High-affinity TCR for identifying AFP antigen
阅读说明：本技术 一种识别afp抗原的高亲和力tcr (High-affinity TCR for identifying AFP antigen ) 是由 李懿 李小林 于 2019-03-08 设计创作，主要内容包括：本发明提供了一种T细胞受体(TCR),其具有结合FMNKFIYEI-HLA A0201复合物的特性；并且所述TCR对所述FMNKFIYEI-HLA A0201复合物的结合亲和力是野生型TCR对FMNKFIYEI-HLA A0201复合物的结合亲和力的至少5倍。本发明还提供了此类TCR与治疗剂的融合分子。此类TCR可以单独使用,也可与治疗剂联用,以靶向呈递FMNKFIYEI-HLA A0201复合物肿瘤细胞。(The present invention provides a T Cell Receptor (TCR) having the property of binding to the FMNKFIYEI-HLA a0201 complex; and the binding affinity of the TCR to the FMNKFIYEI-HLA A0201 complex is at least 5-fold greater than the binding affinity of a wild-type TCR to the FMNKFIYEI-HLA A0201 complex. The invention also provides fusion molecules of such TCRs with therapeutic agents. Such TCRs can be used alone or in combination with therapeutic agents to target FMNKFIYEI-HLA a0201 complex presenting tumor cells.)
1. A T Cell Receptor (TCR) having binding activity to the FMNKFIYEI-HLA A0201 complex, said TCR comprising a TCR a chain variable domain comprising 3 CDR regions and a TCR β chain variable domain, said TCR a chain variable domain comprising 3 CDR regions having the following reference sequences,
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3 α: AVNSGGSNYKLT, and CDR3 α contains at least one of the following mutations:
and/or the variable domain of the beta chain of the TCR is an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO. 2;
preferably, the TCR has CDRs selected from the group consisting of:
2. a TCR as claimed in claim 1 which is selected from the group consisting of:
3. a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in any one of claims 1 to 2.
4. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR according to any one of claims 1-2, or the complement thereof.
5. A vector comprising the nucleic acid molecule of claim 4.
6. A host cell comprising the vector of claim 5 or a nucleic acid molecule of claim 4 integrated into the chromosome.
7. An isolated cell expressing a TCR as claimed in any one of claims 1 to 2.
8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1-2 or a TCR complex according to claim 3 or a cell according to claim 7.
9. A method of treating a disease comprising administering a TCR according to any one of claims 1 to 2, or a TCR complex according to claim 3, or a cell according to claim 7, or a pharmaceutical composition according to claim 8 to a subject in need thereof.
10. Use of a T cell receptor according to any one of claims 1-2, a TCR complex according to claim 3 or a cell according to claim 7 for the preparation of a medicament for the treatment of a tumour.
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 either the α/β chain or the γ 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 hepatocellular carcinoma, expression of AFP is activated. AFP is degraded into small molecule polypeptides after intracellular production and binds to MHC (major histocompatibility complex) molecules to form complexes, which are presented on the cell surface. FMNKFIYEI (SEQ ID NO:25) is a short peptide derived from the AFP antigen, which is a target for the treatment of AFP-related diseases.
Thus, the FMNKFIYEI-HLA A0201 complex provides a marker for targeting of TCR to tumor cells. The TCR capable of combining the FMNKFIYEI-HLA A0201 compound 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, enabling T cells expressing the TCR to destroy tumor cells for administration to patients in a therapeutic 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
The present invention aims to provide a TCR with higher affinity for the FMNKFIYEI-HLA A0201 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) having the activity of binding the FMNKFIYEI-HLAA0201 complex.
In another preferred embodiment, the T Cell Receptor (TCR) has the activity of binding FMNKFIYEI-HLA A0201 complex and comprises a TCR alpha chain variable domain comprising 3 CDR regions and a TCR beta chain variable domain, the 3 CDR regions of the TCR alpha chain variable domain having the following reference sequences,
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3 α: AVNSGGSNYKLT, and CDR3 α contains at least one of the following mutations:
and/or the variable domain of the beta chain of the TCR is an amino acid sequence having at least 90% sequence homology with the amino acid sequence set forth in SEQ ID NO. 2.
In another preferred embodiment, the β chain variable domain of the TCR is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to the amino acid sequence set forth in SEQ ID NO. 2.
In another preferred embodiment, the number of mutations in CDR3 α in the TCR α chain variable domain is 1 to 4.
In another preferred embodiment, the TCR has at least 5-fold greater affinity for the FMNKFIYEI-HLA a0201 complex than a wild-type TCR.
In another preferred embodiment, the α chain variable domain of the TCR comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence set forth in SEQ ID No. 1.
In another preferred embodiment, the TCR β chain variable domain comprises 3 CDR regions, and the amino acid sequences of the 3 CDR regions of the TCR β chain variable domain are as follows:
CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3β：ASSLFGQGREKLF。
in another preferred embodiment, the amino acid sequence of the TCR β chain variable domain is SEQ ID NO: 2.
in another preferred embodiment, the TCR comprises a TCR α chain variable domain comprising CDRs 1 α, CDR2 α and CDR3 α, wherein the amino acid sequence of CDR1 α is DSAIYN and the amino acid sequence of CDR2 α is IQSSQRE; and the TCR β chain variable domain comprises CDR1 β, CDR2 β, and CDR3 β, wherein the amino acid sequence of CDR1 β is SGHVS, the amino acid sequence of CDR2 β is FQNEAQ, and the amino acid sequence of CDR3 β is ASSLFGQGREKLF.
In another preferred example, the TCR comprises a TCR α chain variable domain comprising CDRs 1 α, CDR2 α and CDR3 α, wherein the amino acid sequence of CDR1 α is DSAIYN, the amino acid sequence of CDR2 α is IQSSQRE and the amino acid sequence of CDR3 α is: AV [ 3. alpha. X1] [ 3. alpha. X2] [ 3. alpha. X3] [ 3. alpha. X4] [ 3. alpha. X5] [ 3. alpha. X6] YKLT.
In another preferred embodiment, the [3 α X1] is N or D or E.
In another preferred embodiment, said [3 α X2] is S or D or G or a or W or T or H.
In another preferred embodiment, said [3 α X3] is G or Q or a or V or H or W or Y or M or I.
In another preferred embodiment, said [3 α X4] is G or D or R or P or Q or T or Y.
In another preferred embodiment, the [3 α X5] is S or G or D.
In another preferred embodiment, the [3 α X6] is N or G or D.
In another preferred embodiment, the TCR has CDRs selected from the group consisting of:
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) all or part of a TCR α chain, excluding the transmembrane domain thereof, and (ii) all or part of a TCR β chain, excluding the transmembrane domain thereof, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain.
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 forming the artificial interchain disulfide bond between the constant regions of the TCR α and β chains are substituted at one or more groups of sites selected from:
thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;
tyr10 and TRBC 1x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC 2x 01;
thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;
ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;
arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;
pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01;
and
tyr10 and TRBC1 × 01 of exon 1 of TRAC × 01 or Glu20 of exon 1 of TRBC2 × 01.
In another preferred embodiment, the α chain variable domain amino acid sequence of the TCR is selected from the group consisting of: 11-24 of SEQ ID NO; and/or the amino acid sequence of the beta chain variable domain of the TCR is
In another preferred embodiment, the TCR is selected from the group consisting of:
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 linked by a flexible short peptide sequence (linker).
In another preferred embodiment, the TCR has a conjugate attached to the C-or N-terminus of the alpha and/or beta chain.
In another preferred embodiment, the conjugate to which the TCR is bound is 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 preferred embodiment of the invention, the TCR has at least 5-fold greater affinity for the FMNKFIYEI-HLA a0201 complex than for a wild-type TCR; preferably, at least 10 times; more preferably, at least 50 times.
In another preferred embodiment, the TCR has at least 100-fold greater affinity for the FMNKFIYEI-HLA a0201 complex than a wild-type TCR; preferably, at least 500 times; more preferably, at least 1000 times.
In particular, the dissociation equilibrium constant K of the TCR versus FMNKFIYEI-HLA A0201 complexDLess than or equal to 20 mu M; preferably, 5. mu.M.ltoreq.KD≤10μM。
In another preferred embodiment, the TCR pair FMNKFIYEI-HLA A0201 complex has a dissociation equilibrium constant of 0.1 μ M ≦ KDLess than or equal to 1 mu M; preferably, 1nM ≦ KD≤100nM。
In a preferred embodiment of the invention, the T Cell Receptor (TCR), which has the activity of binding FMNKFIYEI-HLA a0201 complex and comprises a TCR a chain variable domain and a TCR β chain variable domain, is represented by the sequence given in seq id NO:1, the mutated amino acid residue positions comprise one or more of 93N, 94S, 95G, 96G, 97S and 98N, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID No. 1;
preferably, the TCR α chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of: 93D or 93E; 94D or 94G or 94A or 94W or 94T or 94H; 95Q or 95A or 95V or 95H or 95W or 95Y or 95M or 95I; 96D or 96R or 96P or 96Q or 96T or 96Y; 97G or 97D; and 98G or 98D, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO. 1.
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 TCR molecule 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.
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 tumor.
More preferably, the AFP-positive tumor is hepatocellular carcinoma, breast cancer, or germ cell tumor.
In a ninth aspect, the invention provides the use 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, for the manufacture of a medicament for the treatment of a tumour.
Preferably, the tumor is an AFP-positive tumor.
More preferably, the AFP-positive tumor is hepatocellular carcinoma, breast cancer, or germ cell tumor.
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 reiterated herein, but to the extent of space.
Drawings
FIGS. 1a and 1b show the amino acid sequences of the wild-type TCR alpha and beta chain variable domains, respectively, capable of specifically binding to the FMNKFIYEI-HLA A0201 complex.
FIGS. 2a and 2b show the amino acid sequence of the alpha variable domain and the amino acid sequence of the beta variable domain, respectively, of a single-chain template TCR constructed in accordance with the invention.
FIGS. 3a and 3b are the DNA sequences of the α and β chain variable domains, respectively, of a single-chain template TCR constructed in accordance with the invention.
FIGS. 4a and 4b show the amino acid sequence and nucleotide sequence of the linker (linker) of the single-chain template TCR constructed according to the invention.
FIGS. 5a and 5b show the amino acid sequence and DNA sequence, respectively, of a single-stranded template TCR constructed in accordance with the invention.
Fig. 6(1) - (14) show the α chain variable domain amino acid sequences of the heterodimeric TCRs with high affinity for FMNKFIYEI-HLA a0201 complex, respectively, with mutated residues underlined.
FIGS. 7a and 7b show the amino acid sequences of reference TCR alpha and beta chains, respectively, of the invention.
FIGS. 8a and 8b show the wild-type TCR alpha and beta chain amino acid sequences, respectively, capable of binding specifically to the FMNKFIYEI-HLA A0201 complex.
FIG. 9 is a graph of the binding of the reference TCR, i.e., the wild-type TCR, to the FMNKFIYEI-HLA A0201 complex.
FIGS. 10a-f are graphs showing the experimental results of the activation function of effector cells transfected with the high affinity TCR of the invention against T2 cells loaded with specific short peptides.
FIG. 11 is a graph showing the results of an experimental study of the activation function of effector cells transfected with the high affinity TCR of the invention, in relation to tumor cell lines.
FIG. 12 is a graph showing the results of an experiment on the killing function of effector cells transfected with the high affinity TCR of the invention.
FIG. 13 is a graph of the results of in vivo efficacy experiments on T cells transfected with the high affinity TCRs of the invention.
Detailed Description
The present inventors, through extensive and intensive studies, have obtained a high affinity T Cell Receptor (TCR) that recognizes FMNKFIYEI short peptides (derived from the AFP protein), the FMNKFIYEI short peptide being presented as a peptide-HLA a0201 complex. The high affinity TCR has 3 CDR regions in its alpha chain variable domain:
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3 α: AVNSGGSNYKLT;
and, the affinity and/or binding half-life of the inventive TCR after mutation to the FMNKFIYEI-HLA a0201 complex described above is at least 5-fold that of the 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) for
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 connecting region is defined by the unique TRAJ and TRBJ of IMGT, and the TCR constant region is defined by the TRAC and TRBC of IMGT.
Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in a 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; ". 01" indicates allele 1. The beta-strand constant domain has the following symbols: TRBC 1x 01 or TRBC 2x 01, wherein "TR" denotes a T cell receptor gene; "B" represents a beta chain gene; c represents a constant region; ". 01" 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 TCR β chain variable domain 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 alpha chain amino acid sequence and the beta chain amino acid sequence of the wild-type TCR are respectively
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 position-numbered in the order from the N-terminus to the C-terminus, such as TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid is P (proline) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Pro60 in the invention, or TRBC1 × 01 or TRBC2 × 01 exon 1, and as TRBC1 × 01 or TRBC2 × 01, and the 61 st amino acid is Q (glutamine) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2, and as glbc 8201 or TRBC 8536. 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 carcinoma of the lung includes malignant tumors, such as, for example, most cancers of the colon, rectum, renal cell, liver, lung, small cell, small intestine and esophagus. 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 amino acid sequence of the beta chain variable domain of the wild-type TCR capable of binding the antigen short peptide FMNKFIYEI to the HLA a0201 complex (i.e., FMNKFIYEI-HLA a0201 complex) are SEQ ID NO:1 and SEQ ID NO:2, the sequence is discovered by the inventor for the first time. It has the following CDR regions:
alpha chain variable domain CDR1 a: DSAIYN
CDR2α：IQSSQRE
CDR3α：AVNSGGSNYKLT
And the beta chain variable domain CDR1 beta: SGHVS
CDR2β：FQNEAQ
CDR3β：ASSLFGQGREKLF
The invention obtains the high affinity TCR with the affinity of FMNKFIYEI-HLA A0201 complex being at least 5 times that of the wild type TCR and FMNKFIYEI-HLA A0201 complex by carrying out mutation screening on the CDR region.
The present invention provides a T Cell Receptor (TCR) having binding activity to the FMNKFIYEI-HLA A0201 complex.
The T cell receptor comprises a TCR alpha chain variable domain comprising 3 CDR regions and a TCR beta chain variable domain, the reference sequence of the 3 CDR regions of the TCR alpha chain variable domain is as follows,
CDR1α：DSAIYN
CDR2α：IQSSQRE
CDR3 α: AVNSGGSNYKLT, and contains at least one of the following mutations:
and/or, the variable domain of the TCR beta chain comprises 3 CDR regions, the reference sequence of the 3 CDR regions of the variable domain of the TCR beta chain is as follows,
CDR1β：SGHVS
CDR2β：FQNEAQ
CDR3β：ASSLFGQGREKLF。
in more detail, the number of mutations in the CDR regions of the TCR α chain can be 1, 2,3, 4, 5, or 6.
Further, the TCR of the invention is an α β heterodimeric TCR, the α chain variable domain of which comprises at least 85% of the amino acid sequence 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; 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.
Further, the TCR of the invention is a single chain TCR, the α chain variable domain of which comprises at least 85%, preferably at least 90% of the amino acid sequence shown in SEQ ID No. 3; more preferably, at least 92%; most 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; and/or the β chain variable domain of the TCR comprises at least 85%, preferably at least 90% of the amino acid sequence set forth as SEQ ID No. 4; more preferably, at least 92%; most preferably, at least 94%; (e.g., can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of sequence homology.
The wild type TCR alpha chain variable domain SEQ ID NO:1, namely CDR1, CDR2 and CDR3 are located in SEQ ID NO: bits 27-32, 50-56, and 91-102 of 1. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO. 1, 93N is the 3 rd position N of CDR3 alpha, 94S is the 4 th position S of CDR3 alpha, 95G is the 5 th position G of CDR3 alpha, 96G is the 6 th position G of CDR3 alpha, 97S is the 7 th position S of CDR3 alpha, 98N is the 8 th position N of CDR3 alpha.
The invention provides a TCR having the property of binding FMNKFIYEI-HLA a0201 complex and comprising an alpha chain variable domain and a beta chain variable domain, wherein the TCR is as defined in SEQ ID NO:1, and the mutated amino acid residue positions comprise one or more of 93N, 94S, 95G, 96G, 97S and 98N, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID No. 1.
Preferably, the TCR α chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of: 93D or 93E; 94D or 94G or 94A or 94W or 94T or 94H; 95Q or 95A or 95V or 95H or 95W or 95Y or 95M or 95I; 96D or 96R or 96P or 96Q or 96T or 96Y; 97G or 97D; and 98G or 98D, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO. 1.
More specifically, specific forms of the mutations in the alpha chain variable domain include N93/D/E, S94/D/G/A/W/T/H; G95/Q/A/V/H/W/Y/M/I; G96/D/R/P/Q/T/Y; S97/G/D; and one or more of N98/G/D.
The amino acid sequence of the reference TCR, which is shown in figures 7a and 7b, respectively, was obtained by mutating Thr48 of exon 1 of the α chain constant region TRAC 01 of the wild-type TCR to cysteine and Ser57 of exon 1 of the β chain constant region TRBC 1x 01 or TRBC 2x 01 to cysteine, according to site-directed mutagenesis methods well known to those skilled in the art, with the mutated cysteine residues shown in bold letters. The cysteine substitutions described above enable artificial interchain disulfide bonds to be formed between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, enabling more convenient assessment of binding affinity and/or binding half-life between the TCR and the FMNKFIYEI-HLA a2 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 have an effect on the binding affinity and/or binding half-life of the TCR. Therefore, in the present invention, the measured binding affinity between the reference TCR and the FMNKFIYEI-HLA A0201 complex is considered to be the binding affinity between the wild-type TCR and the FMNKFIYEI-HLA A0201 complex. Similarly, if the binding affinity between the inventive TCR and the FMNKFIYEI-HLA A0201 complex is determined to be at least 10 times greater than the binding affinity between the reference TCR and the FMNKFIYEI-HLA A0201 complex, i.e. equivalent to the binding affinity between the inventive TCR and the FMNKFIYEI-HLA A0201 complex being at least 10 times greater than the binding affinity between the wild-type TCR and the FMNKFIYEI-HLA A0201 complex.
Binding affinity (equilibrium constant K to dissociation) can be determined by any suitable methodDInversely proportional) and binding half-life (denoted T)1/2). It will be appreciated that doubling the affinity of the TCR will result in KDAnd (4) halving. T is1/2Calculated as In2 divided by dissociation rate (K)off). Thus, T1/2Doubling can result in KoffAnd (4) halving. 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, these assays are performed using the surface plasmon resonance (BIAcore) method in the examples herein. The method detects the dissociation equilibrium constant K of the reference TCR to the FMNKFIYEI-HLA A2 complexDAt 2.08E-04M, i.e., 208. mu.M, the dissociation equilibrium constant K of the wild-type TCR for the FMNKFIYEI-HLA A2 complex is considered to beDAlso 208. mu.M. Doubling of the affinity due to TCR will result in KDHalving, so if the dissociation equilibrium constant K of the high affinity TCR for the FMNKFIYEI-HLA A0201 complex is detectedDAt 2.08E-05M, i.e., 20.8. mu.M, this indicates that the high affinity TCR has 10-fold greater affinity for the FMNKFIYEI-HLA A0201 complex than the wild type TCR for the FMNKFIYEI-HLA A0201 complex. K is well known to those skilled in the artDThe conversion between the units of values, i.e. 1 μ M to 1000 μ M,1 μ M to 1000nM, 1nM to 1000 pM.
In a preferred embodiment of the invention, the TCR has at least 5-fold greater affinity for the FMNKFIYEI-HLA a0201 complex than for a wild-type TCR; preferably, at least 10 times; more preferably, at least 50 times.
In another preferred embodiment, the TCR has at least 100-fold greater affinity for the FMNKFIYEI-HLA a0201 complex than a wild-type TCR; preferably, at least 500 times; more preferably, at least 1000 times.
In particular, the dissociation equilibrium constant K of the TCR versus FMNKFIYEI-HLA A0201 complexD≤20μM；
In another preferred embodiment, the TCR pair FMNKFIYEI-HLA A0201 complex has a dissociation equilibrium constant of 5 μ M ≦ KDLess than or equal to 10 mu M; preferably, 0.1. mu.M.ltoreq.KDLess than or equal to 1 mu M; more preferably, 1nM ≦ KD≤100nM。
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 Press. 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 a TCR with high affinity for the FMNKFIYEI-HLA-A2 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 TCRs of the invention comprise one of the alpha chain variable domain amino acid sequences
TABLE 1
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 entire long chain (containing both cytoplasmic and transmembrane domains) of an α β heterodimeric TCR can be transfected. The TCRs of the invention are useful 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 TCRs are preferably in soluble form.
For stability, it is disclosed in the prior art that the introduction of an artificial interchain disulfide bond between the α and β chain constant domains of a 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 substitution of Thr48 for exon 1 of TRAC × 01 and a substitution of Ser57 for exon 1 of TRBC1 × 01 or TRBC2 × 01 form disulfide bonds. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC 1x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC 2x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include cysteine residues for the purpose of deleting the native interchain disulphide bond, or by mutating the cysteine residues forming the native interchain disulphide 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 both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native interchain disulfide bonds present in the TCR.
In addition, for stability, patent document 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 TCRs of the invention may also contain 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:
For stability, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that improve 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
More specifically, the TCR with the mutated hydrophobic core region of the invention can be a high stability single chain TCR with a flexible peptide chain connecting the variable domains of the α and β chains 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 template chain for screening high affinity TCR constructed in example 1 of the present invention is the above-described high stability single chain TCR comprising the hydrophobic core mutation. Using a TCR with higher stability, the affinity between the TCR and the FMNKFIYEI-HLA-A0201 complex can be more conveniently assessed.
The CDR regions of the alpha chain variable domain and the beta chain variable domain of the single-chain template TCR are completely identical to the CDR regions of the wild-type TCR. That is, the 3 CDRs of the α chain variable domain are CDR1 α: DSAIYN, CDR2 α: iQSSQRE, CDR3 α: AVNSGGSNYKLT and the 3 CDRs of the β chain variable domain are CDR1 β: SGHVS, CDR2 β: FQNEAQ, CDR3 β: ASSLFGQGREKLF are provided. The amino acid sequence (SEQ ID NO:9) and the nucleotide sequence (SEQ ID NO:10) of the single-chain template TCR are shown in FIGS. 5a and 5b, respectively. Thus, a single-chain TCR composed of an alpha chain variable domain and a beta chain variable domain having high affinity for the FMNKFIYEI-HLA A0201 complex was selected.
The single-chain template TCR alpha chain variable domain SEQ ID NO:3, namely CDR1, CDR2 and CDR3 are located in SEQ ID NO: bits 27-32, 50-56, and 91-102 of 3. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO. 3, 93N is the 3 rd position N of CDR3 alpha, 94S is the 4 th position S of CDR3 alpha, 95G is the 5 th position G of CDR3 alpha, 96G is the 6 th position G of CDR3 alpha, 97S is the 7 th position S of CDR3 alpha, 98N is the 8 th position N of CDR3 alpha.
The α β heterodimer having high affinity for the FMNKFIYEI-HLA-A0201 complex of the present invention was obtained by transferring the CDR regions of the α and β chain variable domains of the selected high affinity single-chain TCR to the corresponding positions of the α chain variable domain (SEQ ID NO:1) and β chain variable domain (SEQ ID NO:2) of the wild-type TCR.
The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.
The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the FMNKFIYEI-HLA-a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.
Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biological toxins (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gil lies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and immunotherapy (Cancer Immunology) 53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004,
Antibodies or fragments thereof that bind to the TCRs of the invention 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 directs effector cells to better 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 of the invention and the anti-CD 3 single chain antibody comprises the TCR alpha chain variable domain amino acid sequence
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 may then be introduced into various existing DNA molecules (or vectors, for example) 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-. 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) NatRev Cancer8 (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.
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) and Val (V); in the present invention, Pro60 or 60P both represent proline at position 60. In addition, the expression of a specific form of the mutation described in the present invention is such that "N93D" represents that N at position 93 is substituted by D, and similarly, "N93D/E" represents that N at position 93 is substituted by D or by E. Others may be analogized.
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 will 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 TCR of the invention, the TCR complex or the TCR-transfected T cell of the invention may 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 may be used alone, or in combination or coupling 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 practical 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 affinity and/or binding half-life of the inventive TCR to the FMNKFIYEI-HLA-a2 complex is at least 5-fold, preferably at least 10-fold that of a wild-type TCR.
(2) The affinity and/or binding half-life of the inventive TCR to the FMNKFIYEI-HLA-a2 complex is at least 100-fold, preferably at least 1000-fold that of the wild-type TCR.
(3) Effector cells transduced with the high affinity TCRs of the invention have a strong killing effect on target cells.
The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.
Materials and methods
The experimental materials used in the examples of the present invention are commercially available as such, unless otherwise specified, wherein e.coli DH5 α is available from Tiangen, e.coli BL21(DE3) is available from Tiangen, e.coli Tuner (DE3) is available from Novagen, and plasmid pET28a is available from Novagen.
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