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CN115490773A - High-affinity T cell receptor aiming at AFP antigen - Google Patents

High-affinity T cell receptor aiming at AFP antigen Download PDF

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CN115490773A
CN115490773A CN202110679998.2A CN202110679998A CN115490773A CN 115490773 A CN115490773 A CN 115490773A CN 202110679998 A CN202110679998 A CN 202110679998A CN 115490773 A CN115490773 A CN 115490773A
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郭姗姗
温桥生
黄姣
翁志明
陈建君
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Xiangxue Life Science Technology Guangdong Co ltd
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Priority to PCT/CN2022/099383 priority patent/WO2022262842A1/en
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Abstract

The invention provides a T Cell Receptor (TCR) having the property of binding to the TSSELMAITR-HLA A1101 complex. The invention also provides multivalent TCR complexes, nucleic acid molecules encoding such TCRs, vectors comprising these nucleic acids, cells expressing such TCRs, and pharmaceutical compositions comprising the foregoing, which are useful for the diagnosis, treatment, and prevention of AFP positive diseases. The invention also provides methods of making such TCRs.

Description

High-affinity T cell receptor aiming at AFP antigen
Technical Field
The present invention relates to the field of biotechnology, and more specifically to T Cell Receptors (TCRs) capable of recognizing polypeptides derived from AFP proteins. The invention also relates to the preparation and use of said receptors.
Background
Only two types of molecules are able to recognize antigens in a specific manner. One of which is an immunoglobulin or antibody; the other is the T Cell Receptor (TCR), which is a cell membrane surface glycoprotein that exists as a heterodimer from the α chain/β chain or the γ chain/δ chain. The composition of the TCR repertoire of the immune system is produced by V (D) J recombination in the thymus, followed by positive and negative selection. In the peripheral environment, TCRs mediate the specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells, and are therefore critical for the cellular immune function of the immune system.
TCRs are the only receptors for specific antigenic peptides presented on the Major Histocompatibility Complex (MHC), and such exogenous or endogenous peptides may be the only signs of cellular abnormalities. 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.
The MHC class I and II molecular ligands corresponding to the TCR are also proteins of the immunoglobulin superfamily but are specific for presentation of antigens, with different individuals having different MHC, and thereby presenting different short peptides of a single protein antigen to the cell surface of the respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.
AFP (alpha Fetoprotein), also called alpha Fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP is expressed at relatively high levels in the yolk sac and liver, and is subsequently inhibited. In liver cancer, expression of AFP is activated. AFP is processed intracellularly into antigenic peptides and bound to MHC (major histocompatibility complex) molecules to form complexes, which are presented to the cell surface. TSSELMAITR is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.
Thus, the TSSELMAITR-HLA a1101 complex provides a marker for the targeting of TCRs to tumor cells. The TCR capable of combining with the TSSELMAITR-HLA A1101 complex has high application value for treating tumors. For example, TCRs capable of targeting the tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or to be transformed into T cells, such that T cells expressing the TCR can destroy tumor cells for administration to a patient in a treatment process known as adoptive immunotherapy. For the former purpose, the ideal TCR is of higher affinity, enabling the TCR to reside on the targeted cell for a long period of time. For the latter purpose, it is preferred to use a medium affinity TCR. Accordingly, those skilled in the art are working to develop TCRs that target tumor cell markers that can be used to meet different objectives.
Disclosure of Invention
It is an object of the present invention to provide a TCR with improved high affinity for the TSSELMAITR-HLA a1101 complex.
It is a further object of the present invention to provide a method for preparing a TCR of the above type and uses thereof.
In a first aspect of the invention, there is provided a T Cell Receptor (TCR) comprising an α chain variable domain and a β chain variable domain, which has binding activity to the TSSELMAITR-HLA a1101 complex, and the amino acid sequence of the TCR α chain variable domain has at least 90% sequence homology to the amino acid sequence set forth in SEQ ID No. 1 and the amino acid sequence of the TCR β chain variable domain has at least 90% sequence homology to the amino acid sequence set forth in SEQ ID No. 2.
In a preferred embodiment, the amino acid sequence of the TCR a chain variable domain and the amino acid sequence of the TCR β chain variable domain are not the amino acid sequence of the wild-type TCR a chain variable domain and the amino acid sequence of the wild-type TCR β chain variable domain at the same time.
In further preferred embodiments, the amino acid sequence of the variable domain of the TCR α chain is not the amino acid sequence shown in SEQ ID NO. 1, or the amino acid sequence of the variable domain of the TCR β chain is not the amino acid sequence shown in SEQ ID NO. 2.
In another preferred embodiment, the α chain variable domain of the TCR comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the sequence set forth in SEQ ID No. 1. Preferably, the amino acid sequence of the variable domain of the TCR alpha chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the β chain variable domain of the TCR is an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the sequence set forth in SEQ ID No. 2. Preferably, the amino acid sequence of the variable domain of the TCR β chain has at least 95% sequence homology with the amino acid sequence set forth in SEQ ID NO. 2.
In another preferred embodiment, CDR1 α in the variable domain of TCR α chain is: DSVNN, and CDR2 α are IPSGT.
In another preferred embodiment, the CDR3 α of the TCR α chain variable domain is selected from the group consisting of SGGSGFRLT, SGGQGYKLT and SGGSNYKLT.
In another preferred embodiment, CDR1 α in the variable domain of TCR α chain is DSVNN, CDR2 α is IPSGT, and CDR3 α is selected from SGGSGFRLT, SGGQGYKLT and SGGSNYKLT.
In another preferred embodiment, the 3 CDRs of the TCR β chain variable domain are: CDR 1. Beta.: SEHNR; CDR 2. Beta. Is: FQNEAQ; and CDR3 β: ASSPGTGVGYT.
In another preferred embodiment, the amino acid sequence of the variable domain of the TCR β chain is SEQ ID NO 2.
In another preferred embodiment, the number of amino acid mutations in the variable domain of the TCR α chain is 1 to 3; preferably 3.
In another preferred embodiment, the amino acid mutation site of the TCR α chain variable domain is the 5 th, 6 th, 7 th position of CDR3 α.
In another preferred embodiment, the amino acid mutation sites of the TCR α chain variable domain are positions 4 and 5 of CDR3 α.
In another preferred embodiment, the reference sequence of the 3 CDR regions (complementarity determining regions) of the variable domain of the TCR alpha chain is as follows,
CDR1α：DSVNN
CDR2α：IPSGT
CDR3 α: SGGSNYKLT, and CDR3 α contains at least one of the following mutations:
residues before mutation Residues after mutation
Position 5N of CDR3 α G
Position 6Y of CDR3 α F
Position 7K of CDR3 alpha R
4 th position S of CDR3 alpha Q
In another preferred embodiment, the amino acid mutation in CDR3 α comprises:
residues before mutation Post-mutation residues
Position 5N of CDR3 α G
In another preferred embodiment, the reference sequence of 3 CDR regions (complementarity determining regions) of the variable domain of TCR β chain is as follows,
CDR1β：SEHNR
CDR2β：FQNEAQ
CDR3. Beta.: ASSPGTGVGYT, and CDR3 β contains at least one of the following mutations:
residues before mutation Post-mutation residues
P4 of CDR3 beta H
Position 5G of CDR3 beta P or Q
6 th position T of CDR3 beta Q or H
V at position 8 of CDR3 beta I
9 th G of CDR3 β Q or R or S or I or L or V
Position 10Y of CDR3 beta Q or F or M
11 th position T of CDR3 β E or Q or H or K or R or L
Preferably, the amino acid mutation in CDR3 β comprises:
residues before mutation Post-mutation residues
V at position 8 of CDR3 beta I
In another preferred embodiment, the TCR has at least 2-fold greater affinity for the TSSELMAITR-HLA a1101 complex than a wild-type TCR.
In another preferred embodiment, the TCR has a mutation in the alpha chain variable domain as shown in SEQ ID NO. 1 selected from one or more of S91Q, N92G, Y93F, K94R, wherein the numbering of the amino acid residues is as shown in SEQ ID NO. 1.
In another preferred embodiment, the TCR has a mutation in the beta chain variable domain shown in SEQ ID NO. 1 selected from one or more of P96H, G97P/Q, T98Q/H, V100I, G101Q/R/S/I/L/V, Y102Q/F/M, T103E/Q/H/K/L/R, wherein the numbering of the amino acid residues is as shown in SEQ ID NO. 2.
In another preferred embodiment, the TCR has CDRs selected from the group consisting of:
Figure BDA0003122492250000041
Figure BDA0003122492250000051
in another preferred embodiment, the TCR is soluble.
In another preferred embodiment, the TCR is an α β heterodimeric TCR comprising an α chain TRAC constant region sequence and a β chain TRBC1 or TRBC2 constant region sequence.
In another preferred embodiment, the TCR comprises (i) a TCR α chain variable domain and all or part of a TCR α chain constant region other than the transmembrane domain; and (ii) a TCR β chain variable domain and all or part of a TCR β chain constant region other than the transmembrane domain.
In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain constant region and the β chain constant region.
In another preferred embodiment, the cysteine residues that form the artificial interchain disulfide bond between the constant regions of the TCR α and β chains replace one or more groups of sites selected from:
thr48 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser57 of TRBC2 × 01 exon 1;
thr45 of TRAC × 01 exon 1 and TRBC1 × 01 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 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1;
ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15;
arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1;
pro89 of TRAC × 01 exon 1 and TRBC1 × 01 or Ala19 of TRBC2 × 01 exon 1;
and Tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu20.
In another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR is one of SEQ ID NOs 1, 5-7; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 8-27.
In another preferred embodiment, the TCR is selected from the group consisting of:
Figure BDA0003122492250000052
Figure BDA0003122492250000061
in another preferred embodiment, the TCR is of human origin.
In another preferred embodiment, the TCR is a single chain TCR.
In another preferred embodiment, the TCR is a single chain TCR consisting of an alpha chain variable domain and a beta chain variable domain, the alpha and beta chain variable domains being linked by a flexible short peptide sequence (linker).
In another preferred embodiment, the TCR comprises an alpha chain constant region and a beta chain constant region, the alpha chain constant region being a murine constant region and/or the beta chain constant region being a murine constant region.
In another preferred embodiment, the C-or N-terminus of the α chain and/or β chain of the TCR is conjugated to a conjugate, preferably a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these.
In another preferred embodiment, the therapeutic agent that binds to the TCR is an anti-CD 3 antibody linked to the C-or N-terminus of the α or β chain of the TCR.
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 a multivalent TCR complex according to the second aspect of the invention, or a complement thereof.
In a fourth aspect of the invention, there is provided a vector comprising the nucleic acid molecule of the third aspect of the invention.
In a fifth aspect of the invention, there is provided a host cell comprising a vector or chromosome of the fourth aspect of the invention and, integrated therein, an exogenous nucleic acid molecule of the third aspect of the invention.
In a sixth aspect of the invention there is provided an isolated cell expressing a TCR according to the first aspect of the invention, preferably the isolated cell is a T cell, NK cell or NKT cell, most preferably the isolated cell is a T 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, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention.
In an eighth 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 TCR according to the first aspect of the invention, or a TCR complex according to the second 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 an AFP-positive tumour, more preferably the tumour is liver cancer.
In a ninth aspect of the invention there is provided the use of a TCR of the first aspect of the invention, or a TCR complex of the second aspect of the invention, or a cell of the sixth aspect of the invention, in the manufacture of a medicament for the treatment of a tumour, preferably the disease is an AFP-positive tumour, more preferably the tumour is liver cancer.
In a tenth aspect of the invention, there is provided a method of preparing a T cell receptor according to the first aspect of the invention, comprising the steps of:
(i) Culturing a host cell according to the fifth aspect of the invention, thereby expressing a T-cell receptor according to the first aspect of the invention;
(ii) Isolating or purifying said T cell receptor.
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 repeated herein, depending on the space.
Drawings
FIGS. 1a and 1b show the amino acid sequences of the variable domains of the wild-type TCR α chain, β chain, respectively, capable of specifically binding to the TSSELMAITR-HLA A1101 complex.
FIGS. 2a and 2b show the amino acid sequences of soluble reference TCR α and β chains, respectively, of the invention.
Figures 3 (1) - (3) show the α chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for the TSSELMAITR-HLA a1101 complex, respectively, with mutated residues underlined.
Figures 4 (1) - (20) show the β chain variable domain amino acid sequence of a heterodimeric TCR with high affinity for the TSSELMAITR-HLA a1101 complex, respectively, with mutated residues underlined.
FIGS. 5a and 5b show the extracellular amino acid sequences of the α and β chains of a wild-type TCR capable of specifically binding to the TSSELMAITR-HLA A1101 complex, respectively.
FIGS. 6a and 6b show the amino acid sequences of the wild-type TCR α and β chains, respectively, capable of specifically binding to the TSSELMAITR-HLA A1101 complex.
FIG. 7 is a graph of the binding of soluble reference TCR, i.e., wild-type TCR, to the TSSELMAITR-HLA A1101 complex.
FIG. 8 is a graph of the results of an experiment on the activation function of effector cells transfected with the high affinity TCR of the invention, in response to T2 cells loaded with short peptides.
FIGS. 9a and 9b are experimental results of the activation function of effector cells transfected with the high affinity TCR of the invention against tumor cell lines.
FIGS. 10a and 10b are experimental results of the killing function of effector cells transfected with the high affinity TCR of the invention against tumor cell lines.
Detailed Description
The present inventors, through extensive and intensive studies, have obtained a T Cell Receptor (TCR) that recognizes TSSELMAITR short peptides (derived from AFP protein) presented as a peptide-HLA a1101 complex. The TCR has 3 CDR regions in its alpha chain variable domain:
CDR1α：DSVNN
CDR2α：IPSGT
CDR3 α: mutations occur in SGGSNYKLT; and/or in the 3 CDR regions of its beta chain variable domain:
CDR1β：SEHNR
CDR2β：FQNEAQ
CDR3. Beta.: mutations occur in ASSPGTGVGYT.
In another preferred embodiment, the affinity and/or binding half-life of the inventive TCR after mutation to the TSSELMAITR-HLA a1101 complex described above is at least 2-fold that of a wild-type TCR.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, 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 belongs.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
Term(s)
T Cell Receptor (TCR)
The TCR may be described using the international immunogenetics information system (IMGT). Native α β heterodimeric TCRs have an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linker region and a constant region, and the beta chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered part of the linker region. The TCR junction region is defined by the TRAJ and TRBJ of the unique IMGT, and the TCR constant region is defined by the TRAC and TRBC of the IMGT.
Each variable region comprises 3 CDRs (complementarity determining regions) CDR1, CDR2 and CDR3, which are chimeric in the framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different types of V α and V β, respectively. In the IMGT system, the α chain constant domain has the following symbols: TRAC 01, wherein "TR" denotes a T cell receptor gene; "A" represents an alpha chain gene; c represents a constant region; "x01" indicates allele 1. The beta-strand constant domain has the following notation: TRBC1 x 01 or TRBC2 x 01, wherein "TR" denotes a T cell receptor gene; "B" represents a beta chain gene; c represents a constant region; "x01" indicates allele 1. The constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes, "C1" and "C2". The constant region gene sequences of the TCR alpha and beta chains can be obtained by those skilled in the art from published IMGT databases.
The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of linked variable regions and linked regions. Thus, in the description and claims of this application, the "TCR α chain variable domain" refers to the linked TRAV and TRAJ regions, and likewise the "TCR β chain variable domain" refers to the linked TRBV and TRBD/TRBJ regions. The 3 CDRs of the TCR α chain variable domain are CDR1 α, CDR2 α and CDR3 α, respectively; the 3 CDRs of the variable domain of the TCR β chain are CDR1 β, CDR2 β and CDR3 β, respectively. The framework sequences of the TCR variable domains of the invention may be murine or human, preferably human. The constant domain of the TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion.
The extracellular amino acid sequence of the alpha chain and the extracellular amino acid sequence of the beta chain of the wild-type TCR are respectively SEQ ID NO 28 and SEQ ID NO 29, as shown in FIG. 5a and FIG. 5 b. The TCR sequences used in the present invention are of human origin. The alpha chain amino acid sequence and the beta chain amino acid sequence of the wild-type TCR are respectively SEQ ID NO. 30 and SEQ ID NO. 31, as shown in figure 6a and figure 6 b. 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, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".
For convenience of description, the amino acid sequences of TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 are numbered sequentially from N to C, for example, in TRBC1 × 01 or TRBC2 × 01, the 60 th amino acid in the sequence from N to C is P (proline), and thus, in the present invention, it may 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 also as the 61 th amino acid in the sequence from N to C is Q (glutamine), and thus, in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01, and also as the Gln of TRBC1 × 61 or TRBC2 × 01 exon 1, and also as the other TRBC1 or TRBC2 × 01, and so forth, the position numbers of TRBC1 × 01 or TRBC2 × 01 may be numbered sequentially from N to C. 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.
Tumor(s)
The term "tumor" is meant to include all types of cancer cell growth or carcinogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or the stage of infection. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignancies of different organ systems, such as sarcomas, squamous carcinomas of the lung and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx. Squamous cell lung cancer includes malignant tumors, such as, for example, a plurality of colon, rectal, renal cell, liver, non-small cell, small intestine, and esophageal cancers. The metastatic lesions of the above-mentioned cancers can likewise be treated and prevented using the methods and compositions of the present invention.
Detailed Description
It is well known that the α chain variable domain and β chain variable domain of a TCR each contain 3 CDRs, similar to the complementarity determining regions of an antibody. CDR3 interacts with antigen short peptides, CDR1 and CDR2 interact with HLA. Thus, the CDRs of the TCR molecule determine their interaction with the antigen short peptide-HLA complex. The amino acid sequences of the alpha chain variable domain and the beta chain variable domain of the wild-type TCR capable of binding the antigen short peptide TSSELMAITR and HLA A1101 complex (i.e., TSSELMAITR-HLA A1101 complex) are SEQ ID NO. 1 and SEQ ID NO. 2, respectively, which were discovered by the inventors for the first time. It has the following CDR regions:
alpha chain variable domain CDR1 α: DSVNN
CDR2α：IPSGT
CDR3α：SGGSNYKLT
And the beta chain variable domain CDR1 β: SEHNR
CDR 2. Beta.: FQNEAQ and
CDR3β：ASSPGTGVGYT。
the invention obtains the TCR with improved affinity with the TSSELMAITR-HLA A1101 compound and improved cell function by carrying out mutation screening on the CDR region.
Further, the TCR of the invention is an α β heterodimeric TCR or a single chain TCR comprising an α chain variable domain and a β chain variable domain, the α chain variable domain of the TCR comprising at least 85% of the amino acid sequence as set forth in SEQ ID No. 1; preferably, at least 90%; more preferably, at least 92%; more preferably, at least 94% (e.g., can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of sequence homology of amino acid sequence; and/or the β chain variable domain of the TCR comprises at least 90%, preferably at least 92%, of the amino acid sequence set forth as SEQ ID No. 2; more preferably, at least 94% (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology) of sequence homology.
The 3 CDRs of the variable domain SEQ ID NO:1 of the wild-type TCR alpha chain of the invention, i.e., CDR1, CDR2 and CDR3, are located at positions 27-31, 49-53 and 88-96 of SEQ ID NO:1, respectively. Accordingly, the amino acid residue numbering is as shown in SEQ ID NO. 1, wherein 91S is the 4 th position S of CDR3 alpha, 92N is the 5 th position N of CDR3 alpha, 93Y is the 6 th position Y of CDR3 alpha, and 94K is the 7 th position K of CDR3 alpha.
In particular, particular forms of said mutations in the alpha chain variable domain include one or more of S91Q, N92G, Y93F, K94R.
The 3 CDRs of the variable domain SEQ ID NO. 2 of the wild-type TCR beta chain of the invention, i.e., CDR1, CDR2 and CDR3, are located at positions 27-31, 49-54 and 93-103 of SEQ ID NO. 2, respectively. Accordingly, the amino acid residue numbering is as shown in SEQ ID NO 1, with 96P being the 4 th P of CDR3 β, 97G being the 5 th G of CDR3 β, 98T being the 6 th T of CDR3 β, 100V being the 8 th V of CDR3 β, 101G being the 9 th G of CDR3 β, 102Y being the 10 th Y of CDR3 β, and 103T being the 11 th T of CDR3 β.
Specifically, the specific form of the mutation in the variable domain of the beta chain includes one or more of P96H, G97P/Q, T98Q/H, V100I, G101Q/R/S/I/L/V, Y102Q/F/M, T103E/Q/H/K/L/R.
It should be understood that the amino acid names herein are given by the international single english letter designation, and the three english letters abbreviation corresponding to the amino acid names are: 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), val (V);
in the present invention, pro60 or 60P both represent proline at position 60. In addition, the present invention describes specific forms of the mutations such as "N92G" representing the substitution of N at position 92 with G, "G97P/Q" representing the substitution of G at position 97 with P or with Q, and so on.
The reference TCR was obtained by mutating Pro89 of exon 1 of the α chain constant region TRAC 01 of the wild-type TCR to cysteine and Ala19 of exon 1 of the β chain constant region TRBC 101 or TRBC 201 to cysteine according to site-directed mutagenesis methods well known to those skilled in the art, the amino acid sequences of which are SEQ ID No. 3 and SEQ ID No. 4, respectively, as shown in fig. 2a and fig. 2b, and the mutated cysteine residues are shown in bold letters. The cysteine substitutions described above enable the formation of an artificial interchain disulfide bond between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, thereby enabling a more convenient assessment of the binding affinity and/or half-life of the TCR with the TSSELMAITR-HLA a1101 complex. It will be appreciated that the CDR regions of the TCR variable region determine their affinity for the pMHC complex and therefore cysteine substitutions in the TCR constant region as described above do not affect the binding affinity and/or binding half-life of the TCR. Thus, in the present invention, the measured binding affinity between the reference TCR and the TSSELMAITR-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the TSSELMAITR-HLA A1101 complex. Similarly, if the binding affinity between the inventive TCR and the TSSELMAITR-HLA a1101 complex is determined to be at least 2-fold greater than the binding affinity between the reference TCR and the TSSELMAITR-HLA a1101 complex, i.e., equivalent to at least 2-fold greater than the binding affinity between the inventive TCR and the TSSELMAITR-HLA a1101 complex relative to the wild-type TCR and the TSSELMAITR-HLA a1101 complex.
Binding affinity (inversely proportional to the dissociation equilibrium constant, KD) and binding half-life (expressed as T) can be determined by any suitable method 1/2 ) For example, surface plasmon resonance. It will be appreciated that doubling the affinity of the TCR will result in a halving of the KD. T is a unit of 1/2 Calculated as In2 divided by the dissociation rate (Koff). Thus, T 1/2 Doubling results in half the Koff. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, e.g. 3 times or more, using the same assay protocol, and the results are averaged. In a preferred embodiment, the surface plasmon resonance (BIAcore) method of the examples herein is used to detect the affinity of soluble TCRs, provided that: the temperature is 25 ℃, and the pH value is 7.1-7.5. The method detects that the dissociation equilibrium constant KD of the reference TCR to the TSSELMAITR-HLA A1101 complex is 4.16E-05M, namely 41.6 mu M, and in the invention, the dissociation equilibrium constant KD of the wild type TCR to the TSSELMAITR-HLA A1101 complex is also considered to be 41.6 mu M. Since doubling the affinity of the TCR will result in a halved KD, a high affinity TCR with a dissociation equilibrium constant KD of 4.16E-06M, i.e. 4.16. Mu.M, for the TSSELMAITR-HLA A1101 complex is detected, indicating that the affinity of the high affinity TCR for the TSSELMAITR-HLA A1101 complex is 4.16. Mu.MThe force was 10 times the affinity of the wild-type TCR for the TSSELMAITR-HLA a1101 complex. The conversion between units of KD values, i.e. 1m =10, is well known to the person skilled in the art 6 μM,1μM＝1000nM。
The mutation may be performed using any suitable method, including but not limited to those based on Polymerase Chain Reaction (PCR), cloning based on restriction enzymes, or Ligation Independent Cloning (LIC) methods. These methods are detailed in a number of standard molecular biology texts. For more details on Polymerase Chain Reaction (PCR) mutagenesis and Cloning by restriction enzymes, see Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (third edition) CSHL publisher. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6).
The method of producing the TCRs of the invention may be, but is not limited to, screening a diverse library of phage particles displaying such TCRs for TCLMAITR-HLA-A1101 complex with high affinity for the TSSELMAITR-HLA-A1101 complex, as described in the literature (Li, et al (2005) Nature Biotech 23 (3): 349-354).
It will be appreciated that genes expressing the α and β chain variable domain amino acids of a wild type TCR, or genes expressing slightly modified α and β chain variable domain amino acids of a wild type TCR, may be used to make a template TCR. The alterations required to produce the high affinity TCRs of the invention are then introduced into the DNA encoding the variable domains of the template TCR.
The high affinity TCR of the invention comprises an alpha chain variable domain amino acid sequence of one of SEQ ID NO 1, 5-7; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 8-27. The amino acid sequences of the α chain variable domain and the β chain variable domain of the heterodimeric TCR molecules of the invention are preferably selected from table 1 below:
TABLE 1
Figure BDA0003122492250000131
Figure BDA0003122492250000141
For the purposes of the present invention, the inventive TCRs are moieties having at least one TCR α and/or TCR β chain variable domain. They typically comprise both a TCR α chain variable domain and a TCR β chain variable domain. They may be α β heterodimers or single chain forms or any other form that is stable. In adoptive immunotherapy, the full-length chain (comprising the cytoplasmic and transmembrane domains) of the α β heterodimeric TCR can be transfected. The inventive TCRs may be used as targeting agents for delivering therapeutic agents to antigen presenting cells or in combination with other molecules to produce bifunctional polypeptides for targeting effector cells, where the TCR is preferably in soluble form.
For stability, it is disclosed in the prior art that introduction of an artificial interchain disulfide bond between the α and β chain constant domains of the TCR enables soluble and stable TCR molecules to be obtained, as described in patent document PCT/CN 2015/093806. Thus, the inventive TCR may be one in which an artificial interchain disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substitution of Pro89 of TRAC × 01 exon 1 and substitution of Ala19 of TRBC1 × 01 or TRBC2 × 01 exon 1. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser77 of TRBC2 × 01 exon 1; tyr10 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Ser17; thr45 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1; ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15; arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1; thr48 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser57 of TRBC2 × 01 exon 1; or Tyr10 of exon 1 TRAC 01 and TRBC 101 or TRBC 201 of Glu20 of exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. Deletion of the native interchain disulfide bonds can be achieved by truncating at most 15, or at most 10, or at most 8 or fewer amino acids at one or more of the C-termini of the TCR constant domains of the invention such that they do not include a cysteine residue, or by mutating a cysteine residue which forms a native interchain disulfide bond to another amino acid.
As described above, the TCRs of the invention may comprise an artificial interchain disulfide bond introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bond introduced as described above between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by the native interchain disulfide bonds present in the TCR.
In addition, for stability, PCT/CN2016/077680 also discloses 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. Thus, the high affinity TCR of the present invention may also comprise an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: 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 exon 1 of TRBC1 x 01 or TRBC2 x 01; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.
For stability, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that increase the stability of the inventive TCRs, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or alpha chain J gene (TRAJ) short peptide amino acid positions 3, 5, 7, and/or beta chain J gene (TRBJ) short peptide amino acid positions 2, 4, 6, wherein the numbering of the amino acid sequence positions is according to the numbering of the positions listed in the International Immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.
More specifically, the TCR with the mutated hydrophobic core region of the present invention may be a high stability single chain TCR comprising a flexible peptide chain connecting the variable domains of the α chain and β chain of the TCR. The CDR regions of the variable region of the TCR determine the affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can stabilize the TCR without affecting the affinity with the short peptide-HLA complex. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains.
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 formed by tetramer formation with the tetrameric domain of p53, or complexes 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, where the TCR is used to detect the presence of cells presenting the TSSELMAITR-HLA-a1101 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 with or coupled to the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, nature 339, 394, epel et al, 2002, cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 etc (Gillies et al, 1992, journal of the national academy of sciences (PNAS) 89, 1428, card et al, 2004, cancer Immunology and Immunotherapy) 53, 345, hain 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 communications (Cancer letters) 239, 36, huang et al, 2006, journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, cancer research) 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, and the like.
Antibodies or fragments thereof that bind to the inventive TCR include anti-T cell or NK-cell determining antibodies, such as anti-CD 3 or anti-CD 28 or anti-CD 16 antibodies, whose binding to the TCR can target effector cells for better targeting of target cells. A preferred embodiment is the binding of a TCR of the invention to an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody. Specifically, the fusion molecule of the TCR and the anti-CD 3 single-chain antibody comprises a TCR alpha chain variable domain amino acid sequence which is one of SEQ ID NO 1 and 5-7; and/or the amino acid sequence of the variable domain of the beta chain of the TCR is one of SEQ ID NO 2, 8-27.
The invention also relates to nucleic acid molecules encoding the inventive TCRs. The nucleic acid molecules of the invention may be in the form of DNA or in the form of RNA. The DNA may be the coding strand or the non-coding strand. For example, a nucleic acid sequence encoding a TCR of the present invention may be identical to or a degenerate variant of a nucleic acid sequence as set out in the figures of the present invention. By way of illustration of the meaning of "degenerate variant", as used herein, is meant a nucleic acid sequence which encodes a protein sequence having SEQ ID NO. 3, but differs from the sequence of SEQ ID NO. 5.
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 can then be introduced into various existing DNA molecules (or e.g., vectors) and cells known in the art.
The invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as genetically engineered host cells with the vectors or coding sequences of the invention.
The invention also includes isolated cells, particularly T cells, expressing a TCR of the invention. There are many methods suitable for T cell transfection using DNA or RNA encoding the high affinity TCRs of the invention (e.g., robbins et al, (2008) j. Immunol.180: 6116-6131). T cells expressing the high affinity 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 Rev Cancer 8 (4): 299-308).
The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention.
The invention also provides a method of treating a disease comprising administering to a subject in need thereof an amount of a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention, or a pharmaceutical composition of the invention.
In the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. Thus, the TCR of the invention also includes TCRs in which up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid (especially outside the CDR regions) of the TCR of the invention has been replaced by amino acids of similar or analogous nature, and still retain its functionality.
The invention also includes TCRs that are slightly modified from the TCRs of the invention. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the inventive TCR such as acetylation or carboxylation. Modifications also include glycosylation, such as those that result from glycosylation modifications made during synthesis and processing or during further processing steps of the inventive TCR. Such modification may be accomplished by exposing the TCR to an enzyme that effects glycosylation, such as mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to improve their resistance to proteolysis or to optimize solubility.
The TCRs of the invention, TCR complexes or TCR-transfected T cells of the invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCRs, multivalent TCR complexes or cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.
In addition, the TCRs of the invention can be used alone, or in combination or conjugation with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. Co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
Pharmaceutically acceptable carriers in therapeutic compositions can comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.
Once formulated, the compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated may be an animal; especially a human.
When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, injections, oral agents and the like are exemplified.
These pharmaceutical compositions may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may be carried out in a conventional manner according to the dosage form.
The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the inventive TCR may be incorporated into a pellet or microcapsule carried by a slow release polymer, which pellet or microcapsule is then surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.
When the pharmaceutical composition of the present invention is used for practical treatment, the TCR or TCR complex of the present invention or the cells presenting the TCR of the present invention as an active ingredient can be determined reasonably according to the body weight, age, sex, degree of symptoms of each patient to be treated, and finally the reasonable amount is decided by a physician.
The main advantages of the invention are:
(1) The inventive TCRs are capable of specifically binding to the TSSELMAITR-HLA a1101, while cells transfected with the inventive high affinity TCRs are capable of being specifically activated.
(2) Effector cells transfected with the high affinity TCRs of the invention have strong specific killing effects.
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 noted 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.
Materials and methods
The experimental materials used in the examples of the present invention are commercially available from commercial sources, as for example, e.coli DH5 α, e.coli BL21 (DE 3), e.coli Tuner (DE 3), novagen, and plasmid pET28a, without specific reference.
Example 1 binding characterization BIAcore analysis
The binding activity of the TCR molecules to the TSSELMAITR-HLA-A1101 complex was measured using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000RU. The conditions are as follows: the temperature is 25 ℃, and the pH value is 7.1-7.5.
And (2) enabling low-concentration streptavidin to flow through the surface of the chip coated with the antibody, then enabling the TSSELMAITR-HLA-A1101 complex to flow through a detection channel, enabling the other channel to serve as a reference channel, and enabling 0.05mM biotin to flow through the chip at the flow rate of 10 mu L/min for 2min to block the remaining binding sites of the streptavidin. The affinity was determined by single cycle kinetic assay by diluting TCR with HEPES-EP buffer (10 mM HEPES,150mM NaCl,3mM EDTA,0.005% P20, pH 7.4) to several different concentrations, sequentially flowing over the chip surface at a flow rate of 30. Mu.L/min for a binding time of 120s for each sample, and dissociating for 600s after the last sample. At the end of each assay run, the chip was regenerated with 10mM Gly-HCl pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software. The binding curve for the soluble reference TCR, i.e., the wild-type TCR, to the TSSELMAITR-HLA a1101 complex is shown in figure 7.
The TSSELMAITR-HLA-A1101 complex is prepared by the following steps:
a. and (3) purification: collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.
Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl with pH of 8.0 to resuspend the inclusion bodies, mixing uniformly, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.
b. Renaturation: the synthesized short peptide TSSELMAITR (Jiangsu Kingsler Biotech Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized using 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. TSSELMAITR peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE was checked for success or failure.
c. And (3) purification after renaturation: the renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least twice to reduce the ionic strength of the solution sufficiently. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric) anion exchange column (5 ml bed volume). The protein was eluted using a linear gradient of 0-400mM NaCl prepared using an Akta purifier (GE general electric) at 20mM Tris pH 8.0, pMHC was eluted at about 250mM NaCl, and the peak fractions were collected and subjected to purity detection using SDS-PAGE.
d. Biotinylation: the purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while replacing the buffer with 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu.M D-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture overnight at room temperature, and SDS-PAGE to determine whether biotinylation was complete.
e. Purification of biotinylated complex: the biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, the HiPrepTM 16/60S200 HR column (GE general electric) was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric), loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal elution at approximately 55 ml. The fractions containing the protein were pooled, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by the BCA method (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ with the addition of the protease inhibitor cocktail (Roche).
Example 2 Generation of high affinity TCR
Phage display technology is one means of generating libraries of TCR high affinity variants to screen for high affinity variants. The TCR phage display and screening methods described by Li et al ((2005) Nature Biotech 23 (3): 349-354) were applied to wild-type TCR templates. The CDR regions of the template strand are mutated using site-directed mutagenesis methods well known to those skilled in the art to create a library of high affinity TCRs and panning. Phage libraries after several rounds of panning have specific binding to corresponding antigens, from which monoclonal antibodies are picked and analyzed.
The affinity of TSSELMAITR-HLA-A1101 complex was determined by BIAcore. The amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in FIG. 1a (SEQ ID NO: 1) and FIG. 1b (SEQ ID NO: 2), respectively.
Extracellular sequence genes of TCR α and β chains to be expressed were synthesized and inserted into expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning a Laboratory Manual (third edition, sambrook and Russell), with upstream and downstream Cloning sites being NcoI and NotI, respectively. Mutations in the CDR regions are introduced by overlap PCR (overlap PCR) which is well known to those skilled in the art. The insert was confirmed by sequencing without error.
Example 3 expression, renaturation and purification of high affinity TCR
The expression vectors for TCR α and β chains were transformed into expression bacteria BL21 (DE 3) by chemical transformation, respectively, the bacteria were grown in LB broth, induced with final concentration 0.5mM IPTG at OD600=0.6, and the inclusion bodies formed after TCR α and β chain expression were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times, and finally the inclusion bodies were dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris (pH 8.1).
The solubilized TCR α and β chains were rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM β -mercapoethylamine (4 ℃) at a mass ratio of 1. After mixing, the solution was dialyzed against 10 times the volume of deionized water (4 ℃ C.), and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. Mu.M filter and then purified by an anion exchange column (HiTrap Q HP,5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently further purified by gel filtration chromatography (HiPrep 16/60, sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA.
Example 4BIAcore analysis results
The affinity of the α β heterodimeric TCR of the high affinity CDRs of the present invention to the TSSELMAITR-HLA-a1101 complex was tested using the method described in example 1.
The amino acid sequences of the α chain and β chain variable domains of the novel TCR obtained by the invention are shown in FIGS. 3 (1) - (3) and FIGS. 4 (1) - (20), respectively. Expression vectors were constructed using the method described in example 2, and the high affinity mutated α β heterodimeric TCRs described above were expressed, renatured and purified using the method described in example 3, and then their affinity for the TSSELMAITR-HLA-a1101 complex was determined using BIAcore T200, as shown in table 2 below.
TABLE 2
Figure BDA0003122492250000221
Figure BDA0003122492250000231
As can be seen from table 2 above, the affinity of the heterodimeric TCR is at least 2-fold greater than the affinity of the wild-type TCR for the TSSELMAITR-HLA-a1101 complex.
Example 5 expression, renaturation and purification of fusions of anti-CD 3 antibodies with high affinity α β heterodimeric TCRs
Fusion molecules were prepared by fusing anti-CD 3 single chain antibodies (scFv) to α β heterodimeric TCR. The scFv of the anti-CD 3 is fused to the β chain of the TCR, which may comprise the β chain variable domain of any of the above-described high affinity α β heterodimeric TCRs, and the TCR α chain of the fused molecule may comprise the α chain variable domain of any of the above-described high affinity α β heterodimeric TCRs.
Construction of fusion molecule expression vectors
1. Construction of alpha chain expression vector: the target gene carrying the alpha chain of the alpha beta heterodimeric TCR is subjected to double enzyme digestion by Nco I and Not I and is connected with a pET28a vector subjected to double enzyme digestion by Nco I and Not I. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli Tuner (DE 3) for expression.
2. Construction of anti-CD 3 (scFv) -beta chain expression vector: by the overlap PCR method, primers are designed to connect the anti-CD 3 scFv and the high-affinity heterodimeric TCR beta chain gene, the middle connecting short peptide (linker) is GGGGS, and the gene segment of the fusion protein of the anti-CD 3 scFv and the high-affinity heterodimeric TCR beta chain is provided with restriction enzyme sites Nco I (CCATGG) and Not I (GCGGCCGC). The PCR amplification product was double-digested with Nco I and Not I, and ligated to pET28a vector double-digested with Nco I and Not I. The ligation product was transformed into E.coli DH 5. Alpha. Competent cells, coated with LB plates containing kanamycin, inverted cultured overnight at 37 ℃, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into E.coli Tuner (DE 3) competent cells for expression.
Expression, renaturation and purification of fusion proteins
The expression plasmids were transformed into E.coli Tuner (DE 3) competent cells, respectively, and LB plates (kanamycin 50. Mu.g/mL) were plated and incubated at 37 ℃ overnight. The next day, the selected clones were inoculated into 10mL of LB liquid medium (kanamycin 50. Mu.g/mL) for 2-3 hours, inoculated into 1L of LB medium at a volume ratio of 1. After 4 hours of induction, cells were harvested by centrifugation at 6000rpm for 10 min. The cells were washed once with PBS buffer and aliquoted, and 200mL of the cells from the bacterial culture were lysed with 5mL of BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000g for 15 min. 4 detergent washes were then performed to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergents and salts. Finally, inclusion bodies were dissolved in a buffer solution containing 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 2mM Tris, pH 8.1, and the inclusion body concentration was measured, and they were stored in portions at-80 ℃ for cryopreservation.
The TCR α chain and the anti-CD 3 (scFv) - β chain after solubilization were separated by a 2:5 in 5M Urea (urea), 0.4M L-arginine (L-arginine), 2mM Tris pH 8.1,3.7mM cystamine,6.6mM β -mer capoethylamine (4 ℃ C.), the final concentrations of α chain and anti-CD 3 (scFv) - β chain were 0.1mg/mL and 0.25mg/mL, respectively.
After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and, after 12 hours, the deionized water was changed to a buffer (10mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. Mu.M filter and then purified by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The eluted peaks contain TCR alpha chain and anti-CD 3 (scFv) -beta chain dimers of which the renaturation was successful TCR alpha chain was confirmed by SDS-PAGE gel. The TCR fusion molecules were subsequently further purified by size exclusion chromatography (S-100/60, GE healthcare) and re-purified on an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The purity of the purified TCR fusion molecule is greater than 90% as determined by SDS-PAGE and the concentration is determined by BCA.
Example 6 activation function experiment of Effector cells transfected with high affinity TCR of the invention against short peptide-loaded T2 cells
IFN- γ is a potent immunomodulatory factor produced by activated T lymphocytes, and therefore this example examines the IFN- γ numbers by ELISPOT assays well known to those skilled in the art to verify the activation function and antigen specificity of cells transfected with the high affinity TCR of the invention. The high affinity TCRs of the invention (TCR numbers and their serial numbers are known from table 2) were transfected into CD3+ T cells isolated from blood of healthy volunteers as effector cells, and CD3+ T cells transfected with other TCRs (A6) from the same volunteer were used as controls. The target cells used were T2-A11 (referred to as HLA-A1101-transfected T2 cells) loaded with AFP antigen short peptide TSSELMAITR, loaded with other antigen short peptides, or unloaded.
Firstly preparing ELISPOT plate, adding 1 × 104 cells/hole of target cell and 2 × 103 cells/hole of effector cell into corresponding hole (calculated according to transfection positive rate), then adding AFP antigen short peptide TSSELMAITR solution into experimental group, adding other antigen short peptide solution into control group and making the final concentration of short peptide be 10 -6 M, blank group was added with equal volume of medium and two duplicate wells were set. Incubation overnight (37 ℃,5% CO) 2 ). On day 2 of the experiment, the plates were washed and subjected to secondary detection and color development, dried, and the spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).
The experimental results are shown in fig. 8, and for the target cells loaded with AFP antigen short peptide TSSELMAITR, the effector cells transfected with the high affinity TCR of the present invention have an obvious activation effect, while the effector cells transfected with other TCRs have no activity; meanwhile, effector cells transfected with the inventive TCR are inactive against other antigen-loaded short peptide or empty target cells.
Example 7 activation function assay of Effector cells transfected with high affinity TCR of the invention against cell lines
This example again demonstrates the activation function and specificity of effector cells transfected with the high affinity TCRs of the invention using tumor cell lines. Again, detection is by ELISPOT assays well known to those skilled in the art. The high affinity TCRs of the invention (TCR numbering and their sequence numbers are known from table 2) were transfected into CD3+ T cells isolated from blood of healthy volunteers as effector cells and CD3+ T cells transfected with other TCRs (A6) or with wild type TCRs (WT-TCRs) were left free (NC) in the same volunteer as controls. The positive tumor cell lines used were HepG2-A11-B2M (HLA-A1101 and. Beta.2M overexpression), SK-MEL-28-AFP (AFP overexpression), and the negative cell lines were HepG2, SK-MEL-28, SNU423, HUCCT1.
The experiment was carried out in two batches, both with the following steps: first, an ELISPOT plate was prepared. ELISPOT plates were ethanol activated coated and incubated overnight at 4 ℃. Day 1 of the experiment, coating was removed, washed and blocked, incubated at room temperature for two hours, blocking solution removed, and the components of the experiment were added to ELISPOT plates: target cells are 2 x 10 4 2 x 10 effector cells per well 3 One/well (calculated as positive rate of transfection) and two duplicate wells were set. Incubation overnight (37 ℃,5% CO) 2 ). On day 2 of the experiment, the plate was washed and subjected to secondary detection and color development, the plate was dried, and spots formed on the membrane were counted using an immuno spot plate READER (ELISPOT READER system; AID20 Co.).
The experimental results are shown in fig. 9a and 9b, and for positive tumor cell lines, the effector cells transfected with the high affinity TCR of the present invention have a more significant activation effect than the effector cells transfected with the wild type, while the effector cells transfected with other TCRs and empty transfection are substantially inactive; at the same time, effector cells transfected with the high affinity TCRs of the invention are essentially inactive against AFP negative cell lines.
Example 8 killing function assay of Effector cells transfected with high affinity TCRs of the invention
This example demonstrates the killing function of cells transfected with the high affinity TCR of the invention by measuring LDH release by non-radioactive cytotoxicity assays well known to those skilled in the art. The test is 51 Colorimetric replacement test for Cr release cytotoxicity test the LDH released after cell lysis was quantitatively determined. LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader. The formula is calculated as% cytotoxicity =100% × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell)Max-target cells are spontaneous).
The LDH experiments of this example were performed by transfecting the high affinity TCRs of the invention (TCR numbers and their sequence numbers are given in Table 2) as effector cells with CD3+ T cells isolated from blood of healthy volunteers, and by transfecting the same volunteers with CD3+ T cells of other TCRs (A6) which were left free to stain (NC) as controls. The following experiments were carried out in two batches (I), (II) one after the other:
(I) The positive tumor cells used were HepG2-A11-B2M and SK-MEL-28-AFP, and the negative cells were HepG2, SK-MEL-28, HUCCT1 and SNU423.
(II) the positive tumor cell line used was HepG2-A11-B2M, and the negative tumor cell line was HepG2.
The following experimental procedures were carried out for both batches: LDH plates were first prepared and the individual components of the assay were added to the plates in the following order: target cell 3X 10 4 Single cell/well, effector cell 3X 10 4 Individual cells/well (calculated as transfection positivity) were added to the corresponding wells and three replicate wells were set. Meanwhile, an effector cell spontaneous hole, a target cell maximum hole, a volume correction control hole and a culture medium background control hole are arranged. Incubation overnight (37 ℃,5% 2 ). On the 2 nd day of the experiment, color development was detected, and after the reaction was terminated, the absorbance was recorded at 490nm using a microplate reader (Biotech).
Experimental results as shown in fig. 10a and 10b, the effector cells transfected with the high affinity TCRs of the invention still showed strong killing efficacy against positive tumor cell lines, whereas the null transfected T cells transfected with other TCRs did not respond; at the same time, the T cells transfected with the high affinity TCR of the invention have essentially no killing of the negative tumor cell line.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Gln Tyr His Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 12
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 12
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Gln Tyr Lys Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 13
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 13
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser His
85 90 95
Gln His Gly Val Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 14
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 14
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Pro Gln Gly Ile Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 15
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 15
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Gln Tyr Arg Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 16
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 16
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Gln Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 17
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 17
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Arg Gln Leu Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 18
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 18
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Arg Phe Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 19
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 19
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Ser Tyr Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 20
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 20
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Val Ile Tyr Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 21
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 21
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Val Gln Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 22
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 22
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Val Arg Tyr Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 23
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 23
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Val Val Tyr Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 24
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 24
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Leu Tyr Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 25
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 25
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Val Tyr Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 26
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 26
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Arg Met Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 27
<211> 113
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 27
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Ile Arg Tyr Gln Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val
<210> 28
<211> 201
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 28
Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu Gly
1 5 10 15
Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu
20 25 30
Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr
35 40 45
Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val
50 55 60
Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr
65 70 75 80
Asp Ser Gly Val Tyr Phe Cys Ser Gly Gly Ser Asn Tyr Lys Leu Thr
85 90 95
Phe Gly Lys Gly Thr Leu Leu Thr Val Asn Pro Asn Ile Gln Asn Pro
100 105 110
Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser
115 120 125
Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser
130 135 140
Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg
145 150 155 160
Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser
165 170 175
Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp
180 185 190
Thr Phe Phe Pro Ser Pro Glu Ser Ser
195 200
<210> 29
<211> 243
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 29
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Val Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp
<210> 30
<211> 248
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 30
Gly Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu Gly
1 5 10 15
Ala Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu
20 25 30
Gln Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe Tyr
35 40 45
Ile Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr Thr Val
50 55 60
Ala Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser Gln Thr Thr
65 70 75 80
Asp Ser Gly Val Tyr Phe Cys Ser Gly Gly Ser Asn Tyr Lys Leu Thr
85 90 95
Phe Gly Lys Gly Thr Leu Leu Thr Val Asn Pro Asn Ile Gln Asn Pro
100 105 110
Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser
115 120 125
Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser
130 135 140
Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg
145 150 155 160
Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser
165 170 175
Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp
180 185 190
Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu
195 200 205
Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val
210 215 220
Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu
225 230 235 240
Met Thr Leu Arg Leu Trp Ser Ser
245
<210> 31
<211> 290
<212> PRT
<213> Artificial sequence
<220>
<223> synthetic polypeptide
<400> 31
Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly
1 5 10 15
Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu
20 25 30
Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr
35 40 45
Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg
50 55 60
Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln
65 70 75 80
Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro
85 90 95
Gly Thr Gly Val Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val
100 105 110
Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu
115 120 125
Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys
130 135 140
Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val
145 150 155 160
Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu
165 170 175
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg
180 185 190
Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg
195 200 205
Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln
210 215 220
Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
225 230 235 240
Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu
245 250 255
Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr
260 265 270
Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys
275 280 285
Asp Phe
290

Claims (10)

1. A T Cell Receptor (TCR) comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, characterised in that it has activity to bind the TSSELMAITR-HLA a1101 complex, and in that the amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence homology with the amino acid sequence set out in SEQ ID No. 1 and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence homology with the amino acid sequence set out in SEQ ID No. 2.
2. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in claim 1.
3. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR as claimed in claim 1, or a complement thereof.
4. A vector comprising the nucleic acid molecule of claim 3.
5. A host cell comprising the vector of claim 4 or a nucleic acid molecule of claim 3 integrated into the chromosome.
6. An isolated cell expressing a TCR as claimed in claim 1.
7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as claimed in claim 1, or a TCR complex as claimed in claim 2, or a cell as claimed in claim 6.
8. A method of treating a disease comprising administering to a subject in need thereof a TCR as claimed in claim 1, or a TCR complex as claimed in claim 2, or a cell as claimed in claim 6, or a pharmaceutical composition as claimed in claim 7; preferably, the disease is an AFP-positive tumor; more preferably, the tumor is liver cancer.
9. Use of a T cell receptor as claimed in claim 1, a TCR complex as claimed in claim 2 or a cell as claimed in claim 6 in the manufacture of a medicament for the treatment of a tumour; preferably, the tumor is an AFP-positive tumor; more preferably, the tumor is liver cancer.
10. A method of preparing a T cell receptor according to claim 1, comprising the steps of:
(i) Culturing the host cell of claim 5 so as to express the T cell receptor of claim 1;
(ii) Isolating or purifying said T cells. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in claim 1.
CN202110679998.2A 2021-06-18 2021-06-18 High-affinity T cell receptor aiming at AFP antigen Pending CN115490773A (en)

Priority Applications (2)

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CN202110679998.2A CN115490773A (en) 2021-06-18 2021-06-18 High-affinity T cell receptor aiming at AFP antigen
PCT/CN2022/099383 WO2022262842A1 (en) 2021-06-18 2022-06-17 High-affinity t-cell receptor for afp antigen

Applications Claiming Priority (1)

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Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101904385B1 (en) * 2013-06-26 2018-10-05 광저우 샹쉐 파마스티컬 컴퍼니 리미티드 High-stability T-cell Receptor and Preparation Method and Application Thereof
CN106519018A (en) * 2015-09-09 2017-03-22 广州市香雪制药股份有限公司 High-affinity T-cell receptors targeting short peptides of RHAMM antigen
WO2021022447A1 (en) * 2019-08-05 2021-02-11 广东香雪精准医疗技术有限公司 T cell receptor capable of recognizing afp antigen-derived short peptide
CN112442119B (en) * 2019-09-05 2023-02-24 香雪生命科学技术（广东）有限公司 High-affinity T cell receptor for identifying SSX2
CN112940106A (en) * 2019-12-11 2021-06-11 中国科学院广州生物医药与健康研究院 High affinity AFPT cellular receptors

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