WO2016124142A1

WO2016124142A1 - High affinity ny-eso t cell receptor

Info

Publication number: WO2016124142A1
Application number: PCT/CN2016/073389
Authority: WO
Inventors: 李懿; 李峰
Original assignee: 广州市香雪制药股份有限公司
Priority date: 2015-02-06
Filing date: 2016-02-03
Publication date: 2016-08-11
Also published as: CN106459177A; CN105985427A; CN106459177B

Abstract

Provided are a T cell receptor (TCR) and a molecule thereof fused with a therapeutic agent. The binding affinity of the TCR with respect to SLLMWITQC-HLA A2 complex is at least twice of that of a wild-type TCR. The TCR either can be used independently or can be used jointly with the therapeutic agent.

Description

High affinity NY-ESO T cell receptor

Technical field

The present invention relates to the field of biotechnology, and more particularly to a T cell receptor (TCR) capable of specifically recognizing a polypeptide derived from a NY-ESO-1 protein. The invention also relates to the preparation and use of said receptors.

Background technique

There are only two types of molecules that recognize antigens in a specific way. One of them is an immunoglobulin or an antibody; the other is a T cell receptor (TCR), which is a glycoprotein on the surface of a cell membrane in the form of a heterodimer of an α chain/β chain or a γ chain/δ chain. The composition of the TCR profile of the immune system is produced by V(D)J recombination in the thymus and then positive and negative selection. In the peripheral environment, TCR mediates the specific recognition of major histocompatibility complex-peptide complex (pMHC) by T cells, and thus it is critical for the cellular immune function of the immune system.

TCR is the only receptor for a specific antigenic peptide presented on the major histocompatibility complex (MHC), which may be the only sign of abnormalities in the cell. In the immune system, the binding of antigen-specific TCR to the pMHC complex triggers direct physical contact between T cells and antigen presenting cells (APC), and then the other cell membrane surface molecules of both T cells and APC interact. This leads to a series of subsequent cell signaling and other physiological responses, allowing different antigen-specific T cells to exert an immune effect on their target cells.

MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily but are specific for antigen presentation, and different individuals have different MHCs, thereby presenting different shortness of one protein antigen Peptides to the surface of the respective APC cells. Human MHC is commonly referred to as the HLA gene or the HLA complex.

The short peptide SLLMWITQC is derived from the NY-ESO-1 protein expressed by various tumor cells (Chen et al., (1997) PNAS USA 94 1914-1918). The type I HLA molecule of tumor cells presents a short peptide derived from NY-ESO-1 including SLLMWITQC. Thus, the SLLMWITQC-HLA A2 complex provides a marker for TCR targeting tumor cells. The TCR capable of specifically binding to the SLLMWITQC-HLA A2 complex has high application value for the treatment of tumors. For example, a TCR capable of targeting the tumor cell marker can be used to deliver a cytotoxic agent or immunostimulatory agent to a target cell, or to a T cell, such that the T cell expressing the TCR can The tumor cells are destroyed to be administered to the patient during a treatment called adoptive immunotherapy. For the former purpose, the ideal TCR has a higher affinity, allowing the TCR to reside on the targeted cells 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 directed to developing TCRs that target tumor cell markers that can be used to meet different purposes.

Summary of the invention

It is an object of the present invention to provide a TCR having a higher affinity for the SLLMWITQC-HLA A2 complex.

It is still another object of the present invention to provide a method of preparing a TCR of the above type and the use of a TCR of the above type.

In a first aspect of the invention, there is provided a T cell receptor (TCR) having the property of binding to a SLLMWITQC-HLA A2 complex and comprising a TCR alpha chain variable domain and/or a TCR beta chain variable domain, relative to having The α chain variable domain amino acid sequence SEQ ID NO: 75 and the β chain variable domain amino acid sequence SEQ ID NO: 76 TCR:

(i) the TCR is mutated in its alpha chain variable domain amino acid as shown in SEQ ID NO: 75 and/or in its beta chain variable domain amino acid as shown in SEQ ID NO: 76;

(ii) The binding affinity of the TCR to the SLLMWITQC-HLA A2 complex is at least twice the binding affinity of the wild-type TCR to the SLLMWITQC-HLA A2 complex.

In another preferred embodiment, the mutation occurs in one or more CDR regions of the alpha chain and/or beta chain.

In another preferred embodiment, the mutation occurs in CDR1 and/or CDR3 of the alpha chain, and/or the mutation occurs in the CDR2 and/or CDR3 of the beta strand.

In another preferred embodiment, the binding affinity of the TCR to the SLLMWITQC-HLA A2 complex is at least 10 times the binding affinity of the wild type TCR to the SLLMWITQC-HLA A2 complex; preferably at least 20 fold; more preferably , at least 100 times.

In another preferred embodiment, the dissociation equilibrium constant K _{D of} the TCR to the SLLMWITQC-HLA A2 complex is ≤ 3.2 μM.

In another preferred embodiment, the dissociation equilibrium constant of the TCR to the SLLMWITQC-HLA A2 complex is 0.5 μM ≤ K _D ≤ 3.2 μM; preferably, 1 μM ≤ K _D ≤ 3.2 μM; more preferably, 1 μM ≤ K _D ≤ 2 μM.

In another preferred embodiment, the TCR dissociation equilibrium constant K _D ≤500nM for solving said SLLMWITQC-HLA A2 complex; preferably, 10pM≤K _{D ≤500nM.} More preferably, 10 pM ≤ K _D ≤ 10 nM.

In another preferred embodiment, the mutation occurs at one or more amino acid residue positions selected from the group consisting of the alpha chain variable domain set forth in SEQ ID NO: 75: 27T, 28S, 29I, 30N, 51S , 53E, 54R, 55E, 91T, 94A, 95G, 96K, 97S and 98T, wherein the amino acid residue numbering is the number shown in SEQ ID NO: 75; and/or

The mutation occurs at one or more amino acid residue sites selected from the group consisting of the β-chain variable domain set forth in SEQ ID NO: 76: 50N, 51N, 52N, 53V, 54P, 95T, 97G, 98A, 99Q, 100P, 101Q and 102H, wherein the amino acid residue numbering is the number shown in SEQ ID NO:76.

In another preferred embodiment, the mutated TCR alpha chain variable domain comprises one or more amino acid residues selected from the group consisting of: 27Y, 27W, 27H, 27F or 27N; 28T, 28Y, 28D, 28P, 28N Or 28W; 29P, 29L, 29T, 29V or 29A; 30Q; 51N; 53S; 54Q; 55T; 91N; 94T, 94N, 94H, 94I or 94S; 95A or 95S; 96R or 96W; 97W; 98N, 98D or 98A Wherein the amino acid residue numbering is the number shown in SEQ ID NO: 75; and/or

The mutated TCR β chain variable domain comprises one or more amino acid residues selected from the group consisting of 50C or 50Y; 51H or 51L; 52G; 53L; 54V or 54I; 95S; 97N; 98G or 98S; 99N or 99L; 100A; 101I; 102V or 102I; wherein the amino acid residue numbering is the number shown in SEQ ID NO:76.

In another preferred embodiment, the amino acid sequence of the alpha chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 90; and/or

The β chain variable domain amino acid sequence of the TCR is selected from the group consisting of: SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.

In another preferred embodiment, the TCR comprises a combination of alpha and beta chain variable domains as shown in the table below:

In another preferred embodiment, the TCR is an αβ heterodimeric TCR having α and β chain constant domain sequences having a cysteine residue in the α and β chain constant domain sequences (the cysteine The amino acid residue may be a native cysteine residue or an artificially introduced cysteine residue) which forms a disulfide between the alpha and beta chain constant domains of the TCR key.

In another preferred embodiment, the disulfide bond is an artificial disulfide bond.

In another preferred embodiment, in the TCR, a cysteine residue forming an artificial disulfide bond replaces one or more sets of sites selected from the group consisting of:

Thr48 of TRAC*01 exon 1 and Ser57 of TRBC1*01 or TRBC2*01 exon 1;

Thr45 of TRAC*01 exon 1 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01;

Tyr10 and TRBC1*01 of exon 1 of TRAC*01 or Ser17 of exon 1 of TRBC2*01;

Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1;

Ser15 and TRBC1*01 of exon 1 of TRAC*01 or Glu15 of exon 1 of TRBC2*01;

Arg53 of TRAC*01 exon 1 and Ser54 of exon 1 of TRBC2*01 or TRBC2*01; Pro89 and TRBC1*01 of exon 1 of TRAC*01 or Ala19 of exon 1 of TRBC2*01;

Tyr10 and TRBC1*01 of exon 1 of TRAC*01 or Glu20 of exon 1 of TRBC2*01.

In another preferred embodiment, the hydrophobic core of the TCR alpha chain variable domain and / or beta chain variable domain is mutated.

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 chain variable domain and the beta chain variable domain consisting of a flexible short peptide sequence (l Inker) connection.

In another preferred embodiment, the hydrophobic core mutation of the TCR occurs in the alpha chain represented by SEQ ID NO:75. One or more amino acid residue sites of the variable domain selected from the group consisting of: 13I, 19A, 21M, and 79S; wherein the amino acid residue numbering is the number shown in SEQ ID NO: 75; and/or

The hydrophobic core mutation occurs at one or more amino acid residue sites selected from the group consisting of 11E, 13T and 82S of the β chain variable domain set forth in SEQ ID NO: 76, wherein the amino acid residue numbering is SEQ ID NO: The number shown in 76.

In another preferred embodiment, the alpha chain variable domain of the TCR after the hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 13V, 19V, 21I and 79V, wherein the amino acid residue numbering is employed The number shown in SEQ ID NO: 75; and/or

The β chain variable domain of the TCR after the hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 11L, 13V and 82V; wherein the amino acid residue numbering is represented by the number shown in SEQ ID NO: 76 .

In another preferred embodiment, the amino acid sequence of the alpha chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 29, 32, 34, 35, 36, 37, 89; and/or

The β chain variable domain amino acid sequence of the TCR is selected from the group consisting of: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33.

In another preferred embodiment, the C- or N-terminus of the alpha chain and/or beta strand of the TCR incorporates a conjugate.

In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modified moiety, or a combination of any of these.

In another preferred embodiment, the therapeutic agent that binds to the T cell receptor is an anti-CD3 antibody linked to the C- or N-terminus of the alpha or beta chain of the TCR.

In another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR that binds to the anti-CD3 antibody is selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 89, 90; and / or

The β chain variable domain amino acid sequence of the TCR that binds to the anti-CD3 antibody is selected from the group consisting of: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 30, 31, 33, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.

In another preferred embodiment, the amino acid sequence of the TCR β chain after binding to the anti-CD3 antibody is selected from the group consisting of SEQ ID NOS: 100 and 101.

In another preferred embodiment, the amino acid sequence of the TCR after binding to the anti-CD3 antibody is selected from the group consisting of SEQ ID NOs: 92, 93, 94, 95, 96, 97, 98 and 99.

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 the TCR of the first aspect of the invention.

In a third aspect of the invention, a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of the first aspect of the invention or the multivalent TCR complex of the second aspect of the invention, or a complement thereof, is provided sequence;

According to a fourth aspect of the invention, there is provided a carrier comprising the third aspect of the invention Said nucleic acid molecule.

According to a fifth aspect of the invention, a host cell comprising the vector of the fourth aspect of the invention or the nucleic acid molecule of the third aspect of the invention integrated with exogenous in the chromosome is provided.

In a sixth aspect of the invention, there is provided an isolated cell expressing the TCR of the first aspect of the invention.

According to a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, Or the cell of the sixth aspect of the invention.

According to an eighth aspect of the present invention, a method for treating a disease, comprising administering an appropriate amount of the TCR according to the first aspect of the present invention, or the TCR complex of the second aspect of the present invention, or the present invention to a subject in need of treatment The cell of the sixth aspect of the invention, or the pharmaceutical composition of the seventh aspect of the invention.

According to a ninth aspect of the invention, the use of the TCR according to the first aspect of the invention, or the TCR complex of the second aspect of the invention, or the cell of the sixth aspect of the invention, for the preparation of a tumor for treatment Drug.

According to a tenth aspect of the invention, a method for the preparation of the T cell receptor of the first aspect of the invention, comprising the steps of:

(i) cultivating the host cell of the fifth aspect of the invention to express the T cell receptor of the first aspect of the invention;

(ii) isolating or purifying the T cell receptor.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figures 1a-I show the alpha chain variable domain amino acid sequence of a single-chain TCR having high affinity for the SLLMWITQC-HLA A2 complex, respectively, and the mutated residues are underlined in bold.

Figures 2a-2q show the β-chain variable domain amino acid sequences of single-chain TCRs with high affinity for the SLLMWITQC-HLA A2 complex, respectively, and the mutated residues are underlined in bold.

Figures 3a-3u show the alpha-chain variable domain amino acid sequence of the αβ heterodimeric TCR having high affinity for the SLLMWITQC-HLA A2 complex, respectively, and the mutated residues are underlined in bold.

Figures 4a-4q show the β-chain variable domain amino acid sequence of the αβ heterodimeric TCR having high affinity for the SLLMWITQC-HLA A2 complex, respectively, and the mutated residues are underlined in bold.

Figures 5a and 5b show the wild-type TCR alpha and beta chain variable domain amino acid sequences that are capable of specifically binding to the SLLMWITQC-HLA A2 complex, respectively.

Figures 6a and 6b show the extracellular amino acid sequences of wild-type TCR alpha and beta chains capable of specifically binding to the SLLMWITQC-HLA A2 complex, respectively.

Figures 7a and 7b show the extracellular amino acid sequences of the reference TCR alpha and beta chains, respectively.

Figure 8a and Figure 8b show the single-chain template TCR alpha chain and beta chain variable domain amino acid sequences, respectively.

Figures 9a and 9b show the single-stranded template TCR alpha chain and beta chain variable domain DNA sequences, respectively.

Figures 10a and 10b show the amino acid sequence and DNA sequence, respectively, showing a flexible linker.

Figure 11a and Figure 11b show the amino acid sequence and DNA sequence of a stable single-chain TCR molecule as a template strand, respectively.

Figure 12 shows the Biacore interaction curve for the reference TCR and SLLMWITQC-HLA A2 complex.

Figures 13a and 13b show the amino acid sequences of fusion molecules fused to anti-CD3 scFv at the N and C terminus of a single-chain TCR molecule (α chain variable domain SEQ ID NO: 2 and β chain variable domain SEQ ID NO: 27), respectively. .

Figures 14a and 14b show the amino acid sequences of fusion molecules fused to anti-CD3 scFv at the N and C terminus of a single-chain TCR molecule (α chain variable domain SEQ ID NO: 1 and β chain variable domain SEQ ID NO: 30), respectively. .

Figures 15a and 15b show the amino acid sequences of fusion molecules fused to anti-CD3 scFv at the N and C terminus of a single-chain TCR molecule (α chain variable domain SEQ ID NO: 89 and β chain variable domain SEQ ID NO: 26), respectively. .

Figures 16a and 16b show the amino acid sequences of fusion molecules fused to anti-CD3 scFv at the N and C terminus of a single-chain TCR molecule (α chain variable domain SEQ ID NO: 89 and β chain variable domain SEQ ID NO: 27), respectively. .

Figure 17 shows the amino acid sequence of a fusion molecule in which the anti-CD3 scFv is fused to the β-strand of the αβ heterodimeric TCR (whose variable domain amino acid sequence is SEQ ID NO: 69).

Figure 18 shows the amino acid sequence of a fusion molecule in which the anti-CD3 scFv is fused to the β-strand of the αβ heterodimeric TCR (whose variable domain amino acid sequence is SEQ ID NO: 70).

Figure 19 and Figure 20 show the activation of effector cells by fusion molecules of the high affinity single chain TCR of the present invention and an anti-CD3 antibody.

Figures 21-25 show the specific activation of effector cells by fusion molecules of the high affinity TCR and anti-CD3 antibodies of the invention.

detailed description

The present inventors obtained a high-affinity T cell receptor (TCR) recognizing a SLLMWITQC short peptide (derived from NY-ESO-1 protein) by peptide-HLA A2 through extensive and intensive research. The form of the complex is presented. The TCR of the invention is mutated in its alpha chain variable domain and/or beta chain variable domain relative to a wild type TCR capable of recognizing the SLLMWITQC-HLA A2 complex, and the affinity of the inventive TCR for the above SLLMWITQC-HLA A2 complex The and/or binding half-life is at least twice that of the wild-type TCR.

the term

T cell receptor (TCR)

The International Immunogenetics Information System (IMGT) can be used to describe TCR. Those skilled in the art are aware of the public database of the system. The native alpha beta heterodimeric TCR has an alpha chain and a beta chain. Broadly speaking, each strand comprises a variable region, a junction region, and a constant region, and the beta strand typically also contains a short polymorphic region between the variable region and the junction region, but the polymorphic region is often considered part of the junction region. The TCR junction region was determined by the unique IMGT TRAJ and TRBJ, and the constant region of the TCR was determined by the TACT and TRBC of IMGT.

Each variable region comprises three CDRs (complementarity determining regions) chimeric in a framework sequence, one of which is a CDR3, which is recombined from a variable region and a junction region, and is referred to as a hypervariable region. Depending on the framework, CDR1 and CDR2 sequences, and partially determined CDR3 sequences, the alpha chain variable regions (Vα) can be divided into several classes, and the beta chain variable regions (Vβ) are also classified into several classes. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to Same as Vα type and Vβ type. In the IMGT system, the alpha chain constant domain has the following symbols: TRAC*01, where "TR" represents the T cell receptor gene; "A" represents the alpha chain gene; C represents the constant region; "*01" represents the allele Gene 1. The β-chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where “TR” represents a T cell receptor gene; “B” represents a β chain gene; C represents a constant region; “*01” represents an allele 1. In the form of the beta strand, there are two possible constant region genes "C1" and "C2".

The alpha and beta chains of TCR are generally considered to have two "domains", ie, a variable domain and a constant domain. The variable domain consists of a connected variable zone and a connection zone. Thus, in the specification and claims of the present application, "TCR alpha chain variable domain" refers to a linked TRAV and TRAJ region, and "TCR alpha chain constant domain" refers to an extracellular TCR alpha chain constant region or a C-terminally truncated TCR alpha chain. Constant zone. Similarly, "TCR beta chain variable domain" refers to the ligated TRBV and TRBD/TRBJ regions, and "TCR beta chain constant domain" refers to the extracellular TCR beta chain constant region or the C-terminally truncated TCR beta chain constant region.

The alpha and beta chain variable regions of the wild type TCR (abbreviated as "wild type TCR" in the present invention) capable of specifically binding to the SLLMWITQC-HLA A2 complex have the following numbers in the IMGT:

Alpha chain: TRAV17

β chain: TRBV12-4

The alpha and beta chain variable domain amino acid sequences of wild type TCR (SEQ ID NO: 75 and SEQ ID NO: 76) are shown in Figure 5, with the alpha and beta chain extracellular amino acid sequences (SEQ ID NO: 77 and SEQ ID). NO: 78) as shown in Figure 6.

Detailed description of the invention

The present invention provides a T cell receptor (TCR) comprising a TCRα variable domain and/or a TCRβ variable domain having the properties of a SLLMWITQC (SEQ ID NO: 91) HLA-A2 complex, relative to having an alpha chain and a beta chain. The TCR of the variable domain amino acid sequence of SEQ ID NO: 75 and SEQ ID NO: 76, characterized in that (i) the TCR is represented by the alpha chain variable domain amino acid of SEQ ID NO: 75 and/or the SEQ ID NO :76 indicates that a mutation occurs in its β-chain variable domain amino acid; and (ii) the TCR has an affinity for the SLLMWITQC-HLA A2 complex that is at least twice the affinity of the wild-type TCR for the SLLMWITQC-HLA A2 complex. . Wherein the mutation in the alpha chain variable domain and/or the beta chain variable domain amino acid residue in (i) above occurs in one or more complementarity determining regions (CDRs) of the alpha and/or beta variable domains, The mutations enable a higher interaction force and/or a slower dissociation rate between the TCR of the invention and the SLLMWITQC-HLA A2 complex.

In a preferred embodiment of the present invention, the T cell receptor (TCR) according to the present invention is SEQ ID NO: 75 and the β chain variable domain amino acid sequence SEQ ID NO: 76 TCR:

The 157T of the extracellular alpha chain constant region of wild-type TCR (ie, 48T of TRAC in IMGT) was mutated to 157C, 169S of the beta-chain constant region (ie, TRBC1 in IMGT) according to methods of site-directed mutagenesis well known to those skilled in the art. The 57S) mutation is 169C, that is, the reference TCR is obtained, and the amino acid sequences thereof are shown in Figures 7a and 7b, respectively, and the mutated cysteine residues are represented by bold letters. The above cysteine substitution can form an artificial interchain disulfide bond between the constant region of the reference TCR and the β chain to form a more stable soluble TCR, thereby making it easier to evaluate the complexation of TCR with SLLMWITQC-HLA A2. Binding affinity and/or binding half-life between the substances. It will be appreciated that the CDR regions of the TCR variable region determine its affinity for the pMHC complex and, therefore, the cysteine substitution of the above TCR constant region does not affect the binding affinity and/or binding half-life of the TCR. Therefore, the binding affinity between the reference TCR and the SLLMWITQC-HLA A2 complex measured in the present invention is the binding affinity between the wild-type TCR and the SLLMWITQC-HLA A2 complex. Similarly, if the binding affinity between the TCR of the present invention and the SLLMWITQC-HLA A2 complex is measured to be at least twice the binding affinity between the reference TCR and the SLLMWITQC-HLA A2 complex, ie equivalent to the TCR and SLLMWITQC of the present invention. The binding affinity between the HLA A2 complex is at least twice the binding affinity between the wild type TCR and the SLLMWITQC-HLA A2 complex.

Binding may be measured by any suitable method, the affinity (dissociation equilibrium constant and inversely proportional to K _D) and half-life of binding (expressed as T _1/2). It should be understood that doubling the affinity of the TCR will result in a halving of K _D . T _{1/2 is} calculated as In2 divided by the dissociation rate (K _off ). Therefore, doubling T _1/2 will cause K _{off to be} halved. Preferably, the same test protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example 3 or more times, and the average of the results is taken. In a preferred embodiment, these measurements are performed using the surface plasmon resonance (BIAcore) method of the examples herein. The method of detecting the reference solution dissociation equilibrium constant K _D of SLLMWITQC-HLA A2 TCR complex ratio of 6.4μM, i.e., wild-type TCR to SLLMWITQC-HLA A2 solution complex dissociation equilibrium constant K _D is also 6.4μM.

Mutations can be performed by any suitable method, including but not limited to, based on polymerase chain reaction (PCR) Those based on restriction enzyme cloning or ligation-independent cloning (LIC) methods. Many standard molecular biology textbooks detail these methods. For more details on polymerase chain reaction (PCR) mutagenesis and cloning based on restriction enzymes, see Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (Third Edition) CSHL Publishing house. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6).

The method of producing the TCR of the present invention may be, but is not limited to, screening for a TCR having high affinity for the SLLMWITQC-HLA-A2 complex from a diverse library of phage particles displaying such TCR, as in the literature (Li, et al). (2005) Nature Biotech 23(3): 349-354).

It will be appreciated that genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing the alpha and beta chain variable domain amino acids of the slightly modified wild-type TCR can be used to prepare template TCRs. The changes required to produce the high affinity TCR of the invention are then introduced into the DNA encoding the variable domain of the template TCR.

In some preferred embodiments of the invention, the TCR of the invention is at the alpha chain variable domain amino acid residues 27T, 28S, 29I, 30N, 51S, 53E, 54R, 55E, 91T, 94A of SEQ ID NO: 75, One or more mutations in 95G, 96K, 97S and 98T (using the numbering shown in SEQ ID NO: 75) and/or the β-chain variable domain amino acid residues 50N, 51N shown in SEQ ID NO: 76 One or more of 52N, 53V, 54P, 95T, 97G, 98A, 99Q, 100P, 101Q and 102H are mutated (using the number shown in SEQ ID NO: 76). For example, the mutated TCR alpha chain variable domain comprises one or more amino acid residues selected from the group consisting of: 27Y, 27W, 27H, 27F or 27N; 28T, 28Y, 28D, 28P, 28N or 28W; 29P, 29L, 29T, 29V or 29A; 30Q; 51N; 53S; 54Q; 55T; 91N; 94T, 94N, 94H, 94I or 94S; 95A or 95S; 96R or 96W; 97W; 98N, 98D or 98A; and/or after mutation The TCR β chain variable domain comprises one or more amino acid residues selected from the group consisting of: 50C or 50Y; 51H or 51L; 52G; 53L; 54V or 54I; 95S; 97N; 98G or 98S; 99N or 99L; 100A; ; 102V or 102I. More specifically, specific forms of the mutation in the alpha chain variable domain include T27Y/W/H/F/N, S28T/Y/D/P/N/W, I29P/L/T/V/A, N30Q One or more groups of S51N, E53S, R54Q, E55T, T91N, A94T/N/H/I/S, G95A/S, K96R/W, S97W or T98N/D/A; β-chain variable domains Specific forms of the mutation include one or more of N50C/Y, N51H/L, N52G, V53L, P54V/I, T95S, G97N, A98G/S, Q99N/L, P100A, Q101I, and H102V/I.

The high affinity TCR of the present invention comprises the alpha chain variable domain amino acid sequences SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, Amino acid sequence of one of 53, 54, 55, 56, 57, 90 and/or β chain variable domain SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, One of 65, 66, 67, 68, 69, 70, 71, 72, 73, 74. Thus, the TCR alpha chain of the alpha chain variable domain amino acid sequence (SEQ ID NO: 75) containing the wild-type TCR can comprise and SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 The TCR β chain of one of 68, 69, 70, 71, 72, 73, 74 is bound. Alternatively, the TCR β chain comprising the β-variable domain amino acid sequence of wild-type TCR (SEQ ID NO: 76) can comprise SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, The TCR alpha chain of one of 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 90 is bound. Or alternatively, comprising the TCR alpha chain variable domain amino acid sequences SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 The TCR alpha chain of one of 56, 57, 90 and the amino acid sequence comprising the TCR β chain variable domain SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 The TCR β chain of one of 71, 72, 73, 74 is bound.

For the purposes of the present invention, a TCR of the invention is a moiety having at least one TCR alpha and/or TCR beta chain variable domain. They usually comprise both a TCR alpha chain variable domain and a TCR beta chain variable domain. They may be alpha beta heterodimers or single stranded forms or any other form that is stable. In adoptive immunotherapy, the full length strand of the alpha beta heterodimeric TCR (including the cytoplasmic and transmembrane domains) can be transfected. The TCR of the present invention can be used as a targeting agent for delivering a therapeutic agent to an antigen presenting cell or in combination with other molecules to prepare a bifunctional polypeptide to direct effector cells, in which case the TCR is preferably in a soluble form.

For stability, in one aspect, the TCR of the invention can be a TCR that introduces an artificial disulfide bond between the residues of its alpha and beta chain constant domains. The cysteine residue forms an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. A cysteine residue can replace other amino acid residues at a suitable position in the native TCR to form an artificial interchain disulfide bond. For example, a Thr248 residue of the exon 1 of TRAC*01 and a cysteine residue of Ser57 of the exon 1 of TRBC1*01 or TRBC2*01 are substituted to form a disulfide bond. Other sites for introducing a cysteine residue to form a disulfide bond may also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 of Tyr10 and TRBC1*01 or TRBC2*01 exon 1 of Ser17; TRAC*01 exon 1 of Thr45 and TRBC1*01 or TRBC2*01 exon 1 of Asp59; TRAC*01 exon 1 Ser15 and TRBC1*01 or TRBC2*01 exon 1 of Glu15; TRAC*01 exon 1 of Arg53 and TRBC1*01 or TRBC2*01 exon 1 of Ser54; TRAC*01 exon 1 of Pro89 and ABC19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 and TRBC1*01 of exon 1 of TRAC*01 or Glu20 of exon 1 of TRBC2*01. That is, a cysteine residue replaces any of the above-mentioned sites in the α and β chain constant domains. A maximum of 15, or a maximum of 10, or a maximum of 8 or fewer amino acids may be truncated at one or more C-termini of the TCR constant domain of the invention such that it does not include a cysteine residue to achieve deletion of the native Disulfide bond The above object can also be achieved by mutating a suitable cysteine residue to another amino acid.

As described above, the TCR of the present invention may comprise an artificial disulfide bond introduced between residues of its α and β chain constant domains. It should be noted that the constant domains may or may not contain the introduced artificial disulfide bonds as described above, and the TCRs of the present invention may each contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence. The TRAC constant domain sequence of TCR and the TRBC1 or TRBC2 constant domain sequence can be joined by a native disulfide bond present in the TCR.

For stability, in another aspect, the TCR of the present invention further comprises a TCR having a mutation in its hydrophobic core region, and the mutation of these hydrophobic core regions is preferably a mutation capable of increasing the stability of the TCR of the present invention, as in the publication number It is described in the patent document of WO2014/206304. Such a TCR can be mutated at its position in the following variable domain hydrophobic core: (alpha and/or beta chain) variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and / Or the α-chain J gene (TRAJ) short peptide amino acid position reciprocal position 3, 5, 7 and/or β chain J gene (TRBJ) short peptide amino acid position reciprocal position 2, 4, 6 where the amino acid sequence position number The location number listed in the International Immunogenetics Information System (IMGT). Those skilled in the art are aware of the above-described international immunogenetic information system and can obtain the position number of the amino acid residues of different TCRs in the IMGT according to the database.

The TCR in which the hydrophobic core region is mutated in the present invention may be a high-stability single-chain TCR composed of a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. It should be noted that the flexible peptide chain of the present invention may be any peptide chain suitable for linking the TCR alpha and beta chain variable domains. The template strand for screening for a high affinity TCR constructed in Example 1 of the present invention is the above single chain TCR containing a hydrophobic core mutation. The α chain variable domain amino acid sequence (SEQ ID NO: 81) and the β chain variable domain amino acid sequence (SEQ ID NO: 82) of the single-stranded template TCR are shown in Figure 8, and the corresponding DNA sequence (SEQ ID NO: 83 and 84) are shown in Figure 9. The amino acid sequence (SEQ ID NO: 85) and DNA sequence (SEQ ID NO: 86) of its flexible linker are shown in FIG. Thus, a single-chain TCR composed of an α-chain variable domain and a β-chain variable domain having high affinity for the SLLMWITQC-HLA-A2 complex was selected.

The αβ heterodimer having high affinity for the SLLMWITQC-HLA-A2 complex of the present invention is obtained by introducing a mutation of the selected CDR region of the high-affinity single-stranded TCR into the wild-type TCRα and β. Obtained from the corresponding position of the chain.

In some embodiments of the invention, the alpha chain variable domain hydrophobic core amino acid residue 13I of the TCR of the invention is employed, using the numbering set forth in SEQ ID NO: 75 (ie, the 13th position of the alpha chain variable region listed in IMGT) ), 19A (ie, the 19th position of the alpha chain variable region listed in IMGT), 21M (ie, the 21st position of the alpha chain variable region listed in IMGT), and 79S (ie, the alpha chain listed in IMGT is variable) One or more of the 94th) Mutations occur and/or the numbering set forth in SEQ ID NO: 76, the TCR β chain variable domain hydrophobic core amino acid residue 11E (ie, the 11th position of the β chain variable region listed in IMGT), 13T (ie IMGT) One or more of the mutations in the β chain variable region (position 13) and 82S (ie, the 94th variable region listed in IMGT) are mutated.

In some preferred embodiments of the invention, the alpha chain variable domain hydrophobic core of the invention comprises one or more of amino acid residues 13V, 19V, 21I or 79V and/or using the numbering set forth in SEQ ID NO:75. Using the numbering set forth in SEQ ID NO: 76, the TCR[beta] variable domain hydrophobic core comprises one or more of amino acid residues 11L, 13V or 82V. More specifically, the mutated form of the TCRα variable domain hydrophobic core comprises one or more of I13V, A19V, M21I or S79V; the mutant form of the TCRβ variable domain hydrophobic core includes E11L, T13V and S82V One or several groups.

The high affinity TCR of the present invention further comprises an alpha chain variable domain amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29 , one of 32, 34, 35, 36, 37, 89 and/or a β chain variable domain amino acid sequence of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, One of 26, 27, 28, 30, 31, 33. Thus, the above highly stable single-chain TCR alpha chain variable domain (SEQ ID NO: 81) and the amino acid sequence are SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 TCR β chain variable domain binding of 26, 27, 28, 30, 31 or 33. Alternatively, the above high stability single-chain TCR β chain variable domain (SEQ ID NO: 82) and the amino acid sequence are SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 TCRα variable domain binding of 12, 13, 14, 29, 32, 34, 35, 36, 37 or 89. Or alternatively, the TCR alpha chain variable domains SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 34, 35, 36, One of 37 or 89 and one of the TCR β chain variable domains SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31 or 33 Combine.

The TCR of the present invention can also be provided in the form of a multivalent complex. The multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention, such as a tetrameric domain of p53 to produce a tetramer, or more A complex formed by combining a TCR of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells that present a particular antigen in vitro or in vivo, as well as intermediates that produce other multivalent TCR complexes for such applications.

The TCR of the present invention may be used singly or in combination with the conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate comprises a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of a cell presenting a SLLMWITQC-HLA-A2 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or any of these Combination or coupling of substances.

Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.

Therapeutic agents that can be combined or coupled to the TCRs of the invention include, but are not limited to: 1. Radionuclides (Koppe et al, 2005, 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, National Academy of Sciences (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al, 2003, Cancer Research 63, 3202); (Mosquera et al, 2005, The Journal Of Immunology 174, 4381); 5. Antibody scFv fragment (Zhu et al, 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles / nanometer Rod (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, Gene Treatment (Ge Ne therapy) 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nanomagnetic particles; 10. prodrug activating enzymes (eg, DT-diaphorase) (DTD) or biphenyl hydrolase-like protein (BPHL); 11. chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.

The antibody or fragment thereof to be combined with the TCR of the present invention includes an anti-T cell or an NK-cell determining antibody, such as an anti-CD3 or an anti-CD28 or an anti-CD16 antibody, and the binding of the above antibody or a fragment thereof to the TCR can effect the effector cell. Orientation to better target target cells. A preferred embodiment is the binding of a TCR of the invention to an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody. Specifically, the TCR-anti-CD3 single-chain antibody fusion of the present invention comprises a TCR alpha chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 89, 90 and a TCR β chain variable domain amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74. More specifically, the amino acid sequence of the TCR and anti-CD3 single chain antibody fusions of the invention may be selected from one of the following amino acid sequences: SEQ ID NOs: 92, 93, 94, 95, 96, 97, 98 and 99. More specifically, the amino acid sequence of the TCR β chain of the present invention and the anti-CD3 single chain antibody fusion may be selected from the following amino acid sequences. One: 100 and 101.

The invention also relates to nucleic acid molecules encoding the TCRs of the invention. The nucleic acid molecule of the invention may be in the form of DNA or in the form of RNA. The DNA can be a coding strand or a non-coding strand. For example, a nucleic acid sequence encoding a TCR of the invention may be the same or a degenerate variant of the nucleic acid sequence set forth in the Figures of the invention. By way of example, the meaning of "degenerate variant", as used herein, "degenerate variant" refers in the present invention to a protein sequence having SEQ ID NO: 81 but to the sequence of SEQ ID NO: 83. Differential nucleic acid sequences.

The full length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification, recombinant methods or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding the TCR (or a fragment thereof, or a derivative thereof) of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

The invention also relates to vectors comprising the nucleic acid molecules of the invention, as well as host cells genetically engineered using the vectors or coding sequences of the invention.

The invention also encompasses isolated cells, particularly T cells, which express the TCR of the invention. There are a number of methods suitable for T cell transfection with DNA or RNA encoding the high affinity TCR of the invention (e.g., Robbins et al., (2008) J. Immunol. 180: 6116-6131). T cells expressing the high affinity TCR of the invention can be used in adoptive immunotherapy. Those skilled in the art will be aware of many suitable methods for performing adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of the present invention, or a TCR complex of the present invention, or a cell which presents the TCR of the present invention.

The invention also provides a method of treating a disease comprising administering to a subject in need of treatment an appropriate 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 in this article are identified by the international common single letter, and the corresponding amino acid names are abbreviated as: 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 addition, the specific form of the mutation in the present invention is expressed as "N30Q" for the 30th The N of the position is substituted by Q. Similarly, "T27Y/W/H/F/N" represents that the T at position 27 is substituted by Y or substituted by W or substituted by H or substituted by F or substituted by N. Others and so on.

In the art, when substituted with amino acids of similar or similar properties, the protein is usually not altered. Quality function. The addition of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the structure and function of the protein. Thus, the TCR of the invention further comprises up to 5, preferably up to 3, more preferably up to 2, optimally 1 amino acid (especially an amino acid located outside the CDR regions) of the TCR of the invention, which is similar in nature Replace the amino acid with a similar amino acid and still be able to maintain its functionality.

The present invention also encompasses a TCR slightly modified for the TCR of the present invention. Modifications (usually without altering the primary structure) include: chemically derivatized forms of the TCRs of the invention, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the TCRs of the invention or in further processing steps. Such modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are TCRs that have been modified to enhance their anti-proteolytic properties or to optimize solubility properties.

The TCR, TCR complexes of the invention or TCR transfected T cells of the invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCR, multivalent TCR complex or cell of the invention is typically provided as part of a sterile pharmaceutical composition, which typically comprises a pharmaceutically acceptable carrier. The pharmaceutical composition can be in any suitable form (depending on the method desired for administration to a patient). It can be provided in unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It can include a plurality of said unit dosage forms.

Furthermore, the TCRs of the invention may be used alone or in combination or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991). Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

The pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid 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.

In general, the therapeutic compositions can be formulated as injectables, such as liquid solutions or suspensions; solid forms such as liquid carriers, which may be formulated in solution or suspension prior to injection.

Once formulated into a composition of the invention, it can be administered by conventional routes including, but not limited to, intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal. External includes 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, a pharmaceutical composition of various dosage forms may be employed depending on the use. Preferably, an injection, an oral preparation, or the like can be exemplified.

These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, and the formulation process can be carried out in a customary manner depending on the dosage form.

The pharmaceutical compositions of the invention may also be administered in the form of sustained release agents. For example, the TCR of the present invention can be incorporated into a pill or microcapsule in which the sustained release polymer is used as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. Examples of the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, and lactic acid polymer. A lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.

When the pharmaceutical composition of the present invention is used for actual treatment, the TCR or TCR complex of the present invention as an active ingredient or the cell presenting the TCR of the present invention may be based on the body weight, age, sex, and degree of symptoms of each patient to be treated. And reasonable to determine, and ultimately the doctor determines the reasonable amount.

The main advantages of the invention are:

(1) The present invention screens a TCR having a high affinity for the SLLMWITQC-HLA-A2 complex using a hydrophobic core-mutated single-chain TCR molecule as a template.

(2) The affinity and/or binding half-life of the TLR of the present invention to the SLLMWITQC-HLA-A2 complex is at least twice that of the wild-type TCR.

(3) The affinity and/or binding half-life of the high affinity TCR of the present invention to the SLLMWITQC-HLA-A2 complex can reach 10 ² -10 ⁵ times or more of the wild type TCR.

(4) The fusion molecule of the high affinity TCR of the present invention and the anti-CD3 antibody activates effector cells much more than the activation of the effector cells by the fusion molecule of the wild type TCR and the anti-CD3 antibody.

(5) The fusion molecule of the high affinity TCR of the present invention and the anti-CD3 antibody has a specific activation effect on effector cells.

The invention is further illustrated by the following specific examples. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental parties not showing specific conditions in the following examples The method is usually 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). The conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Materials and Method

The experimental materials used in the examples of the present invention can be obtained from commercially available channels unless otherwise specified, wherein E. coli DH5α is purchased from Tiangen, E. coli BL21 (DE3) and purchased from Tiangen, E. coli Tuner (DE3). From Novagen, plasmid pET28a was purchased from Novagen.

Example 1 Stability of hydrophobic core mutations Production of single-stranded TCR template strands

The present inventors constructed a stable single-chain TCR molecule composed of a flexible short peptide linked to a TCRα and a β-chain variable domain by a site-directed mutagenesis (see Patent Document WO2014/206304), the amino acid and DNA sequence thereof. SEQ ID NO: 87 and SEQ ID NO: 88, respectively, as shown in FIG. The single-chain TCR molecule was used as a template for screening high affinity TCR molecules. The amino acid sequences of the alpha variable domain (SEQ ID NO: 81) and the beta variable domain (SEQ ID NO: 82) of the template strand are shown in Figure 8; the corresponding DNA sequences are SEQ ID NOs: 83 and 84, respectively. As shown in FIG. 9; the amino acid sequence and DNA sequence of the flexible short linker are SEQ ID NOS: 85 and 86, respectively, as shown in FIG.

The target gene carrying the template strand was digested with NcoI and NotI, and ligated with the pET28a vector digested with NcoI and NotI. The ligation product was transformed into E. coli DH5α, coated with kanamycin-containing LB plate, inverted culture at 37 ° C overnight, and the positive clones were picked for PCR screening. The positive recombinants were sequenced to determine the correct sequence and the recombinant plasmid was extracted. To E. coli BL21 (DE3) for expression.

Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1

The BL21(DE 3) colonies containing the recombinant plasmid pET28a-template strand prepared in Example 1 were all inoculated into LB medium containing kanamycin, and cultured at 37 ° C until the OD ₆₀₀ was 0.6-0.8, and IPTG was added to the end. The concentration was 0.5 mM, and incubation was continued for 4 h at 37 °C. The cell pellet was harvested by centrifugation at 5000 rpm for 15 min, the cell pellet was lysed with Bugbuster Master Mix (Merck), the inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min, and then washed with Bugbuster (Merck) to remove cell debris and membrane fraction, centrifuged at 6000 rpm for 15 min, and collected. body. The inclusion body was dissolved in a buffer (20 mM Tris-HCl pH 8.0, 8 M urea), and the insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by BCA method, and then stored at -80 ° C until use.

To 5 mg of the dissolved single-chain TCR inclusion body protein, 2.5 mL of buffer (6 M Gua-HCl, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA) was added, and DTT was added to a final concentration of 10 mM, and treated at 37 ° C for 30 min. . The above-treated single-chain TCR was added dropwise to a 125 mL refolding buffer (100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM β-mercapthoethylamine, 1.87 mM Cystamine) with a syringe. After stirring at 4 ° C for 10 min, the reconstituted solution was placed in a cellulose membrane dialysis bag with a cut-off amount of 4 kDa, and the dialysis bag was placed in 1 L of pre-cooled water and slowly stirred at 4 ° C overnight. After 17 hours, the dialysate was changed to 1 L of pre-cooled buffer (20 mM Tris-HCl pH 8.0), dialysis was continued for 8 h at 4 ° C, and the dialysate was replaced with the same fresh buffer to continue dialysis overnight. After 17 hours, the sample was filtered through a 0.45 μm filter, and the protein was purified by vacuum degassing through an anion exchange column (HiTrap Q HP, GE Healthcare) in a linear gradient of 0-mM NaCl prepared with 20 mM Tris-HCl pH 8.0. The collected fractions were subjected to SDS-PAGE analysis, and the fractions containing the single-chain TCR were concentrated and further purified by a gel filtration column (Superdex 7510/300, GE Healthcare), and the target components were also subjected to SDS-PAGE analysis.

The eluted fraction for BIAcore analysis was further tested for purity using gel filtration. The conditions were as follows: column Agilent Bio SEC-3 (300A, φ 7.8×300 mm), mobile phase 150 mM phosphate buffer, flow rate 0.5 mL/min, column temperature 25 ° C, UV detection wavelength 214 nm.

Example 3 Binding Characterization

BIAcore analysis

The binding activity of the TCR molecule to the SLLMWITQC-HLA-A2 complex was detected using a BIAcore T200 real-time analysis system. The 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 surface of the chip. Finally, the unreacted activated surface was blocked with a solution of ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000 RU.

A low concentration of streptavidin is passed over the surface of the coated antibody chip, then the SLLMWITQC-HLA-A2 complex is flowed through the detection channel, the other channel is used as a reference channel, and 0.05 mM biotin is then 10 μL/ The flow rate of min flowed through the chip for 2 min, blocking the remaining binding sites of streptavidin. The affinity was determined by single-cycle kinetic analysis. TCR was diluted to several different concentrations with HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) at a flow rate of 30 μL/min. , flowing through the surface of the chip in turn, the bonding time per injection is 120 s, let it dissociate for 600s after the last injection. The chip was regenerated with 10 mM Gly-HCl, pH 1.75, after each round of assay. Kinetic parameters were calculated using BIAcore Evaluation software.

The preparation process of the above SLLMWITQC-HLA-A2 complex is as follows:

Purification

100 ml of E. coli bacterial solution inducing expression of heavy or light chain was collected, and the cells were washed once with 8000 g of PBS at 10 ° C for 10 min, and then resuspended by vigorous shaking with 5 ml of BugBuster Master Mix Extraction Reagents (Merck). Incubate for 20 min at room temperature, then centrifuge at 6000 g for 15 min at 4 ° C, discard the supernatant, and collect inclusion bodies.

The above-mentioned inclusion weight was suspended in 5 ml BugBuster Master Mix, and incubated at room temperature for 5 min; 30 ml of BugBuster diluted 10 times, mixed, centrifuged at 6000 g for 15 min at 4 ° C; the supernatant was discarded, and 30 ml of BugBuster resuspended inclusion body was diluted 10 times. , mix, centrifuge at 6000g for 4min at 4°C for 15min, repeat twice, add 30ml 20mM Tris-HCl pH 8.0, resuspend the inclusion body, mix, centrifuge at 6000g for 15min at 4°C, and finally dissolve the inclusion body with 20mM Tris-HCl 8M urea, SDS- The inclusion body purity was measured by PAGE and the concentration was measured by BCA kit.

b. renaturation

The synthesized short peptide SLLMWITQC (Beijing Saibaisheng Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light and heavy chains were dissolved with 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and further denatured by adding 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA before renaturation. The SLLMWITQC peptide was added to the refolding buffer (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, at 25 mg/L (final concentration), 0.2 mM PMSF, cooled to 4 ° C), then add 20 mg / L light chain and 90 mg / L heavy chain (final concentration, heavy chain added three times, 8h / time), renaturation at 4 ° C for at least 3 days By the time of completion, SDS-PAGE can be used to detect renaturation.

c. renaturation and purification

The renaturation buffer was replaced with 10 volumes of 20 mM Tris pH 8.0 for dialysis, and at least two buffers were exchanged to substantially reduce the ionic strength of the solution. 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 Company) anion exchange column (5 ml bed volume). The protein was eluted using a linear gradient of 0-400 mM NaCl prepared by an Akta Purifier (GE General Electric Company), 20 mM Tris pH 8.0, pMHC was eluted at approximately 250 mM NaCl, peak fractions were collected, and purity was determined by SDS-PAGE.

d. Biotinylation

The purified pMHC molecule was concentrated using a Millipore ultrafiltration tube while the buffer was replaced with 20 mM Tris pH 8.0, followed by biotinylation reagent 0.05M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, 100 μg/ml BirA The enzyme (GST-BirA) was incubated overnight at room temperature and SDS-PAGE was used to determine if biotinylation was complete.

e. Purification of the biotinylated complex

The biotinylated labeled pMHC molecule was concentrated to 1 ml using a Millipore ultrafiltration tube, biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta Purifier (GE General Electric Company). ^{A TM} 16/60 S200 HR column (GE General Electric Company) was loaded with 1 ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. The biotinylated pMHC molecule appeared as a single peak elution at about 55 ml. The protein-containing fractions were pooled, concentrated using a Millipore ultrafiltration tube, protein concentration was determined by BCA method (Thermo), and biotinylated pMHC molecules were dispensed at -80 °C by adding protease inhibitor cocktail (Roche).

Example 4 Production of High Affinity Single-chain TCR

Phage display technology is a means of generating TCR high affinity variant libraries to screen for high affinity variants. The TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3): 349-354) was applied to the single-chain TCR template of Example 1. A library of high affinity TCRs was created and panned by mutating the CDR regions of the template strand. Those skilled in the art can obtain the library construction and screening method by reading the above documents. That is, it is achieved by using a primer having one or more codon changes required and a plasmid containing the relevant DNA as a template. After several rounds of panning, the phage library specifically binds to the corresponding antigen, picks up the monoclonal and performs sequence analysis.

The BIAcore method of Example 3 was used to analyze the interaction of the TCR molecule with the SLLMWITQC-HLA-A2 complex, and the high affinity TCR with affinity and/or binding half-life of at least twice that of the wild-type TCR was screened. high affinity TCR binding solution SLLMWITQC-HLA-A2 complex dissociation equilibrium constant K _D or less of wild-type TCR binding solution SLLMWITQC-HLA-A2 complex equilibrium constant K _D from one of the two points, the results shown in table 1 Shown. Using the above method, the K _D value of the interaction between the reference TCR and the SLLMWITQC-HLA-A2 complex was 6.4 μM, and the interaction curve is shown in Figure 12, that is, the wild type TCR interacts with the SLLMWITQC-HLA-A2 complex. The K _D value was also 6.4 μM.

Specifically, using the numbering shown in SEQ ID NO: 75, the α chain variable domains of these high affinity TCR mutants are mutated in amino acids at one or more of the following positions 27T, 28S, 29I, 30N, 51S, 53E , 54R, 55E, 91T, 94A, 95G, 96K, 97S, 98T and/or using SEQ ID NO: 76 The numbering of the β-chain variable domains of these high-affinity TCR mutants is mutated in amino acids at one or more of the following positions: 50N, 51N, 52N, 53V, 54P, 95T, 97G, 98A, 99Q, 100P, 101Q , 102H.

More specifically, using the numbering set forth in SEQ ID NO: 75, the alpha chain variable domains of these high affinity TCRs comprise one or more amino acid residues 27Y, 27W, 27H, 27F or 27N selected from the group consisting of 28T; , 28Y, 28D, 28P, 28N or 28W; 29P, 29L, 29T, 29V or 29A; 30Q; 51N; 53S; 54Q; 55T; 91N; 94T, 94N, 94H, 94I or 94S; 95A or 95S; 96R or 96W ; 97W; 98N, 98D or 98A; and/or using the numbering set forth in SEQ ID NO: 76, the β-chain variable domains of these high-affinity TCRs comprise one or more amino acid residues 50C or 50Y selected from the group consisting of 51H or 51L; 52G; 53L; 54V or 54I; 95S; 97N; 98G or 98S; 99N or 99L; 100A; 101I; 102V or 102I.

Α-chain variable domains of high-affinity single-chain TCR (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 34, 35, 36, 37, 89) and β chain variable domains (SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30 Specific examples of the amino acid sequences of 31, 33) are shown in Figures 1a-u and 2a-q, respectively.

Table 1

Example 5 Production of High Affinity αβ Heterodimeric TCR

The CDR region mutations of the high affinity single-chain TCRs screened in Example 4 were introduced into the corresponding sites of the variable domain of the wild-type TCR, and their affinity for the SLLMWITQC-HLA-A2 complex was detected by BIAcore. The introduction of high affinity mutation points in the above CDR regions employs a method of site-directed mutagenesis well known to those skilled in the art. The α chain and β chain extracellular amino acid sequences of the above wild type TCR are shown in Figure 6a (SEQ ID NO: 77) and 6b (SEQ ID NO: 78), respectively, and do not contain a leader introduced for efficient initiation of expression in bacteria. Methionine. It should be noted that in order to obtain a more stable soluble TCR for more convenient evaluation of the binding affinity and/or binding half-life between the TCR and the SLLMWITQC-HLA A2 complex, the present invention is in the constant region of the alpha and beta chains of the wild type TCR described above. A cysteine residue is introduced to form an artificial interchain disulfide bond. The amino acid sequences of the extracellular TCRα and β chains after introducing a cysteine residue are shown in Figure 7a (SEQ ID NO: 79) and 7b (SEQ ID NO: 80), respectively, and the introduced cysteine residues are Indicated by bold letters.

The extracellular sequence genes of the TCRα and β chains to be expressed are synthesized and inserted into the expression vector by standard methods described in the Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell). pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI, respectively. Mutations in the CDR regions are introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The insert was sequenced to confirm that it was correct.

Example 6 Expression, renaturation and purification of αβ heterodimeric TCR

The expression vectors of TCRα and β chain were transformed into expression plasmid BL21(DE3) by chemical transformation, respectively, and the bacteria were grown in LB medium. The expression of α and β chain of TCR was induced by LD ₆₀₀ =0.6 with a final concentration of 0.5 mM IPTG. The resulting inclusion bodies were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution. The inclusion bodies were finally dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA). ), in 20 mM Tris (pH 8.1).

The dissolved TCRα and β chains are rapidly mixed in 5M urea at a mass ratio of 1:1, 0.4M fine The final concentration was 60 mg/mL in 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4 ° C). After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ° C), and after 12 hours, deionized water was exchanged for buffer (20 mM Tris, pH 8.0) and dialysis was continued at 4 ° C for 12 hours. The solution after completion of dialysis was filtered through a 0.45 μM filter, and then purified by an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The TCR containing the refolding successful alpha and beta dimers was confirmed by SDS-PAGE gel. The TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purified TCR purity was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.

Example 7 BIAcore analysis results

The affinity of the αβ heterodimeric TCR introduced with the high affinity mutation point and the SLLMWITQC-HLA-A2 complex was detected by the method described in Example 3.

The mutations of the CDR regions in the high-affinity single-chain TCRα and β-chain are introduced into the corresponding positions of SEQ ID NO: 75 and/or SEQ ID NO: 76, respectively, to obtain new TCRα and β-chain variable domain amino acid sequences, respectively As shown in Figure 3 and Figure 4. Since the CDR regions of the TCR molecule determine their affinity for the corresponding pMHC complex, one skilled in the art can expect that the alpha beta heterodimeric TCR incorporating a high affinity mutation also has a high affinity for the SLLMWITQC-HLA-A2 complex. . Select three groups of TCRs for verification. The expression vector was constructed by the method described in Example 5, and the above-mentioned high-affinity mutant αβ heterodimeric TCR was expressed, renatured and purified by the method described in Example 6, and then determined by BIAcore T200 and SLLMWITQC- The affinity of the HLA-A2 complex is shown in Table 2 below.

Table 2

As can be seen from Table 2 above, the αβ heterodimeric TCR introduced with the CDR region mutation maintains a high affinity for the SLLMWITQC-HLA-A2 complex.

Example 8 Expression, renaturation and purification of fusions of anti-CD3 antibodies with high affinity single chain TCR

We selected several high-affinity single-chain TCR molecules with anti-CD3 antibody single-chain antibodies (scFv) Fusion was performed to construct a fusion molecule. Selected high affinity single chain TCR molecules include: (1) a single chain TCR molecule consisting of the alpha chain variable domain SEQ ID NO: 2 and the beta chain variable domain SEQ ID NO: 27, which is anti-CD3 The amino acid sequences of the fusion molecules of the scFv fused at the N and C ends of the single-stranded TCR molecule are shown in Figures 13a and 13b, respectively. (2) a single-chain TCR molecule consisting of the α chain variable domain SEQ ID NO: 1 and the β chain variable domain SEQ ID NO: 30, which is fused to the anti-CD3 scFv at the N and C ends of the TCR molecule. The amino acid sequences of the fusion molecules are shown in Figures 14a and 14b, respectively. (3) A single-chain TCR molecule consisting of the α chain variable domain SEQ ID NO: 89 and the β chain variable domain SEQ ID NO: 26, which is at the N and C positions of the anti-CD3 scFv at the single-stranded TCR molecule The amino acid sequences of the fused fusion molecules are shown in Figures 16a and 16b, respectively. (4) A single-chain TCR molecule consisting of the α chain variable domain SEQ ID NO: 89 and the β chain variable domain SEQ ID NO: 27, which is in the N and C positions of the anti-CD3 scFv at the single-stranded TCR molecule. The amino acid sequences of the fused fusion molecules are shown in Figures 16a and 16b, respectively. The amino acid sequence of the above fusion molecule contains a leading methionine introduced for efficient expression in bacteria. The preparation process of the fusion molecule is as follows:

Fusion protein expression

The expression plasmid was transformed into E. coli strain BL21 (DE3), and LB plate (Kanamycin 50 μg/ml) was applied and cultured at 37 ° C overnight. On the next day, the clones were inoculated into 10 ml of LB liquid medium (kanamycin 50 μg/ml) for 2-3 h, and inoculated into 1 L of LB medium (kanamycin 50 μg/ml) at a volume ratio of 1:100. The culture was carried out until the OD ₆₀₀ was 0.5-0.8, and then the expression of the protein of interest was induced using IPTG at a final concentration of 0.5 mM. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. The cells were washed once in PBS buffer, and the cells were dispensed, and the cells corresponding to 200 ml of the bacterial culture were lysed with 5 ml of BugBuster Master Mix (Novagen), and the inclusion bodies were collected by centrifugation at 6000 g for 15 minutes. A detergent wash was then performed 4 times to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergent and salt. Finally, the inclusion bodies were dissolved in a Tris buffer solution containing 8 M urea, and the inclusion body concentration was measured, and the package was divided and stored at -80 ° C for cryopreservation.

Refolding of fusion proteins

About 10 mg of inclusion bodies were taken out from the -80 ° C ultra-low temperature freezer and thawed, and dithiothreitol (DTT) was added to a final concentration of 10 mM, and incubated at 37 ° C for 30 minutes to 1 hour to ensure complete opening of the disulfide bond. Then, the inclusion body sample solution was separately dropped into 200 ml of 4 ° C pre-cooled refolding buffer (100 mM Tris pH 8.1, 400 mM L-arginine, 2 mM EDTA, 5 M urea, 6.5 mM β-mercapthoethylamine, 1.87 mM Cystamine), 4 ° C Stir slowly for about 30 minutes. The renaturation solution was dialyzed against 8 volumes of pre-cooled H ₂ O for 16-20 hours. It was further dialyzed twice with 8 volumes of 10 mM Tris pH 8.0, and dialysis was continued at 4 ° C for about 8 hours. After dialysis, the sample was filtered and subjected to the following purification.

First step purification of fusion protein

The dialyzed heavy fold (10 mM Tris pH 8.0) was eluted with a gradient of 0-600 mM NaCl using an POROS HQ/20 anion exchange chromatography prepacked column (Applied Biosystems) on an AKTA Purifier (GE Healthcare). Each component was analyzed by Coomassie brilliant blue stained SDS-PAGE and then combined.

Purification of the second step of the fusion protein

The first step of the purified sample solution was concentrated for purification in this step, and the fusion protein was purified by Coomassie blue staining using a Superdex 75 10/300 GL gel filtration chromatography prepacked column (GE Healthcare) pre-equilibrated in PBS buffer. The components of the peak were analyzed by SDS-PAGE and then combined.

Example 9 Expression, renaturation and purification of fusions of anti-CD3 antibodies with high affinity αβ heterodimeric TCRs

A fusion molecule is prepared by fusing an anti-CD3 single-chain antibody (scFv) with an αβ heterodimeric TCR. The anti-CD3 scFv is fused to the β chain of the TCR, and the TCR β chain may comprise the β-chain variable domain of any of the above-mentioned high-affinity αβ heterodimeric TCRs, and SEQ ID NO: 69 and SEQ ID NO are used in this embodiment. : 70 high affinity TCR beta chain variable domain. The TCR α chain of the fusion molecule may comprise the α chain variable domain of any of the above high affinity αβ heterodimeric TCRs, and the α chain variable domain represented by SEQ ID NO: 39 and SEQ ID NO: 90 is used in this embodiment. . The amino acid sequences of the anti-CD3 scFv and TCR β-chain fusion molecules are shown in Figures 17 and 18, respectively, and the amino acid sequence contains a leading methionine introduced for efficient expression in bacteria.

Construction of fusion molecular expression vector

1. Construction of α chain expression vector

The target gene carrying the α chain of the αβ heterodimeric TCR was digested with NcoI and NotI, and ligated with the pET28a vector digested with NcoI and NotI. The ligation product was transformed into E. coli DH5α, plated on LB plate containing kanamycin, and cultured overnight at 37 ° C. The positive clones were picked for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and the recombinant plasmid was extracted. Transformed to E. coli Tuner (DE3) for expression.

2. Construction of anti-CD3 (scFv)-β chain expression vector

Design primers to anti-CD3scFv and high affinity by overlapping PCR methods The dimeric TCRβ chain gene is ligated, the intermediate linker is GGGGS, and the gene fragment of the fusion protein of the anti-CD3 scFv and the high affinity heterodimeric TCRβ chain is subjected to restriction endonuclease The enzyme sites are Nco I (CCATGG) and Not I (GCGGCCGC). The PCR amplification product was digested with Nco I and Not I, and ligated with the pET28a vector digested with Nco I and Not I. The ligation product was transformed into E. coli DH5α competent cells, coated with kanamycin-containing LB plates, and cultured overnight at 37 ° C. Positive clones were picked for PCR screening, positive recombinants were sequenced, and the sequence was determined to be correct. The recombinant plasmid was transformed into E. coli Tuner (DE3) competent cells for expression.

Expression, renaturation and purification of fusion proteins

The expression plasmids were separately transformed into E. coli Tuner (DE3) competent cells, and LB plates (kanamycin 50 μg/mL) were applied and cultured at 37 ° C overnight. On the next day, the clones were inoculated into 10 mL LB liquid medium (kanamycin 50 μg/mL) for 2-3 h, inoculated into 1 L LB medium at a volume ratio of 1:100, and the culture was continued until the OD600 was 0.5-0.8. The final concentration of 1 mM IPTG induced the expression of the protein of interest. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. The cells were washed once in PBS buffer, and the cells were dispensed, and the cells corresponding to 200 mL of the bacterial culture were lysed with 5 mL of BugBuster Master Mix (Merck), and the inclusion bodies were collected by centrifugation at 6000 g for 15 minutes. A detergent wash was then performed 4 times to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergent and salt. Finally, the inclusion bodies were dissolved in a buffer solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris, pH 8.1, and the inclusion body concentration was determined and dispensed. It was then stored frozen at -80 °C.

The dissolved TCRα chain and anti-CD3(scFv)-β chain were rapidly mixed in a mass ratio of 2:5 to 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4 ° C), final concentration α chain and anti-CD3 (scFv)-β chain were 0.1 mg/mL, 0.25 mg/mL, respectively.

After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ° C), and after 12 hours, deionized water was exchanged for buffer (10 mM Tris, pH 8.0) and dialysis was continued at 4 ° C for 12 hours. The solution after completion of dialysis was filtered through a 0.45 μM filter, and then purified by an anion exchange column (HiTrap Q HP 5 ml, GE healthcare). The TCR of the eluted peak containing the reconstituted TCR alpha chain and the anti-CD3 (scFv)-beta chain dimer was confirmed by SDS-PAGE gel. The TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16/60, GE healthcare) and anion exchange column (HiTrap Q HP 5 ml, GE healthcare). The purity of the purified TCR fusion molecule was determined by SDS-PAGE to be greater than 90%, and the concentration was determined by the BCA method.

Example 10 Activation of T cells by fusion molecules of high affinity single chain TCR and anti-CD3 antibody

This example compares the fusion molecule of the high affinity single-chain TCR with an anti-CD3 antibody of the present invention and the fusion molecule of the wild-type TCR and the anti-CD3 antibody to the tumor cell line presenting the SLLMWITQC-HLA-A2 complex, thereby activating The ability of cytotoxic T lymphocytes (CTLs). The production of IFN-γ was measured by ELISPOT assay as a readout value for T cell activation.

The ELISPOT assay described above was carried out by selecting a fusion molecule of the high affinity single-chain TCR of the present invention and an anti-CD3 antibody. Selected fusion molecules include: high affinity single-chain TCR fusion 1 (Vα used is SEQ ID NO: 89; Vβ is SEQ DI NO: 26), high-affinity single-stranded TCR fusion 2 (Vα used is SEQ ID NO: 89 ; Vβ is SEQ DI NO: 27), high-affinity single-stranded TCR fusion 3 (Vα is SEQ ID NO: 2; Vβ is SEQ DI NO: 26), high-affinity single-stranded TCR fusion 4 (Vα used is SEQ) ID NO: 2; Vβ is SEQ DI NO: 27), high-affinity single-stranded TCR fusion 5 (Vα used is SEQ ID NO: 3; Vβ is SEQ DI NO: 26), high-affinity single-stranded TCR fusion 6 ( Vα used is SEQ ID NO: 4; Vβ is SEQ DI NO: 28), high affinity single-chain TCR fusion 7 (Vα used is SEQ ID NO: 3; Vβ is SEQ DI NO: 27), high affinity single-chain TCR Fusion 8 (Vα used is SEQ ID NO: 29; Vβ is SEQ DI NO: 30), and wild-type TCR fusion (Vα used is SEQ ID NO: 75; Vβ is SEQ DI NO: 76).

The reagents used in the ELISPOT assay are as follows: test medium, wash buffer, PBS and human IFN-γ ELISPOT PVDF-enzyme kit, target cells, effector cells, and fusion molecules of high affinity single-chain TCR and anti-CD3 antibody (eg, examples) Preparation of 8 can be diluted with test medium).

The preparation process of the target cells was as follows: The target cells used in this example were IM9 cells (HLA-A2 positive and NY-ESO-1 antigen positive). A sufficient number of target cells (20,000 cells/well) were washed with a 75004250 centrifuge (Thermo Corporation) at 500 g, once every 5 minutes. The cells were then resuspended in test medium at 4 x 10 ⁵ cells/ml.

The preparation process of effector cells is as follows: The effector cells (T cells) used in this example are CD8-positive T cells (obtained from PBL by negative selection (using CD8 negative isolation kit, MACS, catalog number 130-094-156)) . The effector cells were thawed, placed in a test medium, and then washed with a 75004250 centrifuge (Thermo Corporation) at 500 g for 5 minutes by centrifugation. The cells were then resuspended in test medium at 4 times the desired final concentration.

The ELISPOT plate preparation process is as follows: add 50 μl of 35% alcohol per well to pre-wet the bottom of the plate to Dilute 100 μl of anti-IFNγ capture antibody in 10 ml sterile PBS per plate. An aliquot of 100 microliters of diluted capture antibody was then added to each well. The plates were incubated overnight at 4 °C. After incubation, the plates were washed (Procedure 1, Plate Type 2, 96-well plate washer; BioTech) to remove capture antibodies. 1640 medium containing 10% serum was then added to each well at 100 μl/well and the plate was incubated for 2 hours at room temperature to block the plate. The medium was then washed from the plate (Procedure 1, Plate Type 2, 96-well plate washer; BioTech) and any remaining wash buffer was removed by flicking and tapping the ELISPOT plate on a paper towel.

The ELISPOT experimental procedure was as follows: The individual components of the assay were added to the ELISPOT plate in the following order: 50 μl of target cells 4×10 ⁵ cells/ml (total 20,000 target cells/well), 50 μl of reagent (high affinity TCR-anti-CD3) fusion molecules; various concentrations), 50 l medium (assay medium) and 50 l of effector cells (CD8 + 1000 cells / well), plates were then incubated overnight _{(37 ℃, 5% CO 2} ). The plates were then washed and subjected to secondary detection and development, and the plates were dried for 1 hour, and spots formed on the membrane were counted using an immuno spot plate reader (ELISPOT READER system; AID Corporation).

The results of the experiment are shown in Figures 19 and 20, which show that the fusion of the high-affinity single-stranded TCR and anti-CD3 antibody of the present invention activates T cells much more than the fusion of wild-type TCR and anti-CD3 antibodies. The activation of molecules on T cells.

Example 11 Activation of T cells by fusion molecules of high affinity αβ heterodimeric TCR and anti-CD3 antibody

This example compares the fusion molecule of the high affinity αβ heterodimeric TCR with an anti-CD3 antibody of the present invention and the fusion molecule of the wild type TCR and the anti-CD3 antibody to the tumor cell line presenting the SLLMWITQC-HLA-A2 complex. , thereby the ability to activate cytotoxic T lymphocytes (CTLs). The production of IFN-γ was measured by ELISPOT assay as a readout value for T cell activation. For the preparation of reagents, target cells and effector cells used in the ELISPOT experiment, specific experimental procedures can be referred to in Example 10. Among them, a fusion molecule of a high affinity heterodimeric TCR and an anti-CD3 antibody was prepared as described in Example 9, and it can be diluted with a test medium.

The ELISPOT assay described above was carried out by selecting a fusion molecule of the high affinity single-chain TCR of the present invention and an anti-CD3 antibody. Selected fusion molecules include: high affinity heterodimeric TCR fusion 1 (Vα used is SEQ ID NO: 39; Vβ is SEQ DI NO: 69), high affinity heterodimeric TCR fusion 2 (Vα used is SEQ) ID NO: 90; Vβ is SEQ DI NO: 69), and a wild-type TCR fusion (Vα used is SEQ ID NO: 75; Vβ is SEQ DI NO: 76).

The experimental results are shown in Fig. 21. Similarly, the results show the high affinity heterodimeric TCR of the present invention. The activation of T cells by fusion molecules with anti-CD3 antibodies is much higher than the activation of T cells by fusion molecules of wild-type TCR and anti-CD3 antibodies.

Example 12 Specificity Detection of High Affinity TCR Fusion Molecules

This example demonstrates the specific activation of effector cells by fusion molecules of the high affinity TCR and anti-CD3 antibodies of the invention. Several sets of fusion molecules were selected for ELISPOT experiments to detect the production of IFN-γ as a readout for T cell activation.

The reagents used in the ELISPOT assay were as follows: test medium, wash buffer, PBS and human IFN-γ ELISPOT PVDF-enzyme kit, target cells, effector cells, fusion molecules of high affinity single-chain TCR and anti-CD3 antibody (as in the examples) 8 prepared, which can be diluted with the experimental medium) and a fusion molecule of high affinity αβ heterodimeric TCR and anti-CD3 antibody (prepared as described in Example 9, which can be used in experimental medium) Dilute).

The preparation process of the target cells was as follows: The target cells used in this example were IM9 cells (HLA-A2 positive and NY-ESO-1 antigen positive). The target cells of the control group were 293T cells (HLA-A2 positive and NY-ESO-1 antigen negative). A sufficient number of target cells (20,000 cells/well) were washed with a 75004250 centrifuge (Thermo Corporation) at 500 g, once every 5 minutes. The cells were then resuspended in the experimental medium at 4 x 10 ⁵ cells/ml.

Specific examples of the effector cells used and the ELISPOT assay can be referred to in Example 10. Figure 21-25 shows high-affinity single-stranded TCR fusion 2, high-affinity single-stranded TCR fusion 5, high-affinity single-stranded TCR fusion 6, high-affinity single-stranded TCR fusion 7 and high-affinity heterodimeric TCR fusion Figure 2 shows the results of ELISPOT experiment. The above experimental results are graphs showing the specific activation of effector cells by the high affinity TCR fusion molecules of the present invention.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

The T cell receptor according to claim 15, wherein the amino acid sequence of the α chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 34, 35, 36, 37, 89; and / or

The β chain variable domain amino acid sequence of the TCR is selected from the group consisting of: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33;
The T cell receptor according to claim 16, wherein said TCR comprises a combination of alpha and beta chain variable domains as shown in the following table:
A T cell receptor according to any of the preceding claims, wherein the C- or N-terminus of the alpha chain and/or beta chain of the TCR is bound to a conjugate.
The T cell receptor according to claim 18, wherein the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modified moiety or a combination of any of these.
The T cell receptor according to claim 19, wherein the therapeutic agent that binds to the T cell receptor is an anti-CD3 antibody linked to the C- or N-terminus of the α or β chain of the TCR. .
The T cell receptor according to claim 20, wherein the amino acid sequence of the α chain variable domain of the TCR that binds to the anti-CD3 antibody is selected from the group consisting of: SEQ ID NO: 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 89, 90; and/or

The β chain variable domain amino acid sequence of the TCR that binds to the anti-CD3 antibody is selected from the group consisting of: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28, 30, 31, 33, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
The T cell receptor according to claim 21, wherein the amino acid sequence of the TCR β chain after binding to the anti-CD3 antibody is selected from the group consisting of SEQ ID NOS: 100 and 101.
The T cell receptor according to claim 21, wherein the amino acid sequence after binding of the TCR to the anti-CD3 antibody is selected from the group consisting of SEQ ID NO: 92, 93, 94, 95, 96, 97, 98 and 99.
A multivalent TCR complex characterized by comprising at least two TCR molecules, and wherein at least one TCR molecule is the TCR of any of the preceding claims.
A nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule of any of the preceding claims or a complement thereof;
A vector comprising the nucleic acid molecule of claim 25.
A host cell comprising the vector of claim 26 or the nucleic acid molecule of claim 25 integrated with an exogenous source in the host cell.
An isolated cell, characterized in that the cell expresses the TCR according to any one of claims 1-24;
A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 23, or a TCR complex as claimed in claim 24, or a right The cells described in claim 28 are required.
A method for treating a disease, comprising administering an appropriate amount of the TCR according to any one of claims 1 to 23, or the TCR complex according to claim 24, or claim 28 to a subject in need of treatment. Said cell, or the pharmaceutical composition as claimed in claim 29.
Use of a T cell receptor according to any one of claims 1 to 23, a TCR complex as claimed in claim 24 or a cell according to claim 28, for use in the preparation of a medicament for treating a tumor.
A method of preparing a T cell receptor according to any one of claims 1 to 23, comprising the steps of:

(i) cultivating the host cell of claim 27, thereby expressing the T cell receptor of any one of claims 1-23;

(ii) isolating or purifying the T cell receptor.