. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NQ:20 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and nonfunctional variants are well known to a person of ordinary skill in the art.
In one embodiment, the CDR1 of (a) (e.g. within the alpha chain variable region) may have an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NQ:20, whilst retaining the ability to specifically bind to the N terminus of the peptide of SEQ ID NO:1. In other words, a functional CDR1 with one amino acid substitution compared to the sequence of SEQ ID NQ:20 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NQ:20).
In examples where the CDR1 of (a) (e.g. within the alpha chain variable region) has the amino acid sequence of SEQ ID NQ:20, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO:19, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO:19, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
The encoded TCR a chain variable region may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having an amino acid sequence of SEQ ID NO:22, or functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HI_A-A*02:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:22. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:22, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO: 22 that do not specifically bind to HI_A-A*02:01. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:22 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
In one embodiment, the CDR2 of (a) (e.g. within the alpha chain variable region) may have an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:22, whilst retaining the ability to bind to HI_A-A*02:01. In other words, a functional CDR2 with one amino acid substitution compared to the sequence of SEQ ID NO:22 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:22).
In examples where the CDR2 of (a) (e.g. within the alpha chain variable region) has the amino acid sequence of SEQ ID NO:22, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO:21, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO:21, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
The polypeptide of (a) may therefore comprise a TCR alpha chain variable region that comprises the CDRs mentioned in detail above (by SEQ ID specifically, or variants thereof), with appropriate intervening sequences between the CDRs.
The TCR alpha chain variable region of (a) may have an amino acid sequence of SEQ ID NO: 18, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:18. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:18, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO: 18 that do not specifically bind to SEQ ID NO:1. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:18 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
In one embodiment, the TCR alpha chain variable region of (a) may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:18, whilst retaining the ability to specifically bind to the peptide of SEQ ID NO:1. In other words, a functional TCR alpha chain variable region with one or more amino acid substitutions compared to the sequence of SEQ ID NO:18 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:18 may all be in regions of the TCR alpha chain variable region that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 20, SEQ ID NO:22 and/or SEQ ID NO:24, and still have 25% (or less) sequence variability compared to SEQ ID NO:18). In other words, the sequence of the CDRs of SEQ ID NO: 18 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:18).
As an example, the polypeptide of (a) may comprise the CDR3 within a TCR a chain variable region having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO:18, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 24. In this example, the TCR a chain variable region CDR1 may have an amino acid sequence of SEQ ID NQ:20 and the TCR a chain variable region CDR2 may have an amino acid sequence of SEQ ID NO:22.
In examples where the TCR alpha chain variable region of (a) has the amino acid sequence of SEQ ID NO:18, the TCR alpha chain variable region may be encoded by the nucleic acid sequence of SEQ ID NO:17, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (a) may be encoded by the nucleic acid sequence of SEQ ID NO:17, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
For the avoidance of doubt, the polypeptide of (a) may comprise a TCR a chain variable region (as specified above) and a TCR a chain constant region. An example of a suitable constant region is encoded in the MP71-TCR-flex retroviral vector used herein by GenScript. However, the invention is not limited to this specific constant region, and encompasses any appropriate TCR a chain constant region. The constant region may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant regions are well known to a person of skill in the art and are well within their routine capabilities.
By way of example only, the constant region may be encoded by or derived from a vector, such as a lentiviral, retroviral or plasmid vector but also adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus or herpes virus vectors in which murine or human constant regions are pre-cloned. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivies and M Hudecek in Leukemia 2016). Moreover, naked (synthetic) DNA/RNA can also be used to introduce the TCR. As an example, a pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes as described in LV Coren et al., BioTechniques 2015 may be used to provide an appropriate constant region.
Polypeptide (b) - components of the TCR beta chain
In one embodiment, the CDR3 of (b) may have an amino acid sequence of SEQ ID NO:32, or be functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:32. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:32, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO: 32 that do not specifically bind to SEQ ID NO:1. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:32 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
In one embodiment, the CDR3 of (b) may have an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:32, whilst retaining the ability to specifically bind to the peptide of SEQ ID NO:1. In other words, a functional CDR3 with one amino acid substitution compared to the sequence of SEQ ID NO:32 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:32).
In examples where the CDR3 of (b) has the amino acid sequence of SEQ ID NO:32, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO:31, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO:31, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
In one embodiment, the polypeptide of (b) comprises the CDR3 (e.g. the CDR3 of SEQ ID NO:32 or a variant thereof as defined above) within a TCR β chain variable region that specifically binds to the peptide of SEQ ID NO:1. In other words, the polypeptide of (b) comprises a TCR β chain variable region which includes the specified CDR3, wherein the TCR β chain variable region (and the CDR3 within it) specifically binds to the peptide of SEQ ID NO:1. As will be clear to a person of skill in the art, the phrase “TCR β chain variable region” refers to the variable (V) region (extracellular domain) of a TCR beta chain, and thus includes three hypervariable regions (CDR1, CDR2 and the specified CDR3) as well as the intervening sequences, but does not include the constant (C) region of the beta chain, which does not form part of the variable chain.
The encoded TCR β chain variable region may comprise, in addition to the specified CDR3, a CDR1 having an amino acid sequence of SEQ ID NO:28, or functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the C-terminus of the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:28. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:28, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO: 28 that do not specifically bind to the C-terminus of the peptide of SEQ ID NO:1. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:28 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and nonfunctional variants are well known to a person of ordinary skill in the art.
In one embodiment, the CDR1 of (b) (e.g. within the beta chain variable region) may have an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:28, whilst retaining the ability to specifically bind to the C terminus of the peptide of SEQ ID NO:1. In other words, a functional CDR1 with one amino acid substitution compared to the sequence of SEQ ID NO:28 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:28).
In examples where the CDR1 of (b) (e.g. within the beta chain variable region) has the amino acid sequence of SEQ ID NO:28, the CDR1 may be encoded by the nucleic acid sequence of SEQ ID NO:27, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO:27, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
The encoded TCR β chain variable region may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having an amino acid sequence of SEQ ID NQ:30, or functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HI_A-A*02:01). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NQ:30. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NQ:30, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO: 30 that do not specifically bind to HLA-A*02:01. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NQ:30 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
In one embodiment, the CDR2 of (b) (e.g. within the beta chain variable region) may have an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NQ:30, whilst retaining the ability to bind to HLA-A*02:01. In other words, a functional CDR2 with one amino acid substitution compared to the sequence of SEQ ID NQ:30 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NQ:30).
In examples where the CDR2 of (b) (e.g. within the beta chain variable region) has the amino acid sequence of SEQ ID NQ:30, the CDR2 may be encoded by the nucleic acid sequence of SEQ ID NO:29, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO:29, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
The polypeptide of (b) may therefore comprise a TCR beta chain variable region that comprises the CDRs mentioned in detail above (by SEQ ID specifically, or variants thereof), with appropriate intervening sequences between the CDRs.
The TCR beta chain variable region of (b) may have an amino acid sequence of SEQ ID NO:26, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the peptide of SEQ ID NO:1). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:26. The term “variant” also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:26, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
Non-functional variants are amino acid sequence variants of SEQ ID NO:26 that do not specifically bind to SEQ ID NO:1. Non-functional variants will typically contain a nonconservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:26 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
In one embodiment, the TCR beta chain variable region of (b) may have an amino acid sequence having at least 75%, at least 80%, at least 85%, or at least 90% (or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:26, whilst retaining the ability to specifically bind to the peptide of SEQ ID NO:1. In other words, a functional TCR beta chain variable region with one or more amino acid substitutions compared to the sequence of SEQ ID NO:26 is also encompassed. As stated previously, the amino acid substitution may be a conservative amino acid substitution. The variability in sequence compared to SEQ ID NO:26 may all be in regions of the TCR beta chain variable region that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 28, SEQ ID NQ:30 and/or SEQ ID NO:32, and still have 25% (or less) sequence variability compared to SEQ ID NO:11). In other words, the sequence of the CDRs of SEQ ID NO:26 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:26).
As an example, (b) may include the CDR3 within a TCR β chain variable region having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO:26, wherein the CDR3 has an amino acid sequence of SEQ ID NO: 32. In this example, the TCR β chain variable region CDR1 may have an amino acid sequence of SEQ ID NO:28 and the TCR β chain variable region CDR2 may have an amino acid sequence of SEQ ID NQ:30.
In examples where the TCR beta chain variable region of (b) has the amino acid sequence of SEQ ID NO:26, the TCR beta chain variable region may be encoded by the nucleic acid sequence of SEQ ID NO:25, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). Accordingly, the polypeptide of (b) may be encoded by the nucleic acid sequence of SEQ ID NO:25, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
For the avoidance of doubt, (b) may comprise a TCR β chain variable region (as specified above) and a TCR β chain constant region. An example of a suitable constant region is encoded in the MP71-TCR-flex retroviral vector used herein by GenScript. However, the invention is not limited to this specific constant region, and encompasses any appropriate TCR β chain constant region. The constant region may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant regions are well known to a person of skill in the art and are well within their routine capabilities.
By way of example only, the constant region may be encoded by or derived from a vector, such as a lentiviral, retroviral or plasmid vector but also adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus or herpes virus vectors in which murine or human constant regions are pre-cloned. Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivies and M Hudecek in Leukemia 2016). Moreover, naked (synthetic) DNA/RNA can also be used to introduce the TCR. As an example, a MP71-TCR-flex retroviral vector with pre-cloned TCR-Ca and Cb genes as used by the inventors or a pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes as described in LV Coren et al., BioTechniques 2015 may be used to provide an appropriate constant region.
In examples where the nucleic acid molecule encodes both (a) and (b), the polypeptide of (a) may be joined to the polypeptide of (b) via a linker, e.g. a linker that enables expression of two proteins or polypeptides by the same vector. By way of example, a linker comprising a porcine teschovirus-1 2A (P2A) sequence may be used, such as 2A sequences from footand-mouth disease virus (F2A), equine rhinitis A virus (E2A) or Thosea asigna virus (T2A) as published by A.L. Szymczak et al., Nature Biotechnology 22, 589 - 594 (2004) or 2A-like sequences. 2A and 2A-like sequences are linkers that are cleavable once the nucleic acid molecule has been transcribed and translated. Another example of a linker is an internal ribosomal entry sites (IRES) which enables translation of two proteins or polypeptides by the same transcript. Any other appropriate linker may also be used. The identification of appropriate linkers is well within the routine capabilities of a person of skill in the art. As a further example, the nucleic acid sequence encoding (a) and the nucleic acid sequence encoding (b) may be cloned into a vector with dual internal promoters (see e.g. S Jones et al., Human Gene Ther 2009).
Additional appropriate polypeptide domains may also be encoded by the nucleic acid sequence described herein. By way of example only, the nucleic acid sequence may comprise a membrane targeting sequence that provides for transport of the encoded polypeptide to the cell surface membrane of the modified cell. Other appropriate additional domains are well known and are described, for example, in WO2016/071758.
In one example, the nucleic acid sequence may encode a soluble TCR. For example, the nucleic acid sequence may encode (a) and (b), wherein (a) and (b) comprise the variable regions of the TCR alpha and beta chains respectively, and optionally an immune-modulator molecule such as a CD3 agonist (e.g. an anti-CD3 scFv). The CD3 antigen is present on mature human T cells, thymocytes and a subset of natural killer cells. It is associated with the TCR and is involved in signal transduction of the TCR. Antibodies specific for the human CD3 antigen are well known. One such antibody is the murine monoclonal antibody OKT3, which is the first monoclonal antibody approved by the FDA. Other antibodies specific for CD3 have also been reported (see e.g. W02004/106380; U.S. Patent Application Publication No. 2004/0202657; U.S. Pat. No. 6,750,325). Immune mobilising mTCR Against Cancer (ImmTAC; Immunocore Limited, Milton Partk, Abington, Oxon, United Kingdom) are bifunctional proteins that combine affinity monoclonal T-cell receptor (mTCR) targeting with a therapeutic mechanism of action (i.e., an anti-CD3 scFv). In another example, a soluble TCR described herein may be combined with a radioisotope or a toxic drug. Appropriate radioisotopes and/or toxic drugs are well known in the art and are readily identifiable by a person of ordinary skill in the art.
In one example, the nucleic acid sequence described herein may encode a chimeric single chain TCR in which the polypeptide of (a) (e.g. the TCR alpha chain variable region) is linked to the polypeptide of (b) (e.g. the TCR beta chain variable region) and a constant region which is e.g. fused to the CD3 zeta signalling domain. In this example, the linker is noncleavable. In an alternative embodiment, the nucleic acid sequence described herein may encode a chimeric two chain TCR in which the polypeptide of (a) (e.g. the TCR alpha chain variable region) and the polypeptide of (b) (e.g. the TCR beta chain variable region) are each linked to a CD3 zta signalling domain. Methods for preparing such single chain TCRs and two chain TCRs are well known in the art; see for example RA Willemsen et al, Gene Therapy 2000.
As described elsewhere herein, isolated nucleic acid sequences are also provided that encode a target peptide (and corresponding vectors). All general statements herein relating to nucleic acid sequences and vectors apply equally. A person of skill in the art would readily identify suitable nucleic acid sequences and vectors on the basis of the peptide sequences provided herein.
Vectors and modified cells
In one aspect, the invention provides a vector that comprises a nucleic acid sequence described herein (e.g. a nucleic acid sequence that encodes a peptide comprising an amino acid sequence of any one of SEQ ID NO:1 to 16; a nucleic acid sequence encoding a TCR (or portion thereof) as described herein; a CAR protein as described herein, or any other binding agent as described herein).
Any appropriate vector can be used. By way of example only, the vector may be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector. Adenovirus, adenoassociated virus, vaccinia virus, canary poxvirus, herpes virus, minicircle vectors and naked (synthetic) DNA/RNA may also be used (for details on minicircle vectors, see for example non-viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivies and M Hudecek in Leukemia 2016).
Optionally, the vector comprises the nucleic acid sequence operably linked to a promoter.
As used herein, the term “vector” refers to a nucleic acid sequence capable of transporting another nucleic acid sequence to which it has been operably linked. The vector can be capable of autonomous replication or it can integrate into a host DNA. The vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers or suicide genes. The vector can be a nucleic acid sequence in the form of a plasmid, a bacteriophage or a cosmid. Preferably the vector is suitable for expression in a cell (i.e. the vector is an “expression vector”). Preferably, the vector is suitable for expression in a human T cell such as a CD8+ T cell or CD4+ T cell. In certain aspects, the vector is a viral vector, such as a retroviral vector, a lentiviral vector or an adeno-associated vector. Optionally, the vector is selected from the group consisting of an adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or synthetic RNA.
Preferably the (expression) vector is capable of propagation in a host cell and is stably transmitted to future generations.
Operably linked as used herein, refers to a single or a combination of the below-described control elements together with a coding sequence in a functional relationship with one another, for example, in a linked relationship so as to direct expression of the coding sequence.
The vector may comprise regulatory sequences. Regulatory sequences as used herein, refers to, DNA or RNA elements that are capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA- boxes, internal ribosomal entry sites (IRES), attachment sites for transcription factors, transcriptional terminators, polyadenylation sites etc. Optionally, the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct constitutive expression, as well as tissuespecific regulatory and/or inducible sequences.
The vector may comprise a promoter. Promoter, as used herein, refers to the nucleotide sequences in DNA to which RNA polymerase binds to start transcription. The promoter may be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or stimulatory protein. The promoter may be one that is not naturally found in the host cell (e.g. it may be an exogenous promoter). The skilled person in the art is well aware of appropriate promoters for use in the expression of target proteins, wherein the selected promoter will depend on the host cell.
The vector may comprise a transcriptional terminator. “Transcriptional terminator” as used herein, refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA. Preferred transcriptional terminators are characterized by a run of T residues preceded by a GC rich dyad symmetrical region.
The vector may comprise a translational control element. “Translational control element”, as used herein, refers to DNA or RNA elements that control the translation of mRNA. Preferred translational control elements are ribosome binding sites. Preferably, the translational control element is from a homologous system as the promoter, for example a promoter and its associated ribozyme binding site. Preferred ribosome binding sites are known, and will depend on the chosen host cell.
The vector may comprise restriction enzyme recognition sites. Restriction enzyme recognition site as used herein, refers to a motif on the DNA recognized by a restriction enzyme.
The vector may comprise a selectable marker. Selectable marker as used herein, refers to proteins that, when expressed in a host cell, confer a phenotype onto the cell which allows a selection of the cell expressing said selectable marker gene. Generally, this may be a protein that confers a new beneficial property onto the host cell (e.g. antibiotic resistance) or a protein that is expressed on the cell surface and thus accessible for antibody binding. Appropriate selectable markers are well known in the art.
Optionally, the vector may also comprise a suicide gene. “Suicide gene” as used herein, refers to proteins that induce death of the modified cell upon treatment with specific drugs. By way of example, suicide can be induced of cells modified by the herpes simplex virus thymidine kinase gene upon treatment with specific nucleoside analogs including ganciclovir, cells modified by human CD20 upon treatment with anti-CD20 monoclonal antibody and cells modified with inducible Caspase9 (iCasp9) upon treatment with AP1903 (reviewed by BS Jones, LS Lamb, F Goldman, A Di Stasi; Improving the safety of cell therapy products by suicide gene transfer. Front Pharmacol. (2014) 5:254. Appropriate suicide genes are well known in the art.
Preferably the vector comprises those genetic elements which are necessary for expression of the polypeptides described herein by a host cell. The elements required for transcription and translation in the host cell include a promoter, a coding region for the protein(s) of interest, and a transcriptional terminator.
A person of skill in the art will be well aware of the molecular techniques available for the preparation of (expression) vectors and how the (expression) vectors may be transduced or transfected into an appropriate host cell (thereby generating a modified cell as described herein). The (expression) vector of the present invention can be introduced into cells by conventional techniques such as transformation, transfection or transduction. “Transformation”, “transfection” and “transduction” refer generally to techniques for introducing foreign (exogenous) nucleic acid sequences into a host cell, and therefore encompass methods such as electroporation, microinjection, gene gun delivery, transduction with retroviral, lentiviral or adeno-associated vectors, lipofection, superfection etc. The specific method used typically depends on both the type of vector and the cell. Appropriate methods for introducing nucleic acid sequences and vectors into host cells such as human cells are well known in the art; see for example Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637646. Further conventional methods that are suitable for preparing expression vectors and introducing them into appropriate host cells are described in detail in WO2016/071758 for example.
It is understood that it some embodiments, the host cell is contacted with the vector (e.g. viral vector) in vitro, ex vivo, and in some embodiments, the host cell is contacted with the vector (e.g. viral vector) in vivo.
The term host cell includes any cell into which the nucleic acid sequences or vectors described herein may be introduced (e.g. transduced). Once a nucleic acid molecule or vector has been introduced into the cell, it may be referred to as a “modified cell” herein. Once the nucleic acid molecule or vector is introduced into the host cell, the resultant modified cell should be capable of expressing the encoded polypeptide (and e.g. correctly localising the encoded polypeptide for its intended function e.g. transporting the encoded TCR to the cell surface).
The term “modified cell” refers to a genetically altered (e.g. transformed or transfected) cell. The term refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
Although the host cell (and thus the modified cell) may be a bacterial cell, it is typically a eukaryotic cell, and particularly a human cell, more particularly a non-germline human cell (e.g. a T cell such as a CD8+ T cell or a CD4+ T cell, or a mixture thereof),. The host cell (and thus the modified cell) may be an autologous cell (e.g. an autologous T cell such as a CD8+ T cell or a CD4+ T cell, or a mixture thereof), which refers to a cell derived from the same individual to which it is later administered. In other words, the host cell (and thus the modified cell) may be an isolated T cell from a subject to be treated. Suitably, the host cell (and thus the modified cell) may be isolated from a blood sample e.g. by leukaphoresis.
The modified cell may be a dendritic cell.
The modified cell may be a CAR-T cell. CAR T-cells are described elsewhere herein.
The host cell (and thus the modified cell) may be any cell that is able to confer anti-tumour immunity after TCR gene transfer. Non limiting examples of appropriate cells include autologous or allogeneic Natural Killer (NK) cells, NKT cells, gamma-delta T cells, hematopoietic stem cells or other progenitor cells and any other autologous or allogeneic cell or cell line (NK-92 for example or T cell lines) that is able to confer anti-tumor immunity after TCR gene transfer.
Advantageously, the modified cell is capable of expressing the polypeptide encoded by the nucleic acid sequence or vector described herein (e.g. the TCR or TCR component parts, or CAR) such that the modified cell provides an immunotherapy that specifically targets cancerous cells or virally infected cells associated with impaired HLA class I antigen presentation and this can be used to treat or prevent cancer or viral infections associated with impaired HLA class I antigen presentation. More details on this use are given below.
Pharmaceutical compositions
A nucleic acid sequence, vector, modified cell, binding agent, isolated protein or peptide as described herein may be provided as part of a pharmaceutical composition. Advantageously, such compositions may be administered to a human subject in order to treat or prevent a cancer or viral infection associated with impaired HLA class I antigen presentation (e.g. by inducing or enhancing a specific immune response to such cancerous or virally infected cells).
In a particular example, the binding agent is an antibody that specifically binds to a peptide comprising the amino acid sequence of any one of SEQ ID NO:1 to 16. In one example, the antibody specifically binds to an epitope generated by the peptide itself. In another example, the antibody binds to an epitope generated by the combination of the peptide and the HLA molecule that presents it (i.e. an epitope that is generated when the peptide is presented on the cell surface by HLA class I, e.g. HLA*0201).
The terms “pharmaceutical composition” and “composition” are used interchangeably herein, unless the context specifically requires otherwise.
A pharmaceutical composition may comprise a nucleic acid sequence, vector, modified cell, binding agent or isolated protein or peptide described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
For the avoidance of doubt, the nucleic acid sequence, vector, binding agent or isolated peptide may be present in the pharmaceutical composition as part of a cell. In other words, the nucleic acid sequence or vector may be incorporated into a cell; or the binding agent or peptide may be expressed by a cell. The cell may be any suitable cell, for example a bacterial cell, or a eukaryotic cell such as a mammalian cell e.g. a dendritic cell (DC), or a T cell (in such cases, the mammalian cell is typically an ex vivo cell). A pharmaceutical composition comprising a nucleic acid sequence, vector, binding agent or isolated protein or peptide described herein therefore encompasses a pharmaceutical composition comprising a cell (e.g. a bacterial cell, DC, or T cell etc) that encodes the nucleic acid sequence or vector, or is capable of expressing the peptide or binding agent.
Suitably, the cell (e.g. bacterial cell, DC, T cell etc) may be a modified cell that has been modified to introduce into the cell the appropriate nucleic acid sequence/vector (e.g. by transduction, transfection or transformation) such that the modified cell encodes the nucleic acid sequence/vector and becomes capable of expressing the nucleic acid sequence, vector, peptide or binding agent of interest. Such cells may be combined with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier to generate a pharmaceutical composition of the invention. The cell (e.g. DC, T cell etc) may modified ex vivo. For example, it may be an autologous cell that has been derived from the subject that is to be treated with the pharmaceutical composition described herein (e.g. for treating or preventing a cancer or viral infection associated with impaired HLA class I antigen presentation). The cells may be modified ex vivo to introduce e.g. the nucleic acid sequence, or vector into the cell such that the modified cell encodes the nucleic acid sequence/vector and becomes capable of expressing the nucleic acid sequence or vector to generate the peptide or binding agent of interest. The modified cells may then be administered to the subject as a pharmaceutical composition (e.g. as part of an adoptive T cell therapy or equivalent techniques).
Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
As used herein, pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected nucleic acid sequence, vector, modified cell, binding agent or isolated peptide without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a nucleic acid sequence, vector, modified cell, binding agent or isolated peptide as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.
Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
Treatment of a subject
Pharmaceutical compositions described herein may advantageously be used as a medicament. The compositions may be used to treat or prevent a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject. Preferably, the human subject is positive for HLA-A*02:01.
The pharmaceutical compositions for use as a medicament (e.g. in the prevention or treatment of a cancer or a viral infection associated with impaired HLA class I antigen presentation in a human subject) may comprise a nucleic acid sequence, vector, modified cell, binding agent or isolated protein or peptide described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier. As discussed in detail elsewhere herein, this encompasses pharmaceutical compositions comprising cells that encode or express the appropriate nucleic acid sequence, vector, peptide or binding agent.
In a particular example, the binding agent that is useful as a medicament e.g. in the prevention or treatment of a cancer or a viral infection associated with impaired HI_A class I antigen presentation in a human subject) is an antibody that specifically binds to a peptide comprising the amino acid sequence of any one of SEQ ID NO:1 to 16. In one example, the antibody specifically binds to an epitope generated by the peptide itself. In another example, the antibody binds to an epitope generated by the combination of the peptide and the HLA molecule that presents it (i.e. an epitope that is generated when the peptide is presented on the cell surface by HLA class I, e.g. HLA*0201).
The method of treatment or prevention of a cancer or a viral infection associated with impaired HLA class I antigen presentation described herein results in an induced or enhanced immune response (e.g. a cell mediated response) in the subject (e.g. a targeted immune response to cancerous or virally infected cells that present the HLA-A restricted peptide).
The phrase “induced or enhanced immune response” refers to an increase in the immune response (e.g. a cell mediated immune response such as a T cell mediated immune response) of the subject during or after treatment compared to their immune response prior to treatment. An “induced or enhanced” immune response therefore encompasses any measurable increase in the immune response that is directly or indirectly targeted to the cancer or viral infection being treated.
Compositions of the invention may be used to treat or prevent a cancer associated with impaired HLA class I antigen presentation. A person of skill in the art will be fully aware of cancers that are associated with impaired HLA class I antigen presentation and thus may be treated in accordance with the invention.
Suitably, the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma or a lymphoma. Suitably, the cancer is a melanoma.
Compositions of the invention may also be used to treat or prevent a viral infection associated with impaired HLA class I antigen presentation. A person of skill in the art will be fully aware of viral infections that are associated with impaired HLA class I antigen presentation and thus may be treated in accordance with the invention.
As used herein, a cancer or viral infection “associated with impaired HLA class I antigen presentation” refers to a cancer or viral infection that results in a change in the HLA class I antigen presentation pathway in the cancerous or virally infected cell, which results in a reduction in HLA class I antigen presentation in these cells. In this context, a reduction encompasses a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% etc in the presentation of non-TEIPP HLA class l-restricted antigens at the cell surface of these cells (at a given time) compared to control cells (e.g. derived from the same subject that are not cancerous and are not virally infected).
There are several molecular pathways that may be altered in the cancerous or virally infected cell to impair HLA class I antigen presentation. By way of example, it is known that 1-2% of melanomas have deleterious mutations in TAP1 or TAP2, and that a high frequency of metastatic melanomas display low TAP1 expression due to epigenetic silencing 5·7.
A cancer or viral infection associated with impaired HLA class I antigen presentation may therefore be a cancer or viral infection wherein the tumor cells or infected cells have a mutated TAP1 or TAP2 gene. In one example, the mutation reduces TAP1 or TAP2 expression (such that the tumor cell or virally infected cell has low TAP1 or TAP2 expression). In another example, the mutation reduces TAP1 or TAP2 activity in the cell (such that the tumor cell or virally infected cell has reduced/low TAP1 or TAP2 activity). In other example, the mutation reduces TAP1 or TAP2 protein levels in the cell (e.g. the tumor cell or virally infected cell has reduced/low TAP1 or TAP2 protein expression and/or reduced/low TAP1 orTAP2 protein stability).
TAP1 orTAP2 expression may also be reduced/low in a cancerous cell or virally infected cell due to epigenetic silencing. Methods for detecting TAP1 or TAP2 epigenetic silencing are well known in the art.
TAP1 or TAP2 expression, activity, protein level and/or protein stability may also be reduced/low in a cancerous cell or virally infected cell for other reasons than mutation of the TAP1 or TAP2 genes (e.g. due to the cancer/virus altering the molecular machinery and pathways of the cell).
The cancer or viral infection may therefore be a cancer or viral infection associated with reduced (or low) TAP1 orTAP2 protein expression, activity, level, or stability.
Methods for determining the presence of mutations in TAP1 or TAP2 are well known in the art. Furthermore, methods for determining TAP1 or TAP2 expression levels, TAP1 or TAP2 activity levels, TAP1 or TAP2 protein levels, and TAP1 or TAP2 protein stability are well known in the art.
For example, the expression level may be detected by measuring mRNA e.g. using Northern blot analysis or rtPCR). The level of protein may be detected using TAP1 or TAP2 specific antibodies (e.g. with a detectable label) and methods such as enzyme linked immunosorbent assays (ELISAs), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis may be used. Other standard methods for determining these parameters are well known in the art.
As stated above, the cancer or viral infection may be a cancer or viral infection associated with reduced (or low) TAP1 or TAP2 protein expression, activity, level, or stability.
As used herein, “reduced (or low) TAP1 or TAP2 protein expression, activity, level, or stability” refers to a decrease in the protein expression activity, level, or stability compared to a control or a reference level (e.g. at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% decrease). As used herein reference level or control, refers to a cell sample having a normal level of TAP1 or TAP2 protein expression, activity, level, or stability, for example a sample from a healthy subject not having or suspected of having cancer or a viral infection or alternatively a cell sample from the same subject being tested, where the control or reference level cell sample is not (and is not suspected of being) cancerous or virally infected. Alternatively, the reference level may be a TAP1 or TAP2 protein expression, activity, level, or stability value from a reference database, which may be used to generate a pre-determined cut off value, i.e. a diagnostic score that is statistically predictive of a symptom or disease or lack thereof or may be a pre-determined reference level based on a standard population sample, or alternatively, a pre-determined reference level based on a subject's base line level of expression, i.e. prior to developing or being suspected of having cancer or a viral infection. For example, reduced or low protein expression may be determined using immunohistochemistry, using anti-TAP1 or anti-TAP2 antibodies, such as the anti-TAP1 Antibody, clone mAb 148.3 (MABF125 EMD Millipore). In one example, evaluation of TAP1 or TAP2 normal level protein expression in a sample as compared to reduced or low level of expression is determined by the ‘De Ruiter’ evaluation method52. For example, in such a method the sample is a cancer or tumour sample. Alternatively, where the sample is a viral sample, the presence of immune modulatory viral gene products, for example CMV, HSV or BVS, reduces the expression and / or activity of TAP function and the presence of such gene products can be used as a marker of reduced or low TAP1 or TAP2 expression and / or activity.
Other molecular pathways that may be altered in the cancerous or virally infected cell to impair HLA class I antigen presentation include for example a deficiency in tapasin (a chaperone protein involved in TAP-mediated peptide loading of MHC class I molecules) and inhibition of proteasome-mediated degradation of proteins into peptides for MHC class I presentation (see for example US2009/0220534 for more details).
As used herein, the terms “treat”, “treating” and treatment are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (i.e. in this case a cancer or viral infection associated with impaired HLA class I antigen presentation). Accordingly, treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. “Treatment” therefore encompasses a reduction, slowing or inhibition of the amount or concentration of malignant or virally infected cells, for example as measured in a sample obtained from the subject, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the amount or concentration of malignant cells (or virally infected cells) before treatment. Methods of measuring the amount or concentration of malignant cells (or virally infected cells) include, for example, qRT-PCR, and quantification of specific biomarkers, such as peptides comprising the amino acid sequence of one of SEQ ID NO: 1 to 16, in a sample obtained from the subject.
As used here in the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e. a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention. Preferably, the subject is a human subject, preferably a HLA*0201 positive human subject
The compositions described herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.
The compositions described herein may be in any form suitable for the above modes of administration. For example, compositions comprising modified cells may in any form suitable for infusion. As further examples, suitable forms for parenteral injection (including, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion; suitable forms for topical administration include an ointment or cream; and suitable forms for rectal administration include a suppository. Alternatively, the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery. The identification of suitable dosages of the compositions of the invention is well within the routine capabilities of a person of skill in the art.
Advantageously, the compositions of the invention may be formulated for use in T cell receptor (TCR) gene transfer, an approach that is rapid, reliable and capable of generating large quantities of T cells with specificity for the target peptide (e.g. a peptide comprising an amino acid sequence of one of SEQ ID NO: 1 to 16), regardless of the patient’s pre-existing immune repertoire. Using TCR gene transfer, modified autologous cells suitable for infusion may be generated within a few days.
Advantageously, the compositions of the invention may be formulated for use as a vaccine (e.g. a composition comprising one or more peptides comprising an amino acid sequence of one of SEQ ID NO: 1 to 16 (or the corresponding nucleic acid sequence or vector) may be formulated as a pharmaceutical composition that is suitable for use as a peptide vaccine), or a composition comprising a binding agent specific for a peptide comprising an amino acid sequence of one of SEQ ID NO: 1 to 16 may be formulated as a pharmaceutical composition that is suitable for use as a vaccine (e.g. an antibody vaccine). Alternatively, compositions comprising cells may also be formulated as pharmaceutical compositions that are suitable for use as a vaccine. Suitable cell, binding agent (e.g. antibody), peptide and nucleic acid vaccine formulations are well known in the art.
The pharmaceutical composition is preferably for, and therefore formulated to be suitable for, administration to a subject, preferably a human or animal subject. Preferably, the administration is parenteral, e.g. intravenous, subcutaneous, intramuscular, intradermal intracutaneous and/or intratumoral administration, i.e. by injection.
Preferably, the pharmaceutical composition comprises or consists of an amount of active ingredient (e.g. nucleic acid sequence, peptide, vector, binding agent, or cell) that constitutes a pharmaceutical dosage unit. A pharmaceutical dosage unit is defined herein as the amount of active ingredients (i.e. the total amount of peptide in a peptide-based vaccine for example) that is applied to a subject at a given time point. A pharmaceutical dosage unit may be applied to a subject in a single volume, i.e. a single shot, or may be applied in 2, 3, 4, 5 or more separate volumes or shots that are applied preferably at different locations of the body, for instance in the right and the left limb. It is to be understood herein that the separate volumes of a pharmaceutical dosage may differ in composition, i.e. may comprise different kinds or composition of active ingredients and/or adjuvants.
A single injection volume or shot (i.e. volume applied on one location at a certain time point), comprising a total pharmaceutical dosage, or part thereof in case multiple shots applied at substantially the same time point, may between 100 and 2 mL, or between 100 and 1 mL. The single injection volume may be 100 pl, 200 pl, 300 pl, 400 μΙ, 500 μΙ, 600 μΙ, 700 μΙ, 800 μΙ, 900 μΙ, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 3 mL or any value in between.
The pharmaceutical dosage unit, or total amount of active ingredient applied to a subject at a given time point will depend on the type of vaccine (e.g. peptide, cell, nucleic acid etc). As an example, the pharmaceutical dosage unit, or total amount of peptide applied to a subject at a given time point, either in a single or in multiple injections at a certain time point, comprises an amount of peptide in the range from 0.1 pg to 20 mg, such as about 0.1 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 15 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850 pg, 900 pg, 1 mg, 1 ,5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg , 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg , 9 mg , 9.5 mg, 10 mg, 15 mg or about 20 mg or any value in between. Preferred ranges of pharmaceutical dosage units are from 0.1 pg to 20 mg, 1 pg to 10 mg, 10 pg to 5 mg, 0.5 mg to 2 mg, 0.5 mg to 10 mg or I mg to 5 mg or 2 to 4 mg.
The compositions described herein are for administration in an effective amount. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient/subject. For example, the suitable dosage of the composition of the invention for a given patient/subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the composition of the invention for example severity and type of haematological malignancy, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient/subject. Effective dosages may be determined by either in vitro or in vivo methods.
The compositions of the present invention are advantageously presented in unit dosage form.
Methods of generating TCRs
In one aspect, the invention provides a method of generating a T cell receptor that specifically binds to a peptide comprising an amino acid sequence selected from SEQ ID NO: 1 to 16, the method comprising contacting a nucleic acid sequence of the invention (or vector) with a host cell under conditions in which the nucleic acid sequence (or vector) is incorporated and expressed by the cell to generate the T cell receptor.
The method may be carried out on the host cell ex vivo or in vitro. Alternatively, the method may be performed in vivo, wherein the nucleic acid sequence (or vector) is administered to the subject and is contacted with the host cell in vivo, under conditions in which the nucleic acid sequence is incorporated and expressed by the host cell to generate the T cell receptor. In one embodiment, the method is not a method of treatment of the human or animal body.
Appropriate in vivo, in vitro and ex vivo methods for contacting a nucleic acid sequence (or vector) with a host cell under conditions in which the nucleic acid sequence (or vector) is incorporated and expressed by the cell are well known, as described elsewhere herein.
General definitions
As used herein “nucleic acid sequence”, “polynucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably to refer to an oligonucleotide sequence or polynucleotide sequence. The nucleotide sequence may be of genomic, synthetic or recombinant origin, and may be double-stranded or single-stranded (representing the sense or antisense strand). The term nucleotide sequence includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g. mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs.
As used herein, “isolated nucleic acid sequence” refers to a nucleic acid sequence that is not in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. In other words, an isolated nucleic acid sequence is not a native nucleotide sequence, wherein native nucleotide sequence means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment.
As used herein, “specifically binds to FLGPWPAAS” refers to selective binding of the FLGPWPAAS peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to FLGPWPAAS” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to FLGPWPAAS with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell orT cell expressing a TCR specific for FLGPWPAAS). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting FLGPWPAAS in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting FLGPWPAAS in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present FLGPWPAAS in the context of HLA-A*02:01. The selective binding may be in the context of FLGPWPAAS presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to FLGPWPAAS” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to LTLLGTLWGA” refers to selective binding of the LTLLGTLWGA peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to LTLLGTLWGA” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to LTLLGTLWGA with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for LTLLGTLWGA). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting LTLLGTLWGA in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting LTLLGTLWGA in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present LTLLGTLWGA in the context of HLA-A*02:01. The selective binding may be in the context of LTLLGTLWGA presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to LTLLGTLWGA” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to SVLWLGALGL” refers to selective binding of the SVLWLGALGL peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to SVLWLGALGL” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to SVLWLGALGL with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for SVLWLGALGL). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting SVLWLGALGL in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting SVLWLGALGL in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present SVLWLGALGL in the context of HLAA*02:01. The selective binding may be in the context of SVLWLGALGL presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to SVLWLGALGL” may only do so when it is being presented (i.e. it is bound by) HLAA*02:01, or is in an equivalent structural formation as when it is being presented by HLAA*02:01.
As used herein, “specifically binds to VIIKPLVWV” refers to selective binding of the VIIKPLVWV peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to VIIKPLVWV” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to VIIKPLVWV with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for VIIKPLVWV). In assays such as, for example, an assay
100 discussed herein, the modified cell is specifically reactive against a cell presenting VIIKPLVWV in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting VIIKPLVWV in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present VIIKPLVWV in the context of HLA-A*02:01. The selective binding may be in the context of VIIKPLVWV presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to VIIKPLVWV” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to LLALAAGLAV” refers to selective binding of the LLALAAGLAV peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to LLALAAGLAV” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to LLALAAGLAV with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for LLALAAGLAV). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting LLALAAGLAV in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting LLALAAGLAV in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present LLALAAGLAV in the context of HLA-A*02:01. The selective binding may be in the context of LLALAAGLAV presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to LLALAAGLAV” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to FLSELQYYL” refers to selective binding of the FLSELQYYL peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to FLSELQYYL” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to FLSELQYYL with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for FLSELQYYL). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive
101 against a cell presenting FLSELQYYL in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting FLSELQYYL in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present FLSELQYYL in the context of HLA-A*02:01. The selective binding may be in the context of FLSELQYYL presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to FLSELQYYL” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to VLLDHLSLA” refers to selective binding of the VLLDHLSLA peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to VLLDHLSLA” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to VLLDHLSLA with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for VLLDHLSLA). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting VLLDHLSLA in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting VLLDHLSLA in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present VLLDHLSLA in the context of HLA-A*02:01. The selective binding may be in the context of VLLDHLSLA presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to VLLDHLSLA” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to ALFSFVTAL” refers to selective binding of the ALFSFVTAL peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to ALFSFVTAL” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to ALFSFVTAL with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for ALFSFVTAL). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting ALFSFVTAL in the context of HLA-A*02:01. Thus, the modified cell
102 may bind to a cell presenting ALFSFVTAL in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present ALFSFVTAL in the context of HLA-A*02:01. The selective binding may be in the context of ALFSFVTAL presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to ALFSFVTAL” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to VLAVFIKAV” refers to selective binding of the VLAVFIKAV peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to VLAVFIKAV” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to VLAVFIKAV with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for VLAVFIKAV). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting VLAVFIKAV in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting VLAVFIKAV in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present VLAVFIKAV in the context of HLA-A*02:01. The selective binding may be in the context of VLAVFIKAV presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to VLAVFIKAV” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to LLWGRQLFA” refers to selective binding of the LLWGRQLFA peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to LLWGRQLFA” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to LLWGRQLFA with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for LLWGRQLFA). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting LLWGRQLFA in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting LLWGRQLFA in the context of HLA-A*02:01 with at least
103
10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present LLWGRQLFA in the context of HI_A-A*02:01. The selective binding may be in the context of LLWGRQLFA presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to LLWGRQLFA” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to TLLGASLPA” refers to selective binding of the TLLGASLPA peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to TLLGASLPA” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to TLLGASLPA with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for TLLGASLPA). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting TLLGASLPA in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting TLLGASLPA in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present TLLGASLPA in the context of HLA-A*02:01. The selective binding may be in the context of TLLGASLPA presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to TLLGASLPA” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to LLLDVPTAAV” refers to selective binding of the LLLDVPTAAV peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to LLLDVPTAAV” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to LLLDVPTAAV with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for LLLDVPTAAV). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting LLLDVPTAAV in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting LLLDVPTAAV in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a
104 control cell line that does not present LLLDVPTAAV in the context of HLA-A*02:01. The selective binding may be in the context of LLLDVPTAAV presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to LLLDVPTAAV” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to LLSAEPVPA” refers to selective binding of the LLSAEPVPA peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to LLSAEPVPA” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to LLSAEPVPA with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for LLSAEPVPA). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting LLSAEPVPA in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting LLSAEPVPA in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present LLSAEPVPA in the context of HLA-A*02:01. The selective binding may be in the context of LLSAEPVPA presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to LLSAEPVPA” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to FLYPFLSHL” refers to selective binding of the FLYPFLSHL peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to FLYPFLSHL” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to FLYPFLSHL with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for FLYPFLSHL). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting FLYPFLSHL in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting FLYPFLSHL in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present FLYPFLSHL in the context of HLA-A*02:01. The selective binding
105 may be in the context of FLYPFLSHL presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to FLYPFLSHL” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to ILEYLTAEV” refers to selective binding of the ILEYLTAEV peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to ILEYLTAEV” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to ILEYLTAEV with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for ILEYLTAEV). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting ILEYLTAEV in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting ILEYLTAEV in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present ILEYLTAEV in the context of HLA-A*02:01. The selective binding may be in the context of ILEYLTAEV presentation by HLA-A*02:01. In other words, in certain embodiments, a polypeptide that “specifically binds to ILEYLTAEV” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
As used herein, “specifically binds to LSEKLERI” refers to selective binding of the LSEKLERI peptide. Under certain conditions, for example in an immunoassay as described herein, a polypeptide that “specifically binds to LSEKLERI” will selectively bind to this peptide and will not bind in a significant amount to other peptides. Thus the polypeptide may bind to LSEKLERI with at least 10, 20, 30, 40, 50, or 100 fold more affinity than it binds to a control antigenic peptide. Selective binding may also be determined indirectly in the context of a modified cell that expresses a nucleic acid or vector of the invention (i.e. a CAR T cell or T cell expressing a TCR specific for LSEKLERI). In assays such as, for example, an assay discussed herein, the modified cell is specifically reactive against a cell presenting LSEKLERI in the context of HLA-A*02:01. Thus, the modified cell may bind to a cell presenting LSEKLERI in the context of HLA-A*02:01 with at least 10, 20, 30, 40, 50, or 100 fold more reactivity when compared to its reactivity against a control cell line that does not present LSEKLERI in the context of HLA-A*02:01. The selective binding may be in the context of LSEKLERI presentation by HLA-A*02:01. In other words, in certain embodiments,
106 a polypeptide that “specifically binds to LSEKLERI” may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01, or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
A “non-essential” (or “non-critical”) amino acid residue is a residue that can be altered from the wild-type sequence of (e.g., the sequence identified by SEQ ID NO herein) without abolishing or, more preferably, without substantially altering a biological activity, whereas an “essential” (or “critical”) amino acid residue results in such a change. For example, amino acid residues that are conserved are predicted to be particularly non-amenable to alteration, except that amino acid residues within the hydrophobic core of domains can generally be replaced by other residues having approximately equivalent hydrophobicity without significantly altering activity.
A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential (or non-critical) amino acid residue in a protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly, and the resultant mutants can be screened for activity to identify mutants that retain activity.
Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
107 or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50,
108 wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http://www.ncbi.nlm.nih.gov>.
The polypeptides and nucleic acid molecules described herein can have amino acid sequences or nucleic acid sequences sufficiently or substantially identical to the sequences identified by SEQ ID NO. The terms “sufficiently identical” or “substantially identical” are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.
The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.
Examples
In order to identify multiple human TEIPP antigens, the inventors developed a systematic hybrid forward-reversed immunology screen. This approach encompassed an in silico prediction of TEIPP neoantigen-candidates from the whole human proteome, matching of candidates to the cancer-specific peptidome, and an ex vivo screen to confirm the presence of a TEIPP T ceil repertoire in healthy donors. Here, data on 16 identified HI_A-A*02:01 binding TEIPP epitopes and a full characterization of the T ceil reactivity against one of them is presented.
Results:
Strategy for target identification from the complete human proteome
To identify human TEIPP antigens that are presented by TAP-deficient cancer cells, the inventors developed a hybrid forward-reversed immunology identification approach based on alternative antigen processing rules in combination with cancer-specific peptidome database
109 matching (figure 1a). The whole human proteome was chosen as starting point, since TEIPP antigens are nonmutated ‘self’ antigens that are preferentially displayed on cells with deficiency in the peptide transporter TAP. This TAP-independent loading in HLA-I molecules can occur via two known alternative processing pathways: liberation of N-terminal ‘signal peptides’ and C-terminal ‘tail peptides’19'23 (figure 1a-b). A list of signal peptide-containing proteins was selected from the human proteome with the use of a web-based algorithm that predicts the signal peptidase (SP) cleavage site24. Most of these leader peptides need further processing by the proteolytic enzyme signal peptide peptidase (SPP) in order to release them from the ER-membrane and thereby generating peptides of different lengths suitable for loading in HLA-I19'22. This ‘signal peptide’ search yielded 111,525 different 9- and 10-mer TEIPP candidates. Additionally, C-terminal ‘tail peptides’ were selected using information on the topology of transmembrane proteins in the same web-based algorithm, knowing that liberation and TAP-independent processing of C-termini can be exerted by proteases like furin and SPP 21'25’28. This search resulted in 6,674 ‘tail peptides’ (figure 1a-b). Next, these combined peptides were filtered for a predicted high HLA-I binding-affinity (IC50<500 nM) to one of the so-called HLA ‘supertypes’, representing most alleles in the Caucasian population (HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-B*07:02, HLAB*40:01, HLA-A*24:02)29'32. This final list of 13,731 TEIPP candidates was then crossmatched with a large peptidome database, containing peptides eluted from over 200 tumor samples and 150 healthy tissue control samples. Thereby, the inventors focused on those TEIPP candidates that were endogenously processed and exclusively presented by tumor cells (figure 1c). This strategy yielded a short-list of 65 TEIPP neoantigen-candidates of which 40 had a predicted binding to the HLA-A*02:01 allele (figure 2). These 40 HLAA*02:01 binding peptides were chosen to explore the existence of cognate CD8+ T cells (figure 1a).
Detectable frequencies of CD8+ T cells against 16 out of 40 HLA-A*02:01 binding TEIPP neoantigens
To validate that T cells against these TEIPP neoantigens were present in the repertoire of healthy donors, forty different HLA-A*02:01 tetramers containing the TEIPP neoantigens were grouped in eight pools of five peptides each, based on predicted binding affinity (high in ‘peptide pool T to low in ‘peptide pool 8’). Next, CD8+ T cell cultures were started with tetramer-assisted ‘pull-downs’ from total PBMC. These enriched CD8+ T cell pools were stimulated with the five respective synthetic peptides of that particular pool for several rounds and analyzed for presence of peptide-specific T cells by flow cytometry (figure 3a). In an initial screen of three different HLA-A*02:01-positive healthy donors T cell repertoires for 16 out of 40 TEIPP neoantigen candidates were observed (figure 3a). Frequencies of
110 tetramer-stained CD8+ T cells varied from 0.1% up to 78% in these short-term expanded cultures. These percentages differed between the three tested donors, indicating that this protocol enabled the detection of TEIPP-specific CD8+ T cells but that the observed frequencies reflected variation of the in vitro steps of enrichment and expansion. Additional donors were examined for the 16 positive peptides to evaluate the consistency of this finding and against most peptides CD8+ T cells were detected, in the majority of the donors (figure 3b and Table 1). Collectively, these data indicated that the TEIPP identification strategy had a high success rate to identify human antigens to which a CD8+ T cell repertoire existed in healthy individuals.
Most TEIPP-specific CD8+ T cells reside in the naïve repertoire
Previous investigations in the inventors’ mouse tumor model of TEIPP showed that TEIPPspecific T cells remain naive, even in the presence of TAP-deficient tumor cells16. This was shown to be an advantage for their exploitability in therapeutic interventions16. To test this aspect for human TEIPP-specific T cells, tetramer-positive CD8+T cells were enriched from PBMC for the five peptides p14, p29, p34, p35 and p55 that were frequently positive in our screen. Naive CD8+ T cells were directly separated from antigen-experienced CD8+ T cells by flow cytometry sorting, based on differentiation markers CD62L and CD45RA at the cell surface (figure 4a). After this ex vivo sorting, the two populations were expanded by three rounds of in vitro stimulation and analyzed for the presence of tetramer-positive CD8+ T cells (figure 5a-b). Importantly, T cells against p14, p34 and p35 were exclusively found in the sorted naive population in all tested donors, indicating that these specificities had never encountered cognate antigen in these healthy individuals. In addition, T cells against p29 and p55 were found in the naive subset of some donors while in other donors, these specificities were detected in the antigen-experienced repertoire (figure 5b). These data indicated that some TEIPP-specific CD8+ T cells already had been primed in a few healthy individuals, but that the majority of them resides in a naive state. The inventors speculate that this priming could have been triggered by bacterial species in the gut with antigens that show striking sequence similarity with peptide 29 and 55 (figure 4b). In summary, these data demonstrate a universal existence of naive T cells with TEIPP specificity in healthy individuals.
Functional avidity of clonal CD8+ T cell cultures
To functionally study TEIPP-specific CD8+ T cells, clonal cultures against the five TEIPP peptides p14, p29, p34, p35, and p55 were generated. Four of these peptides were derived from N-terminal signal sequences of proteins and one was derived from a C-terminal ‘tail peptide’ (figure 4c). The binding affinity and stability of these five peptides to HLA-A*02:01
111 was evaluated, since these determinants are critically involved in immunogenicity for T cells33’34. Four of the five peptides displayed an intermediate affinity and formed complexes with HI_A-A*02:01 for at least four hours, however, p29 surprisingly failed to show binding in these cellular assays (figure 4d-e). Multiple T cell clones were generated using an antigenindependent expansion protocol of tetramer-positive CD8+ T cells after single cell sorting. Since the functional avidity correlates with high affinity TCRs and thus for in vivo efficacy35-38, the peptide concentrations resulting in half maximum cytokine response (EC50) of the several TEIPP-specific T cell clones when exposed to antigen-presenting cells pulsed with titrated concentrations of cognate peptide were assessed (figure 5c). All isolated T cell clones exhibited a functional avidity between 0.4-66 nM, with the highest avidity for clones recognizing p34 and p55. The other clones, like those against p14, were considered to display moderate avidity. Of note, all clones with the same peptide specificity used different TCRp V-segments, pointing at their independent origin and the availability of a broad repertoire (figure 4f). These results demonstrated that the broad repertoire of TEIPP-specific T cells in healthy individuals exhibits an usual functional avidity for their cognate peptide and is competent to respond upon antigen encounter. To control for artificial CRISPR/CAS9induced immunogenicity, a previously established T cell clone (HSS1 PRAME) that recognizes the TAP-dependent PRAME (SLLQHLIGL) tumor antigen was tested. This PRAME425-433-specific CD8+ T cell only recognized 518A2 WT cells whereas it completely failed to respond towards the TAP KO variant. In contrast, LRPAPl2i-3o-specific T cells displayed clear preference for the TAP KO variant (figure 4g).
CD8+ T cells against p14 recognize TAP-deficient tumors across diverse histological origin In search for TEIPP antigens on immune-escaped cancers, the inventors selected the p14 peptide-epitope with sequence FLGPWPAAS (SEQ ID NO:1) from the ubiquitously expressed LRPAP1 protein (from now on called LRPAP21.30) for in depth examination (Table 1). First, the inventors confirmed specificity for the LRPAP21-30 epitope by testing different peptide length variants (figure 6a). Although unconventional, this examination confirmed the serine amino acid as C-terminus for this HLA-A*02:01 allele. Next, LRPAP1 gene expression data was collected from the TOGA database for different tumor types, revealing that its expression is ubiquitous with exceptionally high expression levels in skin melanomas (figure 7a). The inventors therefore selected the HI_A-A*02:01 positive skin melanoma cell line ‘518A2’ and confirmed the expression of the LRPAP1 gene (figure 7b, figure 6b). For this cell line an TAP1 knock-out variant and an TAP1/antigen double knock-out variant were generated by CRISPR/CAS9 technology and these gene edited variants were tested for recognition by LRPAP2i-3o-specific CD8+ T cell clones (figure 7b, figure 6c-d). Surface
112 display of HLA-A*02:01 strongly decreased after knocking out the gene for the peptide transporter TAP1 (figure 7c). The LRPAP2i-3o-specific CD8+T cells selectively recognized the TAP1-deficient variant of 518A2. This suggested that the LRPAP21-30 signal peptide was only presented by the TAP-deficient tumor variant, despite equal expression of the LRPAP1 gene and lower HLA-A*02:01 levels on these cells (figure 7d). Furthermore, selective knock-out of the epitope (‘LRPAP21-30 Ag-KO’) resulted in complete abrogation of T cell recognition (figure 7d). This confirmed that the applied tetramer-based selection approach yielded functional CD8+ T cells with a bona fide specificity for the antigen from which the TEIPP was derived. Moreover, these data excluded potential cross-reactivity of this T cell clone against other peptides presented by the 518A2 melanoma cells. Finally, antibodies against HLA-I molecules were able to block the tumor recognition by T cells, demonstrating dependency on peptide/HLA-l (figure 7e).
The ubiquitous expression of the LRPAP1 gene in diverse tumor types prompted the inventors to test T cell recognition of other HI_A-A*02:01 positive melanomas and tumors of other histological origin, including renal cell carcinoma and lymphoma. The panel of T1 and T2 lymphoma cells are known for their characterized genomic TAP status (figure 6e)39·40. Equal LRPAP1 gene expression was confirmed by quantitative PCR (figure 6f) and examined recognition by multiple LRPAP2i-3o-specific T cell clones (figure 7f). All tested CD8+ T cell clones selectively recognized the TAP-deficient T2 cells, again displaying a typical TEIPP specificity.
Next, a panel of HI_A-A*02:01 positive TAP-deficient melanomas and renal cell carcinomas was generated by genetically disrupting the TAP1 gene. Tumor cell mRNA levels of the LRPAP1 gene were again comparable as quantified by PCR (figure 6g). Flow cytometry analysis revealed that HI_A-A*02:01 levels varied between the lines, but that these decreased after TAP1 gene knockout (figure 7g). As observed for the 518A2 melanoma and the T2 lymphoma, multiple LRPAP2i-3o-specific T cell clones exhibited a preferential recognition of the TAP1-deficient tumor lines (figure 8). Interestingly, some parental tumors were already recognized even before silencing the TAP1 gene (e.g. melanoma 93.04), implying that the endogenous TAP1 levels were already lower expressed to allow presentation of the LRPAP21-30 antigen. Indeed, quantitative PCR confirmed low TAP1 gene expression in the melanoma 93.04 (figure 6h). In line with this, complete abolishment of the TAP1 gene in tumor line 93.04 did not result in much improved T cell recognition (figure 8). Overall, these data clearly demonstrated that the LRPAP21-30 TEIPP epitope is universally presented in HLA-A*02:01 by cancers of diverse histology when the peptide transporter
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TAP1 is not functional. TEIPP specific CD8+ T cells specific for this TEIPP antigen enables targeting of this shared, nonmutated peptide on immune-escaped HLA-I|OW cancers.
The inventors used quantitative PCR to demonstrate LRPAP1 mRNA expression levels in two colon cancer cell lines, CaCO2 and Colo205 (figure 11a). TAP1 knock-out variants for these cell lines were generated as described above. Flow cytometry analysis revealed that HLA-A*02:01 levels varied between the colon cancer cell lines, but that these decreased after TAP1 gene knockout (figure 11b). As observed for the 518A2 melanoma and the T2 lymphoma, T cell clone 1A8 exhibited a preferential recognition of both of the the TAP1deficient colon carcinoma cell lines (figure 8).
Targeting TEIPP antigens is safe despite ubiquitous expression of their proteins in healthy cells
The fact that human TEIPP antigens derive from ubiquitously expressed nonmutated proteins may rise concerns for auto-immune pathology. Indeed, the LRPAP1 protein is clearly expressed in several critical organs of our body, as revealed by tissue slide staining images available from proteinatlas.org (figure 9a, figure 10a)41. Therefore, two primary melanocyte cultures and two immortalized kidney cell lines were tested for CD8+ T cell recognition. These non-transformed cells contained similar high levels of LRPAP1 transcripts as the 518A2 tumor (figure 9b) and displayed sufficient levels of HLA-A*02:01 at their cell surface (figure 9c). Despite these optimal conditions, LRPAP2i-3o-specific T cells did not respond to these healthy cells (figure 9d). These data confirmed a crucial role for low TAP1 function for the presentation of TEIPP antigens and suggested that TEIPP targeting can be considered a safe therapy, even though these proteins are present in healthy tissues as well. In summary, the data reveal an array of novel nonmutated tumor antigens displayed by tumors with (partial) TAP deficiency (Table I). Some of these are tissue restricted, but others, like the LRPAP1 signal peptide, are ubiquitously expressed and represent universal tumor antigens.
Discussion:
The inventors developed a screening approach which enabled them to identify human antigens belonging to the novel category of non-mutated neoantigens of ‘self’ origin, called TEIPP. The screen yielded 65 potential TEIPP candidates, which are exclusively present in the peptidome of cancer cells. Analysis of the 40 HLA-A*02:01 binding TEIPP candidates revealed a consistent CD8+ T cell repertoire to 16 of these 40 peptides. Additional analyses demonstrated that these CD8+ T cells were present in the naive pool for five peptidecandidates and that they were not affected by central or peripheral tolerance. Monoclonal T
114 cell cultures against the TEIPP epitope LRPAP21-30 selectively recognized TAP1-deficient cancers from different histological origins, including lymphoma, melanoma and renal cell carcinoma, whereas TAP1-proficient primary cells were spared from T cell recognition. This emphasizes the attractiveness of targeting TEIPP neoantigens on HLA-I|OW cancers, namely their universal character.
The inventors observed that knocking out the TAP1 gene rendered all tested cancer cell lines vulnerable for TEIPP-specific T cells. Importantly, TEIPP-specific T cells exhibited moderate to high recognition of some wild-type skin melanomas and renal cell carcinomas (e.g. 93.04, mz1857), and CRISPR/CAS9 mediated knockout of the TAP1 gene only led to modest improvement of cancer cell recognition. In other cancer lines (e.g. melanoma 518A2, 08.11), minimal T cell responses were detected to wild-type cancer cells and knock-out of TAP1 was necessary to induce strong T cell recognition. These data suggested that some cancer cell lines already down modulated TAP1 gene expression in such degree that was already sufficient for the presentation of TEIPP neoantigens. Although only 1-2% of melanomas have deleterious mutations in TAP1 or TAP2, a high frequency of metastatic melanomas display low TAP1 expression due to epigenetic silencing 5·7. The inventors’ previous experiments in mouse models suggested a competition between peptide antigens in the endoplasmic reticulum38·42. The peptide loading complex (PLC) is a multisubunit membrane complex orchestrating the loading and editing of peptides in HLA-I molecules, facilitated by the TAP1/TAP2 channel, and thereby responsible for the translocation of peptides from the cytosol into the PLC complex43. The TAP peptide transporter is incorporated in this efficient peptide loading complex, in close proximity to HLA-I molecules, which could explain why peptides processed through different mechanisms, independent of TAP, will not have the chance to bind in HLA-I molecules. Besides TAP levels, the amount of available antigenic protein also needs to be taken into account. Interestingly, massive overexpression of a TEIPP antigen makes it possible to present its TEIPP peptide in the cell surface even in TAP-proficient cells42. Without wishing to be bound by theory, the inventors believe that these two factors govern the efficiency of TEIPP neoantigen presentation. Low TAP function allows for low antigen expression, whereas moderate TAP function needs higher amounts of antigen expression18·42.
The presence of a TEIPP T cell repertoire in healthy donors indicates that no negative selection has occurred in the thymus, even though gene expression in the thymus was observed for most of the TEIPP neoantigen-candidates (figure 10b). During T cell education in the thymus TEIPP-specific T cells are positively selected for low affinity interaction with peptide/MHC in the cortex of the thymus. To prevent auto-immune responses, negative
115 selection of T cells in the medulla of the thymus is essential to delete self-reactive T cells. This strengthens the hypothesis that TAP-proficient medullary thymic epithelial cells (mTECs) might express the target proteins but do not present TEIPP antigens by their HLA-1 and thus TEIPP T cells will undergo normal thymic selection. Indeed, TEIPP-specific T cells are deleted from the repertoire of TAP-deficient mice15. Moreover, most of the evaluated TEIPP-specific T cells were only found in the naive pool in the circulation and have not encountered antigen yet, signifying that TAP-proficient healthy cells did not trigger TEIPPspecific immunity. These data mirror earlier observations in our mouse model for TEIPP16. Noteworthy, in some donors it was observed that T cells against p29 and p55 were derived from the antigen-experienced pool. The inventors speculate that these TEIPP specificities had been triggered by cross-reactivity to microbiota-derived epitopes leaked from the gut, which can occur after the use of antibiotics or other intestinal injury44·45. Since the tested healthy donors did not suffer from severe auto-immune reactivity, even antigen-experienced TEIPP-specific T cells apparently do not target healthy TAP1-proficient tissues. The safety of TEIPP antigens was confirmed in mouse models in which active tumor targeting towards TEIPP antigens cleared the tumor without any sign of autoimmune reactivity16.
TEIPP neoantigens are selectively presented in cancer cells with deficient function of MHC-I processing machinery. Therefore, the inventors anticipate a complementary role for these antigens to those of other categories, like patient-specific mutanome peptides or cancertestis antigens. A combination of those might prevent immune-escape via shutdown of HLA-I expression due to defects in the antigen processing machinery. A great asset of TEIPP antigens is their non-mutated status and the fact that they are real universal tumor antigens across many cancer types. A few validated TEIPP antigens or gene transfer of their cognate cloned T cell receptors might be sufficient for increased efficacy of immunotherapy through prevention of tumor escape. Future investigations need to reveal safety and applicability of vaccination-based strategies and TCR-based gene transfer approaches for TEIPP. Together, the inventors propose that TEIPP neoantigens are an interesting way to reestablish anti-tumor responses in human immune-escaped cancers.
Material and Methods:
Study design
The aim of this study was to test a systematic hybrid forward-reversed immunology approach to identify TEIPP specific T cells and their cognate antigen (Table I). For this, the whole human proteome dataset from uniprot.org (ID: 9606, Release: 2014_06) was collected. For predictions of the N-terminal “signal” cleavage location and the topology prediction for C-terminal “tail” peptides the web-based topology algorithm Phobius was used
116 (htpp://www.phobius.edu/). For N-terminal peptide candidates, proteins with a predicted signal peptide probability of >0.5 were included. For C-terminal peptide candidates, proteins with a predicted C-terminal “tail” located in the ER were included. Peptide binding affinity to HLA-I molecules were predicted using MHCnet4.0 (http://www.cbs.dtu.dk/). Peptides with a predicted binding affinity <500 nM were included. The peptidome elution library (release 2014_10) of the Tübingen group (Department of Immunology, Tübingen) to select for biologically processed peptides was used. Next, in vitro analysis was performed on forty HLA-A*02:01 TEIPP candidates using healthy donor PBMC. In depth analysis was performed on five TEIPP candidates. To guarantee statistical power, all experiments were performed on at least three different PBMC batches isolated from different donors.
Tumor cell lines
All tumor cell lines were cultured at 37°C, 5% CO2, in DMEM medium (Invitrogen, Carlsbad, CA) containing 8% heat-inactivated FCS, 100 U/mL penicillin, 100 pg/mL streptomycin (Life Technologies, Rockville, MD) and supplemented with 2 mM glutamine (Invitrogen).
Generation of TEIPP specific T-cell clones
Buffy coats from healthy donors were obtained from Sanquin (Sanquin blood facility). PBMC’s were isolated using a ficoll gradient (density 1.077 g/mL). Approximately 500x106 PBMC’s were incubated with PE-pHLA-A*02:01 tetramers for 30 min at 4°C. Anti-PE magnetic beads (MACS) were used to pull out pHLA-A*02:01 tetramer positive cells over a MACS LS column as instructed by the manufacturer (Miltenyi Biotech). T cells were cultured in complete IMDM containing 8% human serum (Sanquin blood facility), and 100 U/mL IL-2 (Proleukine, Novartis). Bulk T cells were stimulated every two weeks with a mixture of T cells (1x106), 10 μΜ synthetic peptide, irradiated PBMCs (1x106 cells, 80Gy) and EBV-JY (1x105 cells, 100Gy) in complete T cell culture medium supplemented with 100 U/mL IL-2 in 24-well plates (Costar). Culture medium was replenished every 2 to 3 days with fresh complete T cell medium. After two rounds of specific stimulation, the polyclonal T cell bulk was incubated with PE labeled tetramers and single cell sorted using flow cytometry to obtain monoclonal T cells. After monoclonal cell sorting, T cell clones were stimulated with a phytohemagglutinin (PHA) mixture (complete T cell medium supplemented with 800 ng/mL PHA (Murex Biotech) and 100 U/mL IL-2) together with irradiated PBMCs (50x105 cells, 80Gy) and EBV-JY cells (10x104 cells, 100Gy) per 96-well.
Generation of pH LA-A*02:01 tetramers
Ultra-pure peptides were synthesized by JPT peptide technology. Recombinant HLAA*02:01 and human β2ιτι proteins were in-house synthesized in Escherichia co//46·47.
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Biotinylated HLA-A*02:01 molecules were produced containing an ultraviolet (UV)-sensitive peptide to enable easy transfer of specific peptide, as previously described48. In brief, HLAA*02:01 complexes were purified by gel filtration using Fast protein liquid chromatography (FPLC). pHLA-A*02:01 complexes were exposed to UV light to facilitate peptide exchange resulting in peptide specific monomers. pHLA-A*02:01 tetramers were formed by coupling biotinylated pHLA-A*02:01 monomers with streptavidin-coupled R-phycoerythrin conjugate (PE, ThermoFisher). Tetramers were stored at -80°C for long term storage or 4°C for short term storage.
Co-culture T cell reactivity assay
IFN-γ (Sanquin Pelikine) or GM-CSF (MabTech) ELISA assays were performed to measure the production of activated T cells. ELISA plates were coated with human IFN-γ or GM-CSF primary antibody overnight at 4°C. T cell conditioned medium was collected incubated in the pre-coated ELISA plates for two hours at room temperature. Next, ELISA plates were washed with PBS-T followed by incubation with the HRP conjugated secondary antibody for one hour. ELISA plates were developed with TMB (3,3,5,5-tetramethyl-benzidine liquid substrate supersensitive, for ELISA; Sigma-Aldrich) and measured at 450 nm using a plate reader (Spectramax id3, Molecular Devices).
Affinity and stability assay
Peptide affinity and stability were determined as previously described3449. In brief, HLAA*02:01+ T2 cells were pulsed with increasing concentrations of peptide (1 uM-100 uM) in serum free RPMI culture medium for 24 hours at 37°C. For MHC:peptide affinity, the maximum HLA-A*02:01 expression was measured by flow cytometry to calculate the EC50 concentration. For MHC:peptide stability, HLA-A*02:01 positive T2 cells were pulsed with 100 uM peptide for 24 hours at 37°C. Maximum HLA-A*02:01 expression was determined and followed over time to calculate the T-1/2 value.
Generation of gene knockouts in tumor cells using CRISPR CAS9 sgRNAs were designed to target exon 1 of the human TAP1 gene (sgTAPI: GCTGCTACTTCTCGCCGACT; SEQ ID NO: 33) or the LRPAPI21-30 epitope located in exon 1 (LRPAPI21.30: AGGGTCAGGTCGTTTCTGCG; SEQ ID NO:34). The sgRNA target sequence was cloned into the lentiCRISPR v2 vector50. Virus particles were generated by co-transfecting sgRNA/CAS9 containing plasmid together with PAX2/pMD2.G packaging vectors into HEK293T cells using lipofectamine 2000 (ThermoFisher). Tumor cells were incubated with medium containing the virus particles for 24 hours and transduced tumor cells
118 were selected with 1-5 ug/mL puromycin (Gibco). TAP1 knockout-efficiency was analyzed by measuring surface HLA-ABC (w6/32, Biolegend) expression using flow cytometry. Polyclonal TAP knockout cell lines were generated by FACS sorting the HLA-I|OW cell population. TAP1 knockout was verified by Western-Blot using anti-TAP1 antibodies (Cell Signaling).
Western Blot
Proteins from cell cultures were extracted in lysis buffer (NP-40), and protein concentrations were assessed using the Bradford Protein Assay Kit (Thermo Fisher). Protein samples were separated on a 4-12% SDS-polyacrylamide gel electrophoresis gel and transferred onto nitrocellulose membranes using semi-dry transfer (BioRad). To prevent a-specific binding, membranes were blocked with 5% milk in TBS-T for one hour at room temperature. Antibodies used: anti-TAP1 (dilution 1:1000, 4°C5 Cell Signaling), anti- β-actin (dilution 1:1000, 4°C, Cell Signaling), HRP linked mouse anti-Rabbit IgG (dilution 1:5000, RT, Cell Signaling).
Flow cytometry analysis
CD3 (SK7), CD4 (SK3), CD8 (SK1), CD45RO (UCHL1), HLA-A*02:01 (bb7.2) (BD Biosciences), and HLA-ABC (MCA81A), CD45RA (H1100), CD62L (DREG-56) (Biolegend) anti-human mAbs were used for flow cytometry analysis. The νβ Repertoire analysis kit was used to determine the TCR νβ of the different monoclonal T cells (Beckman Coulter). Cells were measured using a LSR-II flow cytometer (BD biosciences) and analyzed using Flowjo software version 10 (Tree Star).
mRNA isolation, cDNA synthesis, and qPCR analysis
Cell pellets were washed twice with PBS and snap frozen in liquid nitrogen. RNA was isolated using the RNAeasy kit (Qiagen), according to manufactures protocol. cDNA was synthesized using the High capacity RNA-to-cDNA kit (Applied Biosystems). Quantitative PCR was done using SybrGreen supermix (Bio-Rad).
Statistical analysis
All data are presented as means and SD. Statistical analysis was done using a paired Student’s t test (two-tailed) to determine the statistical significance of the differences.
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# Gene Peptide1 Accession2 HLA-A23 Expression4 T cell
Name Sequence Number Affinity — Repertoire
(IC50)
Tissue5 Normal Cancer
Ubiquitous TEIPP
pl ERGIC3 FLSELQYYL Q9Y282 2 All ++ ++ 4/7
p4 TTYH3 ALFSFVTAL Q9C0H2 10 All + + 1/3
p9 IGJ VLAVFIKAV P01591 52 All ++ ++ 1/3
p!4 LRPAP1 FLGPWPAAS D6REW6 353 All ++ ++ 12/12
p29 ARSA LLALAAGLAV P15289 79 All ++ ++ 9/10
p44 EMILIN2 FLYPFLSHL Q9BXX0 4 All + ++ 4/7
p49 H2AFV ILEYLTAEV E5RJU1 30 All ++ ++ 6/9
p67 SEP1S LSEKLERI 060613 15 All + + 4/8
Tissue Specific TEIPP
p2 IL12A VLLDHLSLA P29459 7 Mixed + + 3/7
pl7 HPR LLWGRQLFA P00739 24 Liver +++ + 6/7
pl8 FSTL4 TLLGASLPA Q6MZW2 27 Mixed + +++ 5/7
p30 IFI30 LLLDVPTAAV M0QZG3 10 Mixed ++ ++ 5/8
p32 CD79S LLLSAEPVPA P40259 41 Lymphoid + ++ 3/7
p34 PCDHGAS LTLLGTLWGA Q9Y5G7 24 Mixed +/- +/- 8/8
p35 C20orfl41 SVLWLGALGL Q9NUB4 161 Testis + + 8/9
p55 APCS VIIKPLVWV P02743 113 Liver +++ +++ 9/10
Table 1: Overview TEIPP neoantigens
Peptide sequence based on predicted signal peptide cleavage site 2 Accession number uniprot.org 3 Predicted peptide affinity binding in HLA-A*02:01 4 Protein expression scores based on data proteinatlas.org 5 Protein expression based on TCGA database score
Nucleic acid and amino acid sequences presented herein are summarised as follows:
SEQ ID NO: 1 - FLGPWPAAS (also referred to as p14 herein)
SEQ ID NO: 2 - LTLLGTLWGA (also referred to as p34 herein)
SEQ ID NO: 3 - SVLWLGALGL (also referred to as p35 herein)
SEQ ID NO: 4 - VIIKPLVWV (also referred to as p55 herein)
SEQ ID NO: 5 - LLALAAGLAV (also referred to as p29 herein)
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SEQ ID NO: 6 - FLSELQYYL (also referred to as p1 herein)
SEQ ID NO: 7 - VLLDHLSLA (also referred to as p2 herein)
SEQ ID NO: 8 - ALFSFVTAL (also referred to as p4 herein)
SEQ ID NO: 9 - VLAVFIKAV (also referred to as p9 herein)
SEQ ID NO: 10 - LLWGRQLFA (also referred to as p17 herein)
SEQ ID NO: 11 - TLLGASLPA (also referred to as p18 herein)
SEQ ID NO: 12 - LLLDVPTAAV (also referred to as p30 herein)
SEQ ID NO: 13 - LLSAEPVPA (also referred to as p32 herein)
SEQ ID NO: 14 - FLYPFLSHL (also referred to as p44 herein)
SEQ ID NO: 15 - ILEYLTAEV (also referred to as p49 herein)
SEQ ID NO: 16 - LSEKLERI (also referred to as p67 herein)
SEQ ID NO: 17 (TCRa) - cggaaggaggtggagcaggatcctggacccttcaatgttccagagggagccactgtcgct ttcaactgtacttacagcaacagtgcttctcagtctttcttctggtacagacaggattgc aggaaagaacctaagttgctgatgtccgtatactccagtggtaatgaagatggaaggttt acagcacagctcaatagagccagccagtatatttccctgctcatcagagactccaagctc agtgattcagccacctacctctgtgtggtgatgggctatggtcagaattttgtctttggt cccggaaccagattgtccgtgctgccct
SEQ ID NO: 18 (TCRa)RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGR FTAQLNRASQYISLLIRDSKLSDSATYLCVVMGYGQNFVFGPGTRLSVLP
SEQ ID NO: 19 (CDR1 TCRa) - aacagtgcttctcagtct
SEQ ID NO: 20 (CDR1 TCRa) - NSASQS
SEQ ID NO: 21 (CDR2 TCRa) - gtatactccagtggtaat
SEQ ID NO: 22 (CDR2 TCRa) - VYSSGN
SEQ ID NO: 23 (CDR3 TCRa) - gtggtgatgggctatggtcagaattttgtc
SEQ ID NO: 24 (CDR3 TCRa) - VVMGYGQNFV
SEQ ID NO: 25 (TCRb) - ggtgctgtcgtctctcaacatccgagctgggttatctgtaagagtggaacctctgtgaag atcgagtgccgttccctggactttcaggccacaactatgttttggtatcgtcagttcccg aaacagagtctcatgctgatggcaacttccaatgagggctccaaggccacatacgagcaa ggcgtcgagaaggacaagtttctcatcaaccatgcaagcctgaccttgtccactctgaca gtgaccagtgcccatcctgaagacagcagcttctacatctgcagtgctatggggagacag agcacagatacgcagtattttggcccaggcacccggctgacagtgctcg
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SEQ ID NO: 26 (TCRb) GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQ GVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAMGRQSTDTQYFGPGTRLTVL
SEQ ID NO: 27 (CDR1 TCRb) - gactttcaggccacaact
SEQ ID NO: 28 (CDR1 TCRb) - DFQATT
SEQ ID NO: 29 (CDR2 TCRb) - tccaatgagggctccaaggcc
SEQ ID NO: 30 (CDR2 TCRb) - SNEGSKA
SEQ ID NO:31 (CDR3 TCRb) - agtgctatggggagacagagcacagatacgcagtat
SEQ ID NO: 32 (CDR3 TCRb) - SAMGRQSTDTQY
SEQ ID NO: 33 (sgTAPI) - GCTGCTACTTCTCGCCGACT
SEQ ID NO: 34 (LRPAP121.30) - AGGGTCAGGTCGTTTCTGCG
SEQ ID NO: 35 (bacterial peptide - ‘Multidrug-efflux transporter’ from Ruminococcus lactaris) - TIIKPLIWV
SEQ ID NO: 36 (bacterial peptide - ‘amino acid adenylation domain-containing protein’ from Mycobacterium abscessus/ Mycobacterium franklinii/ Mycobacterium saopaulense/ Mycobacterium immunogenum) - LLAI_AAGLAV
SEQ ID NO: 37 (p14: LRPAP1 peptide fragment) MAPRRVRSFLRGLPALLLLLLFLGPWPAASHGGK
SEQ ID NO: 38 (p29: ARSA peptide fragment) MGAPRSLLLALAAGLAVARPPNIVLIFADDLGYGD
SEQ ID NO: 39 (p34: PCDHGA6 peptide fragment) MAPPQRHPQRSEQVLLLTLLGTLWGAAAAQIRYSI
SEQ ID NO: 40 (p35: C20ORF141 peptide fragment) GSGSPVRPPVSTWGPSWAQLLDSVLWLGALGLT
SEQ ID NO: 41 (APCS peptide fragment) ILSAYQGTPLPANILDWQALNYEIRGYVIIKPLVWV
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The
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Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms a, an, and the include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
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Claims (42)

CONCLUSIESCONCLUSIONS 1. Geïsoleerde nucleïnezuursequentie die codeert voor:An isolated nucleic acid sequence encoding: a. een polypeptide die een CDR3 van een TCR a ketenpolypeptide omvat die specifiek bindt op een peptide die in het bezit is van de aminozuursequentie FLGPWPAAS (SEQ ID nr.:l); en/ofa. a polypeptide comprising a CDR3 of a TCR a chain polypeptide that specifically binds to a peptide that possesses the amino acid sequence FLGPWPAAS (SEQ ID NO: 1); and / or b. een polypeptide die een CDR3 van een TCR β ketenpolypeptide omvat die specifiek bindt op een peptide die in het bezit is van de aminozuursequentie FLGPWPAAS (SEQ ID nr.:l).b. a polypeptide comprising a CDR3 of a TCR β chain polypeptide that specifically binds to a peptide that possesses the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). 2. Geïsoleerde nucleïnezuursequentie volgens conclusie 1, waarin de nucleïnezuursequentie codeert voor (a) en voor (b), waarin (a) en (b) samen specifiek binden op de peptide FLGPWPAAS (SEQ ID nr.:l).The isolated nucleic acid sequence of claim 1, wherein the nucleic acid sequence encodes (a) and (b), wherein (a) and (b) together specifically bind to the FLGPWPAAS peptide (SEQ ID NO: 1). 3. Geïsoleerde nucleïnezuursequentie volgens conclusie 1 of 2, waarin de CDR3 van (a) in het bezit is van een aminozuursequentie die een sequentie-identiteit vertoont van ten minste 90% ten opzichte van VVMGYGQNFV (SEQ ID nr.:24).An isolated nucleic acid sequence according to claim 1 or 2, wherein the CDR3 of (a) is in possession of an amino acid sequence that exhibits a sequence identity of at least 90% relative to VVMGYGQNFV (SEQ ID NO: 24). 4. Geïsoleerde nucleïnezuursequentie volgens conclusie 3, waarin voor de CDR3 van (a) wordt gecodeerd door de nucleïnezuursequentie met SEQ ID nr.:23.The isolated nucleic acid sequence of claim 3, wherein the CDR3 of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 23. 5. Geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 4, waarin de CDR3 van (b) in het bezit is van een aminozuursequentie die een sequentie-identiteit vertoont van ten minste 90% ten opzichte van SAMGRQSTDTQY (SEQ ID nr.:32).The isolated nucleic acid sequence of any one of claims 1 to 4, wherein the CDR3 of (b) has an amino acid sequence that exhibits a sequence identity of at least 90% relative to SAMGRQSTDTQY (SEQ ID NO: 32) ). 6. Geïsoleerde nucleïnezuursequentie volgens conclusie 5, waarin voor de CDR3 van (b) wordt gecodeerd door de nucleïnezuursequentie met SEQ ID nr.:31.The isolated nucleic acid sequence of claim 5, wherein the CDR3 of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 31. 7. Geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 6, waarin de CDR3 van (a) gelegen is in een TCR α keten variabele zone die specifiek bindt op The isolated nucleic acid sequence of any one of claims 1 to 6, wherein the CDR3 of (a) is located in a TCR α chain variable zone that specifically binds to 127127 FLGPWPAAS (SEQ ID nr.:l), waarin (a) optioneel bovendien een TCR α keten constante zone omvat.FLGPWPAAS (SEQ ID NO: 1), wherein (a) optionally additionally comprises a TCR α chain constant zone. 8. Geïsoleerde nucleïnezuursequentie volgens conclusie 7, waarin de TCR α keten variabele zone in het bezit is van een aminozuursequentie die een sequentie-identiteit vertoont van ten minste 90% ten opzichte van SEQ ID nr.: 18.The isolated nucleic acid sequence of claim 7, wherein the TCR α chain variable zone has an amino acid sequence that exhibits a sequence identity of at least 90% with respect to SEQ ID NO: 18. 9. Geïsoleerde nucleïnezuursequentie volgens conclusie 8, waarin voor de TCR α keten variabele zone van (a) wordt gecodeerd door de nucleïnezuursequentie met SEQ ID nr.:17.The isolated nucleic acid sequence of claim 8, wherein the TCR α chain variable zone of (a) is encoded by the nucleic acid sequence of SEQ ID NO: 17. 10. Geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 9, waarin de CDR3 van (b) gelegen is in een TCR β keten variabele zone die specifiek bindt op FLGPWPAAS (SEQ ID nr.:l), waarin (a) optioneel bovendien een TCR β keten constante zone omvat.The isolated nucleic acid sequence of any one of claims 1 to 9, wherein the CDR3 of (b) is located in a TCR β chain variable zone that specifically binds to FLGPWPAAS (SEQ ID NO: 1), wherein (a) optionally additionally a TCR β chain constant zone. 11. Geïsoleerde nucleïnezuursequentie volgens conclusie 10, waarin de TCR β keten variabele zone in het bezit is van een aminozuursequentie die een sequentie-identiteit vertoont van ten minste 90% ten opzichte van SEQ ID nr.:26.The isolated nucleic acid sequence of claim 10, wherein the TCR β chain variable zone is in possession of an amino acid sequence that exhibits a sequence identity of at least 90% with respect to SEQ ID NO: 26. 12. Geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 9, waarin voor de TCR β keten variabele zone van (b) wordt gecodeerd door de nucleïnezuursequentie met SEQ ID nr.:25.The isolated nucleic acid sequence of any one of claims 1 to 9, wherein the TCR β chain variable region of (b) is encoded by the nucleic acid sequence of SEQ ID NO: 25. 13. Geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 12, waarin de CDR3 van (a) gelegen is in een TCR α keten variabele zone die een sequentieidentiteit vertoont van ten minste 90% ten opzichte van SEQ ID nr.: 18, waarin de CDR3 in het bezit is van een aminozuursequentie die overeenstemt met SEQ ID nr.:24, en waarin optioneel (a) TCR α keten een constante zone omvat.The isolated nucleic acid sequence of any one of claims 1 to 12, wherein the CDR3 of (a) is located in a TCR α chain variable zone that exhibits a sequence identity of at least 90% relative to SEQ ID NO: 18, wherein the CDR3 is in possession of an amino acid sequence corresponding to SEQ ID NO: 24, and wherein optionally (a) TCR α chain comprises a constant zone. 14. Geïsoleerde nucleïnezuursequentie volgens conclusie 13, waarin de TCR a keten variabele zone CDR1 in het bezit is van een aminozuursequentie die overeenstemt met The isolated nucleic acid sequence of claim 13, wherein the TCR a chain variable zone CDR1 is in possession of an amino acid sequence corresponding to 128128 SEQ ID nr. :20, en de TCR α keten variabele zone CDR2 in het bezit is van een aminozuursequentie die overeenstemt met SEQ ID nr. :22.SEQ ID NO: 20, and the TCR α chain variable zone CDR2 is in possession of an amino acid sequence corresponding to SEQ ID NO: 22. 15. Geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 14, waarin de CDR3 van (b) is gelegen in een TCR β keten variabele zone die een sequentieidentiteit vertoont van ten minste 90% ten opzichte van SEQ ID nr.:26, waarin de CDR3 in het bezit is van een aminozuursequentie die overeenstemt met SEQ ID nr.:32; en waarin optioneel (b) een TCR β keten constante zone omvat.The isolated nucleic acid sequence of any one of claims 1 to 14, wherein the CDR3 of (b) is located in a TCR β chain variable zone that exhibits a sequence identity of at least 90% with respect to SEQ ID NO: 26, wherein the CDR3 is in possession of an amino acid sequence corresponding to SEQ ID NO: 32; and wherein optionally (b) comprises a TCR β chain constant zone. 16. Geïsoleerde nucleïnezuursequentie volgens conclusie 15, waarin de TCR β keten variabele zone CDR1 in het bezit is van een aminozuursequentie die overeenstemt met SEQ ID nr.:28, en de TCR β keten variabele zone CDR2 in het bezit is van een aminozuursequentie die overeenstemt met SEQ ID nr.:30.The isolated nucleic acid sequence of claim 15, wherein the TCR β chain variable zone CDR1 is in possession of an amino acid sequence corresponding to SEQ ID NO: 28, and the TCR β chain variable zone CDR2 is in possession of an amino acid sequence that corresponds to with SEQ ID NO: 30. 17. Geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 16, waarin de nucleïnezuursequentie codeert voor een T-celreceptor.The isolated nucleic acid sequence of any one of claims 1 to 16, wherein the nucleic acid sequence encodes a T cell receptor. 18. Geïsoleerde nucleïnezuursequentie die codeert voor een chimere antigeenreceptorproteïne, een doelbindingsgroep omvattende die specifiek bindt op een peptide die de aminozuursequentie FLGPWPAAS (SEQ ID nr.: 1) omvat.An isolated nucleic acid sequence encoding a chimeric antigen receptor protein comprising a target binding moiety that specifically binds to a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). 19. Geïsoleerde nucleïnezuursequentie volgens conclusie 18, waarin voor de doelbindingsgroep wordt gecodeerd door een geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 17.The isolated nucleic acid sequence of claim 18, wherein the target binding moiety is encoded by an isolated nucleic acid sequence of any one of claims 1 to 17. 20. Vector, een nucleïnezuursequentie omvattende volgens een der conclusies 1 tot en metA vector comprising a nucleic acid sequence according to any one of claims 1 to 19.19. 21. Vector volgens conclusie 20, waarin de vector een plasmide of een virale vector is, optioneel waarin de vector geselecteerd is uit de groep die bestaat uit een retrovirus, lentivirus, adeno-geassocieerd virus, adenovirus, vaccinnia-virus, kanariepokkenvirus, herpesvirus, minicirkelvector, en artificieel DNA of RNA.The vector of claim 20, wherein the vector is a plasmid or a viral vector, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinnia virus, canary pox virus, herpes virus, mini circle vector, and artificial DNA or RNA. 129129 22. Gemodificeerde cel, getransfecteerd of getransduceerd met een nucleïnezuursequentie volgens een der conclusies 1 tot en met 19, of een vector volgens conclusie 20 of 21.A modified cell, transfected or transduced with a nucleic acid sequence according to any of claims 1 to 19, or a vector according to claim 20 or 21. 23. Gemodificeerde cel volgens conclusie 22, waarin de gemodificeerde cel is geselecteerd uit de groep die bestaat uit een CD8 T-cel, een CD4 T-cel, een NK-cel, een NKT-cel, een gamma-delta T-cel, een hematopoïetische stamcel, een progenitorcel, een T-cellijn, of een NK-92 cellijn.The modified cell of claim 22, wherein the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NKT cell, a gamma delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line, or an NK-92 cell line. 24. Gemodificeerde cel volgens conclusie 22 of 23, waarin de gemodificeerde cel een menselijke cel is.The modified cell of claim 22 or 23, wherein the modified cell is a human cell. 25. Farmaceutische samenstelling, omvattende:A pharmaceutical composition comprising: a. een geïsoleerde nucleïnezuursequentie volgens een der conclusies 1 tot en met 19;a. an isolated nucleic acid sequence according to any of claims 1 to 19; b. een vector volgens conclusie 20 of 21;b. a vector according to claim 20 or 21; c. een gemodificeerde cel volgens een der conclusies 22 tot en met 24;c. a modified cell according to any of claims 22 to 24; d. een geïsoleerde peptide die de aminozuursequentie FLGPWPAAS (SEQ ID nr.: 1) omvat;d. an isolated peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1); e. een geïsoleerde nucleïnezuursequentie die codeert voor een peptide met de aminozuursequentie FLGPWPAAS (SEQ ID nr.: 1);e. an isolated nucleic acid sequence encoding a peptide with the amino acid sequence FLGPWPAAS (SEQ ID NO: 1); f. een vector die een geïsoleerde nucleïnezuursequentie omvat die codeert voor een peptide met de aminozuursequentie FLGPWPAAS (SEQ ID nr.:l); off. a vector comprising an isolated nucleic acid sequence encoding a peptide with the amino acid sequence FLGPWPAAS (SEQ ID NO: 1); or g. een bindingsmiddel dat specifiek bindt op een peptide die de aminozuursequentie FLGPWPAAS (SEQ ID nr.: 1) omvat;g. a binding agent that specifically binds to a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1); en een farmaceutisch aanvaardbare excipiënt, een farmaceutisch aanvaardbaar adjuvans, verdunningsmiddel, en/of een farmaceutisch aanvaardbare drager.and a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, diluent, and / or a pharmaceutically acceptable carrier. 26. Farmaceutische samenstelling volgens conclusie 25, waarbij de samenstelling een geïsoleerde peptide omvat die de aminozuursequentie FLGPWPAAS (SEQ ID nr.:l) omvat, en waarin de peptide niet meer dan 20 aminozuren omvat.The pharmaceutical composition according to claim 25, wherein the composition comprises an isolated peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1), and wherein the peptide comprises no more than 20 amino acids. 130130 27. Farmaceutische samenstelling volgens conclusie 26, waarin de peptide bestaat uit de aminozuursequentie FLGPWPAAS (SEQ ID nr.: 1).The pharmaceutical composition of claim 26, wherein the peptide consists of the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). 28. Farmaceutische samenstelling volgens conclusie 25, waarbij de samenstelling een geïsoleerde nucleïnezuursequentie omvat die codeert voor een peptide die de aminozuursequentie FLGPWPAAS (SEQ ID nr.:l) omvat, en waarbij de nucleïnezuursequentie mRNA of DNA is.The pharmaceutical composition of claim 25, wherein the composition comprises an isolated nucleic acid sequence encoding a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1), and wherein the nucleic acid sequence is mRNA or DNA. 29. Farmaceutische samenstelling volgens conclusie 25, waarin de samenstelling een bindingsmiddel omvat dat specifiek bindt op een peptide die de aminozuursequentie FLGPWPAAS (SEQ ID nr.: 1) omvat, en waarin het bindingsmiddel een antilichaam is.The pharmaceutical composition of claim 25, wherein the composition comprises a binding agent that specifically binds to a peptide comprising the amino acid sequence FLGPWPAAS (SEQ ID NO: 1), and wherein the binding agent is an antibody. 30. Farmaceutische samenstelling volgens een der conclusies 25 tot en met 29, waarin de samenstelling is geformuleerd als een vaccin.The pharmaceutical composition of any one of claims 25 to 29, wherein the composition is formulated as a vaccine. 31. Farmaceutische samenstelling volgens een der conclusies 25 tot en met 30, voor toepassing als geneesmiddel.Pharmaceutical composition according to any of claims 25 to 30, for use as a medicine. 32. Farmaceutische samenstelling voor toepassing volgens conclusie 31, bij het voorkomen of het behandelen van kanker of van een virale infectie, geassocieerd met een gereduceerde HLA klasse I antigeenpresentatie in een menselijk subject.A pharmaceutical composition for use according to claim 31 in the prevention or treatment of cancer or of a viral infection associated with a reduced HLA class I antigen presentation in a human subject. 33. Werkwijze voor het behandelen van een aandoening in een menselijk subject dat daaraan behoefte heeft, waarbij de werkwijze het aan het subject toedienen omvat van een therapeutisch werkzame hoeveelheid van een farmaceutische samenstelling volgens een der conclusies 25 tot en met 30.A method of treating a condition in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any of claims 25 to 30. 34. Werkwijze volgens conclusie 33, voor het voorkomen of het behandelen van kanker of van een virale infectie, geassocieerd met een gereduceerde HLA klasse I antigeenpresentatie in een menselijk subject.The method of claim 33 for preventing or treating cancer or a viral infection associated with a reduced HLA class I antigen presentation in a human subject. 131131 35. Samenstelling voor toepassing, of werkwijze volgens een der conclusies 31 tot en met 34, waarin de kanker een melanoom, een niercelcarcinoom, een coloncarcinoom, of een lymfoom is.A composition for use, or a method according to any one of claims 31 to 34, wherein the cancer is a melanoma, a renal cell carcinoma, a colon carcinoma, or a lymphoma. 36. Samenstelling voor toepassing, of werkwijze volgens conclusie 35, waarin de kanker een melanoom is.A composition for use, or a method according to claim 35, wherein the cancer is a melanoma. 37. Werkwijze voor het genereren van een T-celreceptor, het in contact brengen omvattende van een nucleïnezuursequentie of van een vector volgens een der conclusies 1 tot en met 21, met een cel, onder voorwaarden waarin de nucleïnezuursequentie of de vector wordt geïncorporeerd in en uitgedrukt wordt door de cel om zodoende de T-celreceptor te genereren die specifiek bindt op een peptide die in het bezit is van de aminozuursequentie FLGPWPAAS (SEQ ID nr.:l).A method for generating a T cell receptor comprising contacting a nucleic acid sequence or a vector according to any of claims 1 to 21 with a cell, under conditions wherein the nucleic acid sequence or vector is incorporated into and is expressed by the cell so as to generate the T cell receptor that specifically binds to a peptide that possesses the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). 38. Werkwijze volgens conclusie 37, waarbij de werkwijze een ex-vivo werkwijze is.The method of claim 37, wherein the method is an ex-vivo method. 39. Toepassing van een peptide als biomarker voor een kanker of voor een virale infectie, geassocieerd met een gereduceerde HLA klasse I antigeenpresentatie in een menselijk subject, waarin de peptide de aminozuursequentie FLGPWPAAS (SEQ ID nr.:l) omvat.39. Use of a peptide as a biomarker for a cancer or for a viral infection, associated with a reduced HLA class I antigen presentation in a human subject, wherein the peptide comprises the amino acid sequence FLGPWPAAS (SEQ ID NO: 1). 40. Werkwijze voor het diagnosticeren van een kanker of van een virale infectie, geassocieerd met een gereduceerde HLA klasse I antigeenpresentatie in een menselijk subject, omvattende:A method for diagnosing a cancer or a viral infection associated with a reduced HLA class I antigen presentation in a human subject, comprising: het bepalen van de aanwezigheid van een peptide in een monster dat is afgenomen bij het subject, waarin de peptide FLGPWPAAS (SEQ ID nr.: 1) is, waarin de aanwezigheid van de peptide in het monster aangeeft dat het subject een kanker of een virale infectie heeft die geassocieerd zijn met een gereduceerde HLA klasse I antigeenpresentatie, en waarin de afwezigheid van de peptide in het monster aangeeft dat het subject geen kanker of een virale infectie heeft die geassocieerd zijn met een gereduceerde HLA klasse I antigeenpresentatie.determining the presence of a peptide in a sample taken from the subject, wherein the peptide is FLGPWPAAS (SEQ ID NO: 1), wherein the presence of the peptide in the sample indicates that the subject is a cancer or a viral has infection associated with a reduced HLA class I antigen presentation, and wherein the absence of the peptide in the sample indicates that the subject has no cancer or a viral infection associated with a reduced HLA class I antigen presentation. 132132 41. Werkwijze voor het behandelen van kanker of van een virale infectie die geassocieerd zijn met een gereduceerde HLA klasse I antigeenpresentatie in een menselijk subject, waarbij de werkwijze omvat:A method of treating cancer or a viral infection associated with a reduced HLA class I antigen presentation in a human subject, the method comprising: i. het bepalen van de aanwezigheid van een peptide in een monster dat is afgenomeni. determining the presence of a peptide in a sample that has been taken 5 bij het subject, waarin de peptide FLGPWPAAS (SEQ ID nr.: 1) is; en ii. het aan het subject toedienen van een therapeutisch werkzame hoeveelheid van een farmaceutische samenstelling volgens een der conclusies 25 tot en met 30.5 to the subject, wherein the peptide is FLGPWPAAS (SEQ ID NO: 1); and ii. administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any one of claims 25 to 30. 42. Farmaceutische samenstelling volgens een der conclusies 25 tot en met 30, voorA pharmaceutical composition according to any of claims 25 to 30 10 toepassing bij het behandelen of het voorkomen van een kanker of van een virale infectie die geassocieerd zijn met een gereduceerde HLA klasse I antigeenpresentatie in een menselijk subject, waarin van het subject werd vastgesteld dat het een kanker of een virale infectie heeft die geassocieerd is met een gereduceerde HLA klasse I antigeenpresentatie, door de aanwezigheid van een peptide in een monster dat is 15 afgenomen bij het subject, waarin de peptide FLGPWPAAS (SEQ ID nr.:l) is.Use in the treatment or prevention of a cancer or of a viral infection associated with a reduced HLA class I antigen presentation in a human subject, wherein the subject was determined to have a cancer or a viral infection associated with a reduced HLA class I antigen presentation, due to the presence of a peptide in a sample taken from the subject, wherein the peptide is FLGPWPAAS (SEQ ID NO: 1). 1/10 n-terminai signa! peptides1/10 n-terminai signa! peptides A^<?rittnk p^dictions ;··: cytöiol';A ^ <title> p ^ dictions; ··: cytoliol; Human Proteome n~&1383 f ÉR lumen I j Tumor I x n—7780A '' ƒ Signal \ i Peptide |Human Proteome n & 1383 f lumen I y Tumor I x n — 7780A '' ƒ Signal \ i Peptide | TopologyTopology 1^=1181091 ^ = 118109 Peptidome η=Ί ίΠ71Peptidome η = Ί ίΠ71 TE1PP selection π·· 65TE1PP selection π ·· 65 HLA class I binding affinity n—13731HLA class I binding affinity n-13731 Healthy ri-VUftfcHealthy RI-VUftfc HLA class I presented peptides . n-111171 pHLA-A2 tetramer \ screen § n- 40HLA class I presented peptides. n-111171 pHLA-A2 tetramer screen § n- 40 T cell repertoire m; wT cell repertoire m; w Validation : ÉR lumenValidation: ÉR lumen
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