T cell receptor for identifying AFP antigen
阅读说明：本技术 一种识别afp抗原的t细胞受体 (T cell receptor for identifying AFP antigen ) 是由 李懿 胡静 李骏 孙含丽 于 2018-07-30 设计创作，主要内容包括：本发明提供了一种能够特异性结合衍生自AFP抗原的短肽FMNKFIYEI的T细胞受体(TCR),所述抗原短肽FMNKFIYEI可与HLA A0201形成复合物并一起被呈递到细胞表面。本发明还提供了编码所述TCR的核酸分子以及包含所述核酸分子的载体。另外,本发明还提供了转导本发明TCR的细胞。(The present invention provides a T Cell Receptor (TCR) capable of specifically binding short peptide FMNKFIYEI derived from AFP antigen, said antigen short peptide FMNKFIYEI being capable of forming a complex with HLA a0201 and being presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.)
1. A T Cell Receptor (TCR) capable of binding to the FMNKFIYEI-HLA A0201 complex, preferably the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, wherein the CDR3 of the TCR α chain variable domain has the amino acid sequence AVNSGGSNYKLT (SEQ ID NO:12) and/or the CDR3 of the TCR β chain variable domain has the amino acid sequence ASSLFGQGREKLF (SEQ ID NO: 15);
more preferably, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
αCDR1-DSAIYN(SEQ ID NO:10)
αCDR2-IQSSQRE(SEQ ID NO:11)
α CDR3-AVNSGGSNYKLT (SEQ ID NO:12), and/or
The 3 complementarity determining regions of the variable domain of TCR β chain are:
βCDR1-SGHVS(SEQ ID NO:13)
βCDR2-FQNEAQ(SEQ ID NO:14)
βCDR3-ASSLFGQGREKLF(SEQ ID NO:15)。
2. a TCR as claimed in claim 1 which comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1 and/or the TCR β chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 5.
3. A TCR as claimed in claim 1 wherein a conjugate is attached to the C-or N-terminus of the α chain and/or β chain of the TCR, preferably wherein the conjugate which binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these, preferably wherein the therapeutic agent is an anti-CD 3 antibody.
4. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in any one of the preceding claims.
5. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to any preceding claim, or the complement thereof;
preferably, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 2 or SEQ ID NO 33 encoding the variable domain of the TCR α chain and/or
The nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 6 or SEQ ID NO 35 encoding the variable domain of the TCR β chain.
6. A vector comprising the nucleic acid molecule of claim 5; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
7. An isolated host cell comprising the vector of claim 6 or a nucleic acid molecule of claim 5 integrated into the chromosome.
8. A cell which transduces the nucleic acid molecule of claim 5 or the vector of claim 6; preferably, the cell is a T cell or a stem cell.
9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1-3, a TCR complex according to claim 4, a nucleic acid molecule according to claim 5, or a cell according to claim 8.
10. Use of a T cell receptor according to any one of claims 1 to 3, or a TCR complex according to claim 4 or a cell according to claim 8, for the preparation of a medicament for the treatment of a tumour or an autoimmune disease.
Technical Field
The present invention relates to TCRs capable of recognizing short peptides derived from the AFP antigen, to AFP-specific T cells obtained by transduction of such TCRs, and to their use in the prevention and treatment of AFP-related diseases.
Background
AFP (α Fetoprotein), also called α Fetoprotein, is a protein expressed during embryonic development and is a major component of embryonic serum, during development, AFP has relatively high expression levels in the yolk sac and liver, and is subsequently inhibited, in hepatocellular carcinoma, AFP expression is activated (Butterfield et al.J. Immunol.,2001, Apr 15; 166(8): 5300-8). AFP, after being produced intracellularly, is degraded into small molecules and bound to MHC (major histocompatibility Complex) molecules to form complexes, which are presented to the cell surface. FMNKFIYEI (SEQ ID NO:9) is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
T cell adoptive immunotherapy is the transfer of reactive T cells specific for a target cell antigen into a patient to act on the target cell. The T Cell Receptor (TCR) is a membrane protein on the surface of T cells that recognizes a corresponding short peptide antigen on the surface of a target cell. In the immune system, the direct physical contact between T cells and Antigen Presenting Cells (APC) is initiated by the binding of antigen short peptide specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then other cell membrane surface molecules of the T cells and APC interact to cause a series of subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells. Accordingly, those skilled in the art have focused on isolating TCRs specific for short AFP antigen peptides and transducing the TCRs into T cells to obtain T cells specific for short AFP antigen peptides, thereby allowing them to function in cellular immunotherapy.
Disclosure of Invention
The invention aims to provide a T cell receptor for recognizing AFP antigen short peptide.
In a first aspect of the invention, there is provided a T Cell Receptor (TCR) capable of binding to the FMNKFIYEI-HLAA0201 complex.
In another preferred embodiment, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the amino acid sequence of the CDR3 of the TCR α chain variable domain is AVNSGGSNYKLT (SEQ ID NO:12), and/or the amino acid sequence of the CDR3 of the TCR β chain variable domain is ASSLFGQGREKLF (SEQ ID NO: 15).
In another preferred embodiment, the 3 Complementarity Determining Regions (CDRs) of the TCR α chain variable domain are:
αCDR1-DSAIYN(SEQ ID NO:10)
αCDR2-IQSSQRE(SEQ ID NO:11)
α CDR3-AVNSGGSNYKLT (SEQ ID NO:12), and/or
The 3 complementarity determining regions of the variable domain of TCR β chain are:
βCDR1-SGHVS(SEQ ID NO:13)
βCDR2-FQNEAQ(SEQ ID NO:14)
βCDR3-ASSLFGQGREKLF(SEQ ID NO:15)。
in another preferred embodiment, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1, and/or the TCR β chain variable domain being an amino acid sequence having at least 90% sequence identity to SEQ ID No. 5.
In another preferred embodiment, the TCR comprises the α chain variable domain amino acid sequence SEQ ID NO 1.
In another preferred embodiment, the TCR comprises the β chain variable domain amino acid sequence
In another preferred embodiment, the TCR is an αβ heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC1 01 or TRBC2 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO. 3 and/or the β chain amino acid sequence of the TCR is SEQ ID NO. 7.
In another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is single chain.
In another preferred embodiment, the TCR is comprised of the α chain variable domain linked to the β chain variable domain by a peptide linker sequence.
In another preferred embodiment, the TCR has one or more mutations in α chain variable region
In another preferred embodiment, the α chain variable domain amino acid sequence of the TCR comprises SEQ ID NO. 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID NO. 34.
In another preferred embodiment, the amino acid sequence of the TCR is SEQ ID NO 30.
In another preferred embodiment, the TCR comprises (a) all or part of a TCR α chain except for the transmembrane domain and (b) all or part of a TCR β chain except for the transmembrane domain;
and (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of the TCR chain constant domain.
In another preferred example, cysteine residues form an artificial disulfide bond between the α and β chain constant domains of the TCR.
In another preferred embodiment, the cysteine residues forming the artificial disulfide bond in the TCR are substituted at one or more groups of sites selected from the group consisting of:
thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;
ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;
arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;
pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and
tyr10 and TRBC1 × 01 of exon 1 of TRAC × 01 or Glu20 of exon 1 of TRBC2 × 01.
In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO. 26 and/or the β chain amino acid sequence of the TCR is SEQ ID NO. 28.
In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain variable region and the β chain constant region.
In another preferred embodiment, the cysteine residues that form the artificial interchain disulfide bond in the TCR replace one or more groups of sites selected from the group consisting of:
amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;
amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or
Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01.
In another preferred embodiment, the TCR comprises α and β chain variable domains and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the α chain constant domain, the α chain variable domain of the TCR forming a heterodimer with the β chain.
In another preferred embodiment, the TCR has a conjugate attached to the C-or N-terminus of chain α and/or chain β.
In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these. Preferably, the therapeutic agent is an anti-CD 3 antibody.
In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one of the TCR molecules is a TCR according to the first aspect of the invention.
In a third aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention, or the complement thereof.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 2 or SEQ ID NO 33 encoding the variable domain of the TCR α chain.
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO 6 or
In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO. 4 encoding the TCR α chain and/or comprises the nucleotide sequence SEQ ID NO. 8 encoding the TCR β chain.
In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
In a fifth aspect of the invention, there is provided an isolated host cell comprising a vector according to the fourth aspect of the invention or a genome into which has been integrated an exogenous nucleic acid molecule according to the third aspect of the invention.
In a sixth aspect of the invention, there is provided a cell which transduces a nucleic acid molecule according to the third aspect of the invention or a vector according to the fourth aspect of the invention; preferably, the cell is a T cell or a stem cell.
In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth aspect, the invention provides the use of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention, for the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.
In a ninth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a T cell receptor according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, a vector according to the fourth aspect of the invention, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention;
preferably, the disease is a tumor, preferably the tumor is hepatocellular carcinoma.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1a, FIG. 1b, FIG. 1c, FIG. 1d, FIG. 1e and FIG. 1f are the TCR α chain variable domain amino acid sequence, TCR α chain variable domain nucleotide sequence, TCR α chain amino acid sequence, TCR α chain nucleotide sequence, TCR α chain amino acid sequence with leader sequence and TCR α chain nucleotide sequence with leader sequence, respectively.
FIG. 2a, FIG. 2b, FIG. 2c, FIG. 2d, FIG. 2e and FIG. 2f are the TCR β chain variable domain amino acid sequence, TCR β chain variable domain nucleotide sequence, TCR β chain amino acid sequence, TCR β chain nucleotide sequence, TCR β chain amino acid sequence with leader sequence and TCR β chain nucleotide sequence with leader sequence, respectively.
FIG. 3 is CD8 of monoclonal cells +And tetramer-PE double positive staining results.
Fig. 4a and 4b are the amino acid and nucleotide sequences, respectively, of a soluble TCR α chain.
Fig. 5a and 5b are the amino acid and nucleotide sequences, respectively, of a soluble TCR β chain.
Figure 6 is a gel diagram of the soluble TCR obtained after purification. The leftmost lane is reducing gel, the middle lane is molecular weight marker (marker), and the right lane is non-reducing gel.
FIGS. 7a and 7b are the amino acid and nucleotide sequences, respectively, of a single-chain TCR.
FIGS. 8a and 8b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single-chain TCR α chain.
FIGS. 9a and 9b are the amino acid and nucleotide sequences, respectively, of the variable domain of the single-chain TCR β chain.
FIGS. 10a and 10b are the amino acid and nucleotide sequences, respectively, of a single-chain TCR linker sequence (linker).
FIG. 11 is a gel diagram of the soluble single chain TCR obtained after purification. The left lane is the molecular weight marker (marker) and the right lane is the non-reducing gel.
FIG. 12 is a BIAcore kinetic profile of binding of soluble TCRs of the invention to the FMNKFIYEI-HLA A0201 complex.
FIG. 13 is a BIAcore kinetic profile of binding of soluble single chain TCRs of the invention to the FMNKFIYEI-HLA A0201 complex.
FIG. 14 shows the results of functional verification of the ELISPOT activation of the resulting T cell clones.
FIG. 15 is a graphical representation of the results of functional confirmation of ELISPOT activation of effector cells transduced with the TCRs of the invention.
Detailed Description
The present inventors have extensively and intensively studied to find a TCR capable of specifically binding to AFP antigen short peptide FMNKFIYEI (SEQ ID NO:9), which antigen short peptide FMNKFIYEI can form a complex with HLA A0201 and be presented together on the cell surface. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention.
Term(s) for
MHC molecules are proteins of the immunoglobulin superfamily, which may be MHC class I or class II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.
The T Cell Receptor (TCR), is the only receptor for a specific antigenic peptide presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.
TCR is a glycoprotein on the surface of cell membranes which consists of α/β or γ/δ chains in the form of heterodimers, in 95% of T cells TCR heterodimers consist of β and β 1 chains, while 5% of T cells have TCRs consisting of γ and δ chains native β 3 β heterodimer TCR has β and β chains, β and β chains constituting subunits of α β heterodimeric TCR. broadly, each of α and β chains comprises a variable region, a connecting region and a constant region, β chains usually also contain a short variable region between the variable and connecting regions, but the variable region is usually considered part of the connecting region. each variable region comprises 3 CDRs (complementarity determining regions) which are chimeric in a framework structure (work domains), CDR1, CDR2 and CDR 3. CDR regions determine the binding of the TCR complex to pMHC, wherein the variable region and CDR 29 are composed of variable and constant regions, known as imk domains, and the variable region is known as the TCR α -variable region, TCR 80 domain, the variable region is usually found as a TCR domain of a TCR α -variable region linked to a TCR-variable region, and TCR-variable domain of a TCR-variable domain which is known as a TCR-variable domain linked to a TCR-variable domain.
In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.
Natural interchain disulfide bond and artificial interchain disulfide bond
A set of disulfide bonds exists between the membrane proximal region C α and the C β chain of a native TCR, and are referred to herein as "native interchain disulfide bonds.
For convenience of description of the positions of disulfide bonds, the positions of the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are numbered in the order from the N-terminus to the C-terminus, such as in TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid in the order from the N-terminus to the C-terminus is P (proline), and thus in the present invention it can be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, and as in 737bc 3 × 01 or TRBC2 × 01, and the 61 th amino acid in the order from the N-terminus to the C-terminus is Q (glutamine), and thus in the present invention it can be described as TRBC1 × 01 or TRBC 6301 × 01, or TRBC 8501, and similarly as TRBC 8261 or glbc 891. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.
Detailed Description
TCR molecules
Thus, in a first aspect of the invention, there is provided a TCR molecule which is capable of binding to the FMNKFIYEI-HLA A0201 complex, preferably the TCR molecule is isolated or purified, the chains α and β of the TCR each having 3 Complementarity Determining Regions (CDRs).
In a preferred embodiment of the invention, the α chain of the TCR comprises CDRs having the amino acid sequence:
αCDR1-DSAIYN(SEQ ID NO:10)
αCDR2-IQSSQRE(SEQ ID NO:11)
α CDR3-AVNSGGSNYKLT (SEQ ID NO:12), and/or
The 3 complementarity determining regions of the variable domain of TCR β chain are:
βCDR1-SGHVS(SEQ ID NO:13)
βCDR2-FQNEAQ(SEQ ID NO:14)
βCDR3-ASSLFGQGREKLF(SEQ ID NO:15)。
the TCR molecules of the invention are thus defined as TCR molecules comprising the α and/or β chain CDR region sequences and any suitable framework structure, the TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID NO:1, and/or the TCR β chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID NO: 5.
In a preferred embodiment of the invention, the TCR molecule of the invention is a heterodimer of α and β chains, in particular, the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain on the one hand and the α chain variable domain amino acid sequence comprises the CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11) and CDR3(SEQ ID NO:12) of the α chain described above, preferably, the TCR molecule comprises the α chain variable domain amino acid sequence SEQ ID NO:1, more preferably, the α chain variable domain amino acid sequence of the TCR molecule is SEQ ID NO:1, and on the other hand, the β chain of the heterodimeric molecule comprises a variable domain and a constant domain, the β chain variable domain amino acid sequence comprises the CDR1(SEQ ID NO:13), CDR 48 (SEQ ID NO: 3914) and CDR3 (TCR ID NO:15) of the β chain, preferably, the TCR molecule comprises the variable domain amino acid sequence 5965 chain variable domain of the variable domain, more preferably, the TCR molecule is SEQ ID NO: 5965 chain variable domain.
In a preferred embodiment of the invention, the TCR molecule of the invention is a single chain TCR molecule consisting of part or all of the α chain and/or part or all of the β chain the description of single chain TCR molecules can be found in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654. 12658 from the literature, the skilled person can readily construct single chain TCR molecules comprising the CDRs regions of the invention.
The α chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11) and CDR3(SEQ ID NO:12) of the above-mentioned α chain preferably the single chain TCR molecule comprises α chain variable domain amino acid sequence SEQ ID NO:1 more preferably the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO:1 the β chain variable domain amino acid sequence of the single chain TCR molecule comprises CDR1(SEQ ID NO:13), CDR2(SEQ ID NO: 14) and CDR3(SEQ ID NO:15) of the above-mentioned β chain preferably the single chain TCR molecule comprises β chain variable domain amino acid sequence SEQ ID NO:5 more preferably the single chain TCR β chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO: 5.
For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", the constant domain sequence of the β chain of the TCR molecules can be "TRBC 1" 01 "or" TRBC2 "01", the amino acid sequence given in TRAC 01 of the IMGT at position 53 is Arg, here indicated as Arg53 of TRAC 01 exon 1, and the rest of the way around, preferably, the amino acid sequence of the α chain of the TCR molecules of the invention is SEQ ID NO:3, and/or the amino acid sequence of the β chain is SEQ ID NO: 7.
Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD 3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. The present invention also provides soluble TCRs with specificity for AFP antigen short peptides.
In order to obtain a soluble TCR, the TCR of the invention may be a TCR in which an artificial disulfide bond is introduced between residues of a constant domain of the TCR, a cysteine residue may be substituted for another amino acid residue at a suitable site in the native TCR to form an artificial interchain disulfide bond, e.g., a cysteine residue substituted for Thr of exon 1 of TRAC 01 and a Ser of exon 1 of TRBC 01 or TRBC 01 to form a disulfide bond, and the other site in which a cysteine residue is introduced to form a disulfide bond may be a truncated Thr of exon 1 of TRAC 01 and a Ser of exon 1 of TRBC 01 or TRBC 01, a Tyr of exon 1 of TRAC 01 and a Ser of exon 1 of TRBC 01 or TRBC 01, a Thr of exon 1 of TRAC 01 and a TRBC 01 or a set of multiple amino acid residues of the TrBC 01, a multiple TrBC1 or Ser of the same, a TrBC 01, a TrBC1, a C01, a C1, a TrBC1, a C01, a TrBC1, a C1, a TrBC1, a C1, a TrBC, a C1, a T1, a C1, a T5, a T1, a C1, a T1, a C1.
As noted above, the TCRs of the invention may comprise an artificial disulfide bond introduced between the residues of the constant domains of its α and β chains it is noted that the constant domains may or may not contain the artificial disulfide bond introduced as described above and that the TCRs of the invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence.
To obtain a soluble TCR, on the other hand, the TCR of the invention also includes a TCR having mutations in its hydrophobic core region, preferably mutations that increase the stability of the soluble TCR of the invention, as described in the patent publication WO2014/206304, such a TCR may have mutations in its following variable domain hydrophobic core positions (chain α and/or β) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or α chain J gene (TRAJ) short peptide
The TCR with the mutated hydrophobic core region can be a stable soluble single-chain TCR formed by connecting α of the TCR and a variable domain of a β chain through a flexible peptide chain, and the flexible peptide chain can be any peptide chain suitable for connecting TCR α and β chain variable domains, for example, the single-chain soluble TCR constructed in the
In addition, for stability, it is disclosed in 201680003540.2 that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR, and therefore, an artificial interchain disulfide bond can also be included between the β chain variable region and the β chain constant region of the high affinity TCR of the present invention, specifically, the cysteine residue forming the artificial interchain disulfide bond between the β chain variable region and the β chain constant region of the TCR replaces amino acid 46 of TRAV and amino acid 60 of TRBC1 or TRBC2 exon 1, amino acid 47 of TRAV and amino acid 61 of TRBC 1.01 or TRBC2 exon 1, amino acid 46 of TRAV and amino acid 61 of TRBC 1.01 or TRBC2 exon 1, and amino acid 47 of TRAV and amino acid 61 of TRBC2 or TRBC 5801 exon 1, or the TCR 47 of TRAV and TRBC 24 or TRBC1 or the transmembrane domain may include at least part of a transmembrane variable domain (preferably, but may include a variant domain 867 or a portion of the variable domain of the variable region of the chain, and a variable domain of the variable region 865, preferably include a transmembrane chain, and a portion of the variable domain of the variable region of the chain of the β, which may include at least one of the transmembrane region 867 chain, preferably a portion of a transmembrane chain, and a variable region of the variable region 867 chain, and a variable region of the variable chain of the variable region of the chain (preferably, particularly, which may include a chain of the variable region of chain of the variable region of the.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the FMNKFIYEI-HLA a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biological toxins (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and immunotherapy (Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research (Cancer Research)63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004,
In addition, the TCRs of the invention may also be hybrid TCRs comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the inventive TCR may comprise a human variable domain and a murine constant domain. The drawback of this approach is the possibility of eliciting an immune response. Thus, there should be a regulatory regimen to immunosuppresse when it is used for adoptive T cell therapy to allow for the engraftment of murine expressing T cells.
It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).
Nucleic acid molecules
A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a portion thereof which may be a variable domain of one or more of the CDRs, <tt translation = α ">t α &/t >t and/or β chains, and the α chain and/or β chain.
The nucleotide sequence encoding the α chain CDR regions of the TCR molecule of the first aspect of the invention is as follows:
αCDR1-gatagcgctatttacaac(SEQ ID NO:16)
αCDR2-attcagtcaagtcagagagag(SEQ ID NO:17)
αCDR3-gctgtgaatagtggaggtagcaactataaactgaca(SEQ ID NO:18)
the nucleotide sequence encoding the β chain CDR regions of the TCR molecule of the first aspect of the invention is as follows:
βCDR1-tcgggtcatgtatcc(SEQ ID NO:19)
βCDR2-ttccagaatgaagctcaa(SEQ ID NO:20)
βCDR3-gccagcagcttattcgggcagggacgggaaaaactgttt(SEQ ID NO:21)
thus, the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR α chain of the invention comprises SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the TCR β chain of the invention comprises SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21.
Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a variable domain of the TCR α chain of the invention comprises SEQ ID NO 2 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a variable domain of the TCR β chain of the invention comprises SEQ ID NO 6 or the nucleotide sequence of the nucleic acid molecule of the invention encoding a variable domain of the TCR α chain of the invention comprises SEQ ID NO 33 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a variable domain of the TCR β chain of the invention comprises
It will be appreciated that, due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Thus, the nucleic acid sequence encoding the TCR of the present invention may be identical to or a degenerate variant of the nucleic acid sequences shown in the figures of the present invention. As illustrated by one of the examples herein, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 2.
The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for various other organisms are well known to those skilled in the art.
The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.
Carrier
The invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages and animal and plant viruses.
Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.
Preferably, the vector can transfer the nucleotide of the invention into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the AFP antigen. Ideally, the vector should be capable of sustained high level expression in T cells.
Cells
The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises a vector of the invention or has integrated into its chromosome a nucleic acid molecule of the invention. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.
In addition, the invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4 +Helper T cell or CD8 +Cytotoxic T cells. The cell may be in CD4 +Helper T cell/CD 8 +A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD 3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.
Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.
There are many methods suitable for T cell transfection using DNA or RNA encoding the TCR of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the TCRs of the invention may be used for adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Revcancer8 (4): 299-308).
AFP antigen associated diseases
The present invention also relates to a method for the treatment and/or prevention of a disease associated with AFP in a subject, comprising the step of adoptive transfer of AFP-specific T cells to the subject. The AFP-specific T cells recognize FMNKFIYEI-HLA A0201 complex.
The AFP-specific T-cells of the invention can be used to treat any AFP-related disease presenting an AFP antigen short peptide FMNKFIYEI-HLAA0201 complex. Including but not limited to hepatocellular carcinoma.
Method of treatment
Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the AFP antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating an AFP-related disease comprising infusing into a patient an isolated T cell expressing a TCR of the invention, preferably, the T cell is derived from the patient itself. Generally, this involves (1) isolating T cells from the patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.
The main advantages of the invention are:
(1) the inventive TCR was capable of binding to the AFP antigen short peptide complex FMNKFIYEI-HLA a0201, while cells transduced with the inventive TCR were capable of being specifically activated.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
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