CN106432475B

CN106432475B - high affinity NY-ESO T cell receptor

Info

Publication number: CN106432475B
Application number: CN201610894646.8A
Authority: CN
Inventors: 李懿; 李小林
Original assignee: Guangdong Xiangxue Precision Medical Technology Co Ltd
Current assignee: Xiangxue Life Science Technology (Guangdong) Co.,Ltd.
Priority date: 2015-10-19
Filing date: 2016-10-13
Publication date: 2018-06-01
Anticipated expiration: 2036-10-13
Also published as: CN106432475A

Abstract

The present invention provides a kind of T cell receptor (TCR), have the characteristic with reference to SLLMWITQC HLA A2 compounds；And the TCR is wild type TCR to the binding affinity of the SLLMWITQC HLA A2 compounds at least 10 times of the binding affinity of SLLMWITQC HLA A2 compounds.The present invention also provides the fusion molecules of such TCR and therapeutic agent.Such TCR can be used alone, and can be also combined with therapeutic agent, and SLLMWITQC HLA A2 compound tumour cells are presented with targeting.

Description

High affinity ny-eso T cell receptor

Technical field

The present invention relates to biological technical fields, relate more specifically to identify the T derived from NY-ESO-1 polypeptides Cell receptor (T cell receptor, TCR).The invention further relates to the preparation and uses of the receptor.

Background technology

Only the molecule there are two types of type can identify antigen in a manner of specificity.One of which be immunoglobulin or Antibody；Another kind is T cell receptor (TCR), it is the cell as existing for α chains/β chains or γ chains/δ chains in the form of heterodimer The glycoprotein of film surface.The composition of the TCR scores of immune system is to be recombinated in thymus gland by V (D) J, then carry out it is positive and Solid phase and generate.In peripheral ring border, TCR has mediated T cell to main histocompatibility complex-peptide complexes (pMHC) specific recognition, therefore it is vital to the cellular immune function of immune system.

TCR is the unique receptor for presenting specific antigen peptide in main histocompatibility complex (MHC), this external source Peptide or endogenous peptide may be that cell abnormal unique sign occurs.In immune system, by the TCR of antigentic specificity with The combination of pMHC compounds triggers T cell to be directly physically contacted with antigen presenting cell (APC), then both T cell and APC Other cell membrane surface molecules just interact, this just causes a series of subsequent cell signals and transfers and other lifes Reason reaction, so that the T cell of different antigentic specificities plays immunological effect to its target cell.

MHC I classes corresponding with TCR and II quasi-molecule ligands be also the protein of immunoglobulin superfamily but for The presentation of antigen has specificity, and different individuals has different MHC, so as to present small peptide different in a kind of proteantigen To respective APC cell surfaces.The MHC of the mankind is commonly referred to as HLA genes or HLA complexs.

Small peptide SLLMWITQC be derived from by kinds of tumor cells expression NY-ESO-1 albumen (Chen et al., (1997)PNAS USA 94 1914-1918).I type HLA molecular presentations being derived from including SLLMWITQC of tumour cell In the small peptide of NY-ESO-1.Therefore, SLLMWITQC-HLA A2 compounds provide a kind of TCR can targets neoplastic cells mark Note.The TCR that SLLMWITQC-HLA A2 compounds can be combined has very high application value to the treatment of tumour.For example, energy The TCR for enough targeting tumour cell mark can be used for cytotoxic agent or immunostimulant are delivered to target cell or are converted Enter T cell, the T cell of the expression TCR is enable to destroy tumour cell, to be referred to as the therapeutic process of adoptive immunotherapy In give patient.For previous purpose, preferable TCR is that have higher affinity, so that the TCR can be resident for a long time On the cell targeted.For latter purpose, then it is preferable to use the TCR of medium affinity.Therefore, those skilled in the art It is directed to the TCR that exploitation can be used for the targets neoplastic cells mark for meeting different purposes.

The content of the invention

It is an object of the invention to provide a kind of TCR to SLLMWITQC-HLA A2 compounds with higher affinity.

Another object of the present invention is to provide the preparation method of the above-mentioned type TCR a kind of and the purposes of the above-mentioned type TCR.

The first aspect of the present invention provides a kind of T cell receptor (TCR), has and combines SLLMWITQC-HLA A2 The activity of compound.

In of the invention one preferably embodiment, TCR receptors according to the present invention,

Comprising TCR α chains variable domains and TCR β chain variable domains, the TCR α chains variable domain includes 3 CDR regions, the TCR α The consensus sequence of 3 CDR regions of chain variable domain is as follows,

CDR1α：ATGYPS

CDR2α：ATKADDK

CDR3α：ALTLNNAGNMLT, and contain at least one following mutation：

Residue before mutation	Residue after mutation
		The 3rd K of CDR2 α	A, R, M or T
The 4th A of CDR2 α	P
		The 5th D of CDR2 α	G
The 6th D of CDR2 α	Q or E
		The 7th K of CDR2 α	T, V, R or I
The 2nd L of CDR3 α	R
		The 7th A of CDR3 α	S
The 8th G of CDR3 α	A
		The 9th N of CDR3 α	S or G
The 10th M of CDR3 α	S, P, H or F
		The 12nd T of CDR3 α	I

In another preference, the TCR β chains variable domain includes 3 CDR regions, 3 CDR of the TCR β chain variable domains The consensus sequence in area is as follows,

CDR1β：SNHLY

CDR2β：FYNNEI

CDR3β：ASLDPRAGTDTQY, and contain at least one following mutation：

Residue before mutation	Residue after mutation
		The 1st S of CDR1 β	P, H or L
The 2nd N of CDR1 β	G
		The 3rd H of CDR1 β	S or A
The 4th L of CDR1 β	I, M or P
		The 5th Y of CDR1 β	A or G
The 4th D of CDR3 β	G
		The 5th P of CDR3 β	F or I
The 6th R of CDR3 β	L
		The 7th A of CDR3 β	H or P
The 8th G of CDR3 β	I
		The 9th T of CDR3 β	Y。

In another preference, the mutation numbers of the TCR α chain CDR regions can be 1,2,3,4,5,6 It is a, 7,8,9,10 or 11.

In another preference, the mutation numbers of the TCR β chain CDR regions can be 1,2,3,4,5,6 It is a, 7,8,9,10 or 11.

In another preference, the TCR includes TCR α chains variable domains and TCR β chain variable domains, the TCR β chain variable domains Comprising CDR1 β, CDR2 β and CDR3 β, wherein the CDR2 β include sequence：FYNNEI.

In another preference, the CDR3 β include sequence：

ASL [3 β X1] [3 β X2] [3 β X3] [3 β X4] [3 β X5] [3 β X6] DTQY, wherein, [3 β X1], [3 β X2], [3 β X3], [3 β X4], [3 β X5] and [3 β X6] be respectively independently selected from arbitrary native amino acid residues.

In another preference, [the 3 β X1] is G.

In another preference, [the 3 β X2] is F or I.

In another preference, [the 3 β X3] is L.

In another preference, [the 3 β X4] is H or P.

In another preference, [the 3 β X5] is I.

In another preference, [the 3 β X6] is Y.

In another preference, the CDR3 β include sequence selected from the group below：

ASLDFLHIYDTQY, ASLDFLHIYDTQY, ASLDPRAGYDTQY and ASLDPRAGYDTQY.

In another preference, the CDR1 β include sequence:IA.

In another preference, the CDR1 β include sequence：[1 β X1] [1 β X2] [1 β X3], wherein, [1 β X1], [1 β X2], [1 β X3] independently selected from arbitrary native amino acid residues.

In another preference, [the 1 β X1] is P, H or L.

In another preference, [the 1 β X2] is G or N.

In another preference, [the 1 β X3] is S or A.

In another preference, the CDR1 β include sequence selected from the group below：

PGSIA, SNHLY, PGAIA, PGAIG, SGSIA, SGSPA, HGSIA and LNSMG.

In another preference, T cell receptor (TCR) according to the present invention can comprising TCR α chains variable domains and TCR β chains Variable domain, the TCR α chains variable domain include CDR1 α, CDR2 α and CDR3 α.

In another preference, the CDR1 α include sequence:ATGYPS.

In another preference, the CDR3 α include sequence：

A [3 α X1] TLNN [3 α X2] [3 α X3] [3 α X4] [3 α X5] L [3 α X6], wherein, [3 α X1], [3 α X2], [3 α X3], [3 α X4], [3 α X5], [3 α X6] are independently selected from arbitrary native amino acid residues.

In another preference, [the 3 α X1] is R or L.

In another preference, [the 3 α X2] is S or A.

In another preference, [the 3 α X3] is A or G.

In another preference, [the 3 α X4] is S, G or N.

In another preference, [the 3 α X5] is S, P, H, F or M.

In another preference, [the 3 α X6] is I or T.

In another preference, the CDR3 α include sequence selected from the group below：

ALTLNNSGSFLT、ALTLNNAGNMLT、ALTLNNAGGPLI、ALTLNNSASFLT、ALTLNNSASSLT、 ALTLNNSGGHLT, ALTLNNSGNFLT, ARTLNNAGNMLT and ARTLNNSGNFLT.

In another preference, the CDR2 α include sequence：

AT [2 α X1] [2 α X2] [2 α X3] [2 α X4] [2 α X5], wherein, [2 α X1], [2 α X2], [2 α X3], [2 α X4], [2 α X5] independently selected from arbitrary native amino acid residues.

In another preference, [the 2 α X1] is A, R, M, T or K.

In another preference, [the 2 α X2] is P or A.

In another preference, [the 2 α X3] is G or D.

In another preference, [the 2 α X4] is Q, E or D.

In another preference, [the 2 α X5] is T, V, R, I or K.

In another preference, the CDR2 α include sequence selected from the group below：

ATAPGQT, ATAPGQI, ATKADDK, ATKPGET, ATMPGEI, ATRPGQR, ATRPGQV and ATTPGEV.

In another preference, following CDR is included during the TCR α chain variable domain differences of the TCR：

CDR1α：ATGYPS；CDR2α：ATKADDK；With CDR3 α：ALTLNNAGNMLT.

In another preference, following CDR is included during the TCR β chain variable domain differences of the TCR：

CDR1β：SNHLY；CDR2β：FYNNEI；With CDR3 β：ASLDPRAGTDTQY.

In another preference, the TCR has CDR selected from the group below：

In another preference, the TCR is in SEQ ID NO：It undergos mutation in α chain variable domains shown in 40, it is described prominent The acid residues sites of change include one in 52K, 53A, 54D, 55D, 56K, 92L, 97A, 98G, 99N, 100M or 102T Or it is multiple, wherein, numbering amino acid residues use SEQ ID NO:Number shown in 40.

In another preference, the TCR is in SEQ ID NO：It undergos mutation in β chain variable domains shown in 41, it is described prominent The acid residues sites of change include one in 27S, 28N, 29H, 30L, 31Y, 96D, 97P, 98R, 99A, 100G or 101T Or it is multiple, wherein, numbering amino acid residues use SEQ ID NO:Number shown in 41.

In another preference, the TCR α chains variable domain after mutation includes one or more amino acid selected from the group below Residue：52A, 52R, 52M or 52T；53P；54G；55Q or 55E；56T, 56V, 56R or 56I；92R；97S；98A；99S or 99G； 100S, 100P, 100H or 100F；102I；Wherein, numbering amino acid residues use SEQ ID NO:Number shown in 40.

In another preference, the TCR β chains variable domain after mutation includes one or more amino acid selected from the group below Residue：27P, 27H or 27L；28G；29S or 29A；30I, 30M or 30P；31A or 31G；96G；97F or 97I；98L；99H or 99P；100I；101Y；Wherein, numbering amino acid residues use SEQ ID NO:Number shown in 41.

In another preference, the TCR is the heterogeneous dimerization TCR of α β.

In another preference, the α chains variable domain of the TCR includes and SEQ ID NO：Amino acid sequence tool shown in 40 Have at least 90% (preferably, at least 92%；It is highly preferred that at least 94%；Most preferably, sequence homology at least 96%) Amino acid sequence.

In another preference, the β chains variable domain of the TCR includes and SEQ ID NO：Amino acid sequence tool shown in 41 At least 90% (preferably, at least 92%；It is highly preferred that at least 94%；Most preferably, the ammonia of sequence homology at least 97%) Base acid sequence.

In another preference, the TCR include (I) all or part of TCR α chains in addition to its transmembrane domain and The all or part of TCR β chains of (II) in addition to its transmembrane domain, wherein (I) and (II) variable domain and extremely comprising TCR chains Few a part of constant domain.

In another preference, the α chain variable domain amino acid sequences of the TCR are selected from：SEQ ID NO:5-26.

In another preference, the β chain variable domain amino acid sequences of the TCR are selected from：SEQ ID NO:27-39.

In another preference, the TCR is the heterogeneous dimerization TCR of α β, the α chains variable region and β chains constant region of the TCR it Between contain artificial interchain disulfide bond.

In another preference, artificial interchain disulfide bond is formed between the α chains variable region of the TCR and β chain constant regions Cysteine residues instead of selected from following one or more groups of sites：

The 46th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；

The 47th amino acids of TRAV and 61 amino acids of TRBC1*01 or TRBC2*01 exons 1s；

The 46th amino acids of TRAV and the 61st amino acids of TRBC1*01 or TRBC2*01 exons 1s；Or

The 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s.

In another preference, the TCR containing artificial interchain disulfide bond includes α chains between α chains variable region and β chain constant regions Variable domain and β chains variable domain and all or part of β chains constant domain in addition to transmembrane domain, but it does not contain α chains are constant Domain, α chains variable domain and the β chains of the TCR form heterodimer.

In another preference, the TCR containing artificial interchain disulfide bond includes (I) between α chains variable region and β chain constant regions The all or part of TCR β of all or part of TCR α chains and (II) in addition to its transmembrane domain in addition to its transmembrane domain Chain, wherein (I) and (II) includes the variable domain of TCR chains and at least a portion constant domain.

In another preference, the TCR is the heterogeneous dimerization TCR of α β, complete in addition to its transmembrane domain it includes (I) The all or part of TCR β chains of portion or part TCR α chains and (II) in addition to its transmembrane domain, wherein (I) and (II) includes The variable domain of TCR chains and at least a portion constant domain contain artificial interchain disulfide bond between α chains constant region and β chain constant regions.

In another preference, half Guang ammonia of artificial interchain disulfide bond is formed between the TCR α and the constant region of β chains Sour residue is instead of selected from following one or more groups of sites：

The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 extrons The Ala19 of 1 Pro89 and TRBC1*01 or TRBC2*01 exons 1；With

The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s.

In another preference, the TCR is single-stranded TCR.

In another preference, single-stranded TCR that the TCR is made of α chains variable domain and β chain variable domains, the α chains can Variable domain and β chains variable domain are connected by a flexible short peptide sequence (linker).

In another preference, the hydrophobic core of the TCR is undergone mutation.

In another preference, the hydrophobic core of the TCR α chains variable domain and/or β chain variable domains is undergone mutation.

In another preference, TCR that the hydrophobic core is undergone mutation is made of single-stranded α variable domains and β variable domains TCR, the α variable domains and β variable domains are connected by a flexible short peptide sequence (linker).

In another preference, the hydrophobic core mutation of the TCR is happened at SEQ ID NO:The choosing of the variable domains of α chains shown in 40 From one or more acid residues sites of the following group：11V, 13L, 19L, 77K and 80V；Wherein, numbering amino acid residues use SEQ ID NO:Number shown in 40；And/or

The hydrophobic core mutation is happened at SEQ ID NO:One or more ammonia selected from the group below of the variable domains of β chains shown in 41 Base acid Residue positions：11Q, 13T and 82T, wherein, numbering amino acid residues use SEQ ID NO:Number shown in 41.

In another preference, the α chains variable domain of the TCR after hydrophobic core mutation includes one selected from the group below or more A amino acid residue：11L, 13V, 19V, 77I and 80I；And/or the β chains variable domain of the TCR after hydrophobic core mutation includes choosing From one or more amino acid residues of the following group：11L, 13V and 82V.

In another preference, the TCR is soluble.

In another preference, the TCR has CDR selected from the group below：

In another preference, the TCR is selected from the group：

In another preference, the mutation is happened in α chains and/or one or more CDR regions of β chain variable domains.

In another preference, the mutation is happened in the CDR2 and/or CDR3 of α chains and/or the mutation is happened at In the CDR1 and/or CDR3 of β chains.

In another preference, the α chain variable domain amino acid sequences of the TCR are selected from：SEQ ID NO:44-65 and 96- 101；And/or the β chain variable domain amino acid sequences of the TCR are selected from：SEQ ID NO:66-78.

In another preference, the affinity of the TCR and SLLMWITQC-HLA A2 compounds be wild type TCR extremely It is 2 times few；Preferably, at least 5 times；It is highly preferred that at least 10 times.

In another preference, the affinity of the TCR and SLLMWITQC-HLA A2 compounds be wild type TCR extremely It is 50 times few；Preferably, at least 100 times；It is highly preferred that at least 500 times；Most preferably, at least 1000 times, such as 10⁴Or 10⁵Times.

In another preference, the TCR is to the Dissociation equilibrium constant K of SLLMWITQC-HLA A2 compounds_D≤9μM；

In another preference, the TCR is to the 0.5 μM≤K of Dissociation equilibrium constant of SLLMWITQC-HLA A2 compounds_D ≤3.6μM；Preferably, 100nM≤K_D≤500nM；

In another preference, the TCR is to Dissociation equilibrium constant 10nM≤K of SLLMWITQC-HLA A2 compounds_D ≤100nM.Preferably, 0.1nM≤K_D≤10nM。

In another preference, the α chains of the TCR and/or C- the or N- ends of β chains are combined with conjugate.

In another preference, the conjugate combined with the TCR is detectable, therapeutic agent, PK modified parts Or the combination of these any substances.

In another preference, with C- or the N- end that the TCR therapeutic agents combined are α the or β chains for being connected to the TCR The anti-CD 3 antibodies at end.

In another preference, the α chain variable domain amino acid sequences of the TCR combined with anti-CD 3 antibodies are selected from：SEQ ID NO:5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、44、45、46、 47th, 48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,96,97,98,99,100 and 101；And/or the β chain variable domain amino acid sequences of the TCR combined with anti-CD 3 antibodies are selected from：SEQ ID NO:27、28、 29th, 30,31,32,33,34,35,36,37,38,39,66,67,68,69,70,71,72,73,74,75,76,77 and 78.

In another preference, high-affinity TCR of the present invention and the amino acid sequence of anti-CD 3 antibodies fusion molecule are preferred From：SEQ ID NO:83-93.

The second aspect of the present invention provides a kind of multivalent TCR complex, comprising at least two TCR molecules, and wherein At least one TCR molecules be the TCR described in first aspect present invention.

The third aspect of the present invention provides a kind of nucleic acid molecules, and the nucleic acid molecules, which include, encodes first party of the present invention The nucleotide sequence or its complementary series of the multivalent TCR complex described in TCR molecules or second aspect of the present invention described in face；

The fourth aspect of the present invention, provides a kind of carrier, and the carrier contains the institute described in third aspect present invention The nucleic acid molecules stated.

The fifth aspect of the present invention provides a kind of host cell, contains present invention four directions in the host cell The nucleic acid molecules described in the third aspect present invention of external source are integrated in carrier or chromosome described in face.

The sixth aspect of the present invention, provides a kind of separated cell, and the cell is expressed described in first aspect present invention TCR.

The seventh aspect of the present invention, provides a kind of pharmaceutical composition, and the composition contains pharmaceutically acceptable load The 6th side of TCR compounds or the present invention described in TCR or second aspect of the present invention described in body and first aspect present invention Cell described in face.

The eighth aspect of the present invention provides a kind of method for treating disease, including giving object application in need for the treatment of suitable The TCR compounds or sixth aspect present invention described in TCR or second aspect of the present invention described in the first aspect present invention of amount Pharmaceutical composition described in the cell or seventh aspect present invention.

The ninth aspect of the present invention is provided described in TCR or the second aspect of the present invention described in first aspect present invention The purposes of cell described in TCR compounds or sixth aspect present invention is used to prepare the drug for the treatment of tumour.

The tenth aspect of the present invention provides a kind of method for preparing the T cell receptor described in first aspect present invention, bag Include step：

(i) host cell described in fifth aspect present invention is cultivated, so as to express the T cell described in first aspect present invention Receptor；

(ii) isolated or purified goes out the T cell receptor.

It is to be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, so as to form new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 a and Fig. 1 b are respectively the amino acid sequence and DNA sequence dna for the single-stranded template TCR that the present invention is built.

Fig. 2 a and Fig. 2 the b respectively amino acid sequence of the α variable domains for the single-stranded template TCR that the present invention is built and β chains are variable The amino acid sequence in domain.

Fig. 3 a and Fig. 3 b are respectively the DNA sequence dna of α variable domains of single-stranded template TCR and β chain variable domains that the present invention is built DNA sequence dna.

Fig. 4 a and Fig. 4 b are respectively the amino acid sequence of the connection small peptide (linker) for the single-stranded template TCR that the present invention is built And nucleotide sequence.

The α chains that Fig. 5 a-v respectively illustrate the single-stranded TCR for having high-affinity to SLLMWITQC-HLA A2 compounds can Domain amino acid sequence, the residue of mutation are represented with underlining.

The β chains that Fig. 6 a-m respectively illustrate the single-stranded TCR for having high-affinity to SLLMWITQC-HLA A2 compounds can Domain amino acid sequence, the residue of mutation are represented with underlining.

Fig. 7 a and Fig. 7 b respectively illustrate the wild type TCR that can be specifically bound to SLLMWITQC-HLA A2 compounds α and β chain variable domain amino acid sequences.

Fig. 8 a and Fig. 8 b respectively illustrate the amino acid sequence of reference TCR α and β chains in the present invention.

Fig. 9 a-v respectively illustrate the α for the heterogeneous dimerization TCR for having high-affinity to SLLMWITQC-HLA A2 compounds Chain variable domain amino acid sequence, the residue of mutation are represented with underlining.

Figure 10 a-m respectively illustrate the β for the heterogeneous dimerization TCR for having high-affinity to SLLMWITQC-HLA A2 compounds Chain variable domain amino acid sequence, the residue of mutation are represented with underlining.

Figure 11 a-k are the amino acid sequence of the fusion molecule of the single-stranded TCR of high-affinity and anti-CD 3 antibodies.

α chains variable domain and the extracellular amino acid sequence of β chains before Figure 12 a and 12b are respectively the CDR region for introducing high-affinity, The cysteine residues of disulfide bond are formed in α chains variable region and β chains constant region to represent with the letter with underscore.

α chains of Figure 13 a-f in the heterogeneous dimerization TCR of high-affinity α β that α chains variable region and β chains constant region form disulfide bond can Domain amino acid sequence.

Figure 14 is wild type TCR (i.e. reference TCR) and the affinity curve to SLLMWITQC-HLA A2 compounds.

Figure 15 a and Figure 15 b respectively illustrate the amino acid sequence of wild type TCR α and β chains in the present invention.

Figure 16 is the Elispot experimental result pictures for the effector cell for transfecting high-affinity TCR of the present invention.

Figure 17 is high-affinity TCR of the present invention and the fusion protein pairing effect cell of anti-CD 3 antibodies redirects Elispot experimental result pictures.

Specific embodiment

For the present invention by extensive and in-depth study, a kind of identification SLLMWITQC small peptides of acquisition (are derived from NY-ESO-1 eggs High-affinity T cell receptor (TCR) in vain), the SLLMWITQC small peptides are rendered in the form of peptide-HLA A2 compounds.Institute State 3 CDR regions of the high-affinity TCR in its α chain variable domain

CDR1α：ATGYPS

CDR2α：ATKADDK

CDR3α：It undergos mutation in ALTLNNAGNMLT；And/or 3 CDR regions in its β chain variable domain

CDR1β：SNHLY

CDR2β：FYNNEI

CDR3β：It undergos mutation in ASLDPRAGTDTQY；Also, TCR of the present invention is to above-mentioned SLLMWITQC-HLA after being mutated The affinity of A2 compounds and/or combination half-life period are at least 10 times of wild type TCR.

Before describing the present invention, it should be understood that the invention is not restricted to the specific method and experiment condition, because this Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and And it is not intended to restricted, the scope of the present invention will be limited only by the claims which follow.

Unless otherwise defined, otherwise whole technologies used herein are respectively provided with scientific terminology such as fields of the present invention The normally understood identical meanings of those of ordinary skill.

Although it can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention And material, herein place enumerate preferred method and material.

Term

T cell receptor (T cell receptor, TCR)

International immunogenetics information system (IMGT) may be employed to describe TCR.Natural α β heterodimerics TCR has α Chain and β chains.In a broad sense, each chain includes variable region, bonding pad and constant region, and β chains are usually also between variable region and bonding pad Containing short variable region, but the variable region is often regarded as a part for bonding pad.It is determined by the TRAJ and TRBJ of unique IMGT The bonding pad of TCR determines the constant region of TCR by the TRAC and TRBC of IMGT.

Each variable region includes 3 CDR (complementary determining region) being entrenched in Frame sequence, CDR1, CDR2 and CDR3. In IMGT nomenclatures, the different numbers of TRAV and TRBV refer to different V α types and the type of V β respectively.In IMGT systems, α Chain constant domain has following symbol：TRAC*01, wherein " TR " represents T cell receptor gene；" A " represents α chain genes；C Represent constant region；" * 01 " represents allele 1.β chains constant domain has following symbol：TRBC1*01 or TRBC2*01, Wherein " TR " represents T cell receptor gene；" B " represents β chain genes；C represents constant region；" * 01 " represents allele 1.α chains Constant region uniquely determines, and in the form of β chains, there are two possible constant region genes " C1 " and " C2 ".This field skill Art personnel can obtain the constant region gene sequences of TCR α and β chains by disclosed IMGT databases.

α the and β chains of TCR generally regard that each there are two " structural domain " i.e. variable domain and constant domains as.Variable domain is by connecting Variable region and bonding pad form.Therefore, in the description and claims of this application, " TCR α chains variable domain " refers to connection TRAV and TRAJ areas, similarly, " TCR β chains variable domain " refers to TRBV the and TRBD/TRBJ areas of connection.The 3 of TCR α chain variable domains A CDR is respectively CDR1 α, CDR2 α and CDR3 α；3 CDR of TCR β chain variable domains are respectively CDR1 β, CDR2 β and CDR3 β.This It can be mouse source or people source to invent the Frame sequence of TCR variable domains, be preferably people source.The constant domain of TCR includes Intracellular part, transmembrane region and extracellular portion.To obtain sTCR, to measure TCR and SLLMWITQC-HLA A2 compounds Between affinity, TCR of the present invention preferably do not include transmembrane region.It is highly preferred that the amino acid sequence of TCR of the present invention refers to The extracellular amino acid sequence of TCR.

The α chain amino acid sequences and β chain amino acid sequences of heretofore described " wild type TCR " are respectively SEQ ID NO: 102 and SEQ ID NO：103, as shown in figs. 15 a and 15b.The α chain amino acid sequences and β chains of heretofore described " reference TCR " Amino acid sequence is respectively SEQ ID NO:42 and SEQ ID NO：43, as shown in figs. 8 a and 8b.In the present invention, it can combine α and β the chain variable domain amino acid sequence of the wild type TCR of SLLMWITQC-HLA A2 compounds is respectively SEQ ID NO:40 Hes SEQ ID NO：41, as illustrated in figs. 7 a and 7b.In the present invention, term " polypeptide of the present invention ", " TCR of the invention ", " present invention T cell receptor " be used interchangeably.

In one, ground of the invention preferably embodiment, the TCR includes TCR α chains variable domains and TCR β chain variable domains, The TCR β chains variable domain includes CDR1 β, CDR2 β and CDR3 β, wherein the CDR2 β include sequence：FYNNEI.

In another preference, the CDR3 β include sequence：

One in the present invention is preferably carried out in mode, and [the 3 β X1] is G.

One in the present invention is preferably carried out in mode, and [the 3 β X2] is F or I.

One in the present invention is preferably carried out in mode, and [the 3 β X3] is L.

One in the present invention is preferably carried out in mode, and [the 3 β X4] is H or P.

One in the present invention is preferably carried out in mode, and [the 3 β X5] is I.

One in the present invention is preferably carried out in mode, and [the 3 β X6] is Y.

One in the present invention is preferably carried out in mode, and the CDR3 β include sequence selected from the group below：

ASLDFLHIYDTQY, ASLDFLHIYDTQY, ASLDPRAGYDTQY and ASLDPRAGYDTQY.

One in the present invention is preferably carried out in mode, and the CDR1 β include sequence:IA.

One in the present invention is preferably carried out in mode, and the CDR1 β include sequence：[1βX1][1βX2][1βX3], Wherein, [1 β X1], [1 β X2], [1 β X3] are independently selected from arbitrary native amino acid residues.

One in the present invention is preferably carried out in mode, and [the 1 β X1] is P, H or L.

One in the present invention is preferably carried out in mode, and [the 1 β X2] is G or N.

One in the present invention is preferably carried out in mode, and [the 1 β X3] is S or A.

One in the present invention is preferably carried out in mode, and the CDR1 β include sequence selected from the group below：

PGSIA, SNHLY, PGAIA, PGAIG, SGSIA, SGSPA, HGSIA and LNSMG.

One in the present invention is preferably carried out in mode, and T cell receptor (TCR) according to the present invention includes TCR α chains Variable domain and TCR β chain variable domains, the TCR α chains variable domain include CDR1 α, CDR2 α and CDR3 α.

One in the present invention is preferably carried out in mode, and the CDR1 α include sequence:ATGYPS.

One in the present invention is preferably carried out in mode, and the CDR3 α include sequence：A[3αX1]TLNN[3αX2][3α X3] [3 α X4] [3 α X5] L [3 α X6], wherein, [3 α X1], [3 α X2], [3 α X3], [3 α X4], [3 α X5], [3 α X6] are independently selected From arbitrary native amino acid residues.

One in the present invention is preferably carried out in mode, and [the 3 α X1] is R or L.

One in the present invention is preferably carried out in mode, and [the 3 α X2] is S or A.

One in the present invention is preferably carried out in mode, and [the 3 α X3] is A or G.

One in the present invention is preferably carried out in mode, and [the 3 α X4] is S, G or N.

One in the present invention is preferably carried out in mode, and [the 3 α X5] is S, P, H, F or M.

One in the present invention is preferably carried out in mode, and [the 3 α X6] is I or T.

One in the present invention is preferably carried out in mode, and the CDR3 α include sequence selected from the group below：

One in the present invention is preferably carried out in mode, and the CDR2 α include sequence：AT[2αX1][2αX2][2αX3] [2 α X4] [2 α X5], wherein, [2 α X1], [2 α X2], [2 α X3], [2 α X4], [2 α X5] are independently selected from arbitrary native amino Sour residue.

One in the present invention is preferably carried out in mode, and [the 2 α X1] is A, R, M, T or K.

One in the present invention is preferably carried out in mode, and [the 2 α X2] is P or A.

One in the present invention is preferably carried out in mode, and [the 2 α X3] is G or D.

One in the present invention is preferably carried out in mode, and [the 2 α X4] is Q, E or D.

One in the present invention is preferably carried out in mode, and [the 2 α X5] is T, V, R, I or K.

One in the present invention is preferably carried out in mode, and the CDR2 α include sequence selected from the group below：

ATAPGQT, ATAPGQI, ATKADDK, ATKPGET, ATMPGEI, ATRPGQR, ATRPGQV and ATTPGEV.

One in the present invention is preferably carried out in mode, comprising as follows during the TCR α chain variable domain differences of the TCR CDR：

CDR1α：ATGYPS；CDR2α：ATKADDK；With CDR3 α：ALTLNNAGNMLT.

CDR1β：SNHLY；CDR2β：FYNNEI；With CDR3 β：ASLDPRAGTDTQY.

Native interchain disulfide bond and artificial interchain disulfide bond

There is one group of disulfide bond in membrane-proximal region C α and the C β interchains of natural TCR, referred to as " two sulphur of native interchain in the present invention Key ".In the present invention, by what is be artificially introduced, the position interchain covalent disulfide bonds different from the position of native interchain disulfide bond claim For " artificial interchain disulfide bond ".

For convenience of description, the Position Number of TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequences is pressed in the present invention Order successively carries out Position Number from N-terminal to C-terminal, in TRBC1*01 or TRBC2*01, by successively suitable from N-terminal to C-terminal The 60th amino acid of sequence is P (proline), then can describe it as TRBC1*01 or TRBC2*01 exons 1s in the present invention Pro60 can also be stated that the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s, for another example TRBC1*01 or In TRBC2*01, it is Q (glutamine) by the 61st amino acid of the order from N-terminal to C-terminal successively, then can be retouched in the present invention It states as the Gln61 of TRBC1*01 or TRBC2*01 exons 1s, can also be stated that TRBC1*01 or TRBC2*01 exons 1s The 61st amino acids, other and so on.In the present invention, the Position Number of the amino acid sequence of variable region TRAV and TRBV, According to the Position Number listed in IMGT.Such as some amino acid in TRAV, the Position Number listed in IMGT is 46, then this hair The 46th amino acids of TRAV are described it as in bright, other and so on.In the present invention, the Sequence position numbers of other amino acid There is specified otherwise, then by specified otherwise.

Tumour

Term " tumour " refers to including all types of growth of cancer cells or oncogenic process, and metastatic tissue or vicious transformation are thin Born of the same parents, tissue or organ, no matter histological type or the stage infected.The embodiment of tumour includes without limitation：Solid tumor, soft group Knit knurl and metastasis (metastases).The embodiment of solid tumor includes：The malignant tumour of Different Organs system, such as sarcoma, lung squamous cancer and Cancer.Such as：The prostate of infection, lung, breast, lymph, stomach (such as：Colon) and genitourinary tract (such as：Kidney, on Chrotoplast), pharynx.Lung squamous cancer includes malignant tumour, for example, most colon cancers, the carcinoma of the rectum, clear-cell carcinoma, liver cancer, lung Non-small cell carcinoma, carcinoma of small intestine and cancer of the esophagus.Above-mentioned cancer metastasis venereal disease become can equally with method and composition of the invention come It treats and prevents.

Detailed description of the invention

It is well known that the α chains variable domain of TCR respectively contains 3 CDR with β chains variable domain, the complementation similar to antibody determines Area.CDR3 interacts with antigen small peptide, and CDR1 and CDR2 and HLA interact.Therefore, the CDR of TCR molecules determine its with The interaction of antigen small peptide-HLA compounds.The present inventor has found that antigen small peptide SLLMWITQC and HLA can be combined for the first time The α chains variable domain amino acid sequence and β chains of the wild type TCR of A2 compounds (that is, SLLMWITQC-HLA A2 compounds) is variable Domain amino acid sequence is respectively SEQ ID NO:40 and SEQ ID NO：41.It is with following CDR region：

α chain variable domain CDR CDR1 α：ATGYPS

CDR2α：ATKADDK

CDR3α：ALTLNNAGNMLT

With β chain variable domain CDR CDR1 β：SNHLY

CDR2β：FYNNEI

CDR3β：ASLDPRAGTDTQY

The present invention obtains the parent with SLLMWITQC-HLA A2 compounds by carrying out screen mutation to above-mentioned CDR region It is the high-affinity TCR of at least 10 times of wild type TCR and SLLMWITQC-HLA A2 compounds affinity with power.

The present invention provides a kind of T cell receptor (TCR), have the work with reference to SLLMWITQC-HLA A2 compounds Property.

The TCR of the present invention includes α chains variable domain and β chain variable domains, and TCR α chains variable domain includes 3 CDR regions and TCR β chains Variable domain includes 3 CDR regions, and the TCR is in 3 CDR region CDR1 α of α chain variable domains：ATGYPS

CDR2α：ATKADDK

CDR3α：Contain at least one following mutation in ALTLNNAGNMLT：

And/or the TCR is in 3 CDR regions of β chain variable domains

CDR1β：SNHLY

CDR2β：FYNNEI

CDR3β：Contain at least one following mutation in ASLDPRAGTDTQY：

Residue before mutation	Residue after mutation
		The 1st S of CDR1 β	P, H or L
The 2nd N of CDR1 β	G
		The 3rd H of CDR1 β	S or A
The 4th L of CDR1 β	I, M or P
		The 5th Y of CDR1 β	A or G
The 4th D of CDR3 β	G
		The 5th P of CDR3 β	F or I
The 6th R of CDR3 β	L
		The 7th A of CDR3 β	H or P
The 8th G of CDR3 β	I
		The 9th T of CDR3 β	Y

In more detail, the mutation number of the TCR α chain CDR regions is 3 or 4 or 6 or 7 or 8 or 9 or 11 It is a；And/or the mutation number of the TCR β chain CDR regions is 2 or 3 or 5 or 6 or 7 or 8 or 9.

Further, TCR of the present invention is the heterogeneous dimerization TCR of α β, and the α chains variable domain of the TCR includes and SEQ ID NO:Amino acid sequence shown in 40 has at least 90% (preferably, at least 92%；It is highly preferred that at least 94%；Most preferably, until Few 96%；Such as, can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) Sequence homology amino acid sequence；And/or the β chains variable domain of the TCR includes and SEQ ID NO:Amino shown in 41 Acid sequence has at least 90% (preferably, at least 92%；It is highly preferred that at least 94%；Most preferably, at least 97%；It such as, can be with At least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology Amino acid sequence.

Further, TCR of the present invention is single-stranded TCR, and the α chains variable domain of the TCR includes and SEQ ID NO:Shown in 2 Amino acid sequence have at least 90% (preferably, at least 92%；It is highly preferred that at least 94%；Most preferably, at least 96%； Such as, can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence The amino acid sequence of homology；And/or the β chains variable domain of the TCR includes and SEQ ID NO:Amino acid sequence shown in 3 has At least 90% (preferably, at least 92%；It is highly preferred that at least 94%；Most preferably, at least 97%；Such as, can be at least 91%th, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) sequence homology amino acid Sequence.

Preferably, the TCR includes all or part of TCR α chains and (II) of (I) in addition to its transmembrane domain and removes it All or part of TCR β chains beyond transmembrane domain, wherein (I) and (II) includes the variable domain and at least a portion of TCR chains Constant domain.

Wild type TCR α chains variable domain SEQ ID NO in the present invention：40 3 CDR, that is, CDR1, CDR2 and CDR3 difference position In SEQ ID NO：27-32,50-56 and 91-102 of 40.Accordingly, numbering amino acid residues use SEQ ID NO:Number shown in 40,52K are the 3rd K of CDR2 α, and 53A is the 4th A of CDR2 α, and 54D is the 5th of CDR2 α D, 55D are the 6th D of CDR2 α, and 56K is the 7th K of CDR2 α, and 92L is the 2nd L of CDR3 α, and 97A is CDR3 α The 7th A, 98G is the 8th G of CDR3 α, and 99N is the 9th N of CDR3 α, and 100M is the 10th M of CDR3 α, 102T is the 12nd T of CDR3 α.

Similarly, wild type TCR β chain variable domain SEQ ID NO in the present invention：41 3 CDR, that is, CDR1, CDR2 and CDR3 It is located at SEQ ID NO respectively：27-31,49-54 and 93-105 of 41.Therefore, numbering amino acid residues use SEQ ID NO:Number shown in 41,27S are the 1st S of CDR1 β, and 28N is the 2nd N of CDR1 β, and 29H is CDR1 β The 3rd H, 30L is the 4th L of CDR1 β, and 31Y is the 5th Y of CDR1 β, and 96D is the 4th D of CDR3 β, and 97P is For the 5th P of CDR3 β, 98R is the 6th R of CDR3 β, and 99A is the 7th A of CDR3 β, and 100G is the 8 of CDR3 β Position G, 101T is the 9th T of CDR3 β.

The present invention is provided with the characteristic with reference to SLLMWITQC-HLA A2 compounds, and comprising TCR α chains variable domains and TCR β chain variable domains, which is characterized in that the TCR is in SEQ ID NO：It undergos mutation in α chain variable domains shown in 40, it is described prominent The acid residues sites of change include one in 52K, 53A, 54D, 55D, 56K, 92L, 97A, 98G, 99N, 100M or 102T Or it is multiple, wherein, numbering amino acid residues use SEQ ID NO:Number shown in 40；And/or the TCR is in SEQ ID NO： Undergo mutation in β chain variable domains shown in 41, the acid residues sites of the mutation include 27S, 28N, 29H, 30L, 31Y, One or more of 96D, 97P, 98R, 99A, 100G or 101T, wherein, numbering amino acid residues use SEQ ID NO:41 Shown number.

In the preferred embodiment of the present invention, the TCR α chains variable domain after mutation includes selected from the group below One or more amino acid residues：52A, 52R, 52M or 52T；53P；54G；55Q or 55E；56T, 56V, 56R or 56I；92R； 97S；98A；99S or 99G；100S, 100P, 100H or 100F；102I；And/or the TCR β chain variable domains after mutation include One or more amino acid residue selected from the group below：27P, 27H or 27L；28G；29S or 29A；30I, 30M or 30P；31A or 31G；96G；97F or 97I；98L；99H or 99P；100I；101Y.

It, will be outside wild type TCR α chain constant regions TRAC*01 according to the method for rite-directed mutagenesis well known to those skilled in the art The Thr48 of aobvious son 1 sports cysteine, and the Ser57 of β chain constant region TRBC1*01 or TRBC2*01 exons 1s sports half Cystine is to get to reference TCR, and respectively as shown in figs. 8 a and 8b, cysteine residues after mutation are to add for amino acid sequence Thick letter represents.Above-mentioned cysteine substitution can make to form artificial interchain disulfide bond between the constant region of the α and β chains of reference TCR, To form more stable sTCR, so as to more easily assess TCR and SLLMWITQC-HLA A2 compounds it Between binding affinity and/or with reference to half-life period.It is to be understood that the CDR region of TCR variable regions determines it between pMHC compounds Affinity, therefore, the substitution of the cysteines of above-mentioned TCR constant regions can't partly decline to the binding affinity of TCR and/or combine Phase has an impact.So in the present invention, the combination parent between reference TCR and SLLMWITQC-HLA the A2 compounds measured And power is the binding affinity being considered between wild type TCR and SLLMWITQC-HLA A2 compounds.Similarly, if measured Binding affinity between TCR and SLLMWITQC-HLA A2 compounds of the present invention is reference TCR and SLLMWITQC-HLA A2 At least 10 times of binding affinity between compound, that is, be equal to TCR of the present invention and SLLMWITQC-HLA A2 compounds it Between binding affinity be at least 10 times of the binding affinity between wild type TCR and SLLMWITQC-HLA A2 compounds.

Binding affinity can be measured by any suitable method (with Dissociation equilibrium constant K_DBe inversely proportional) and combine partly decline Phase (is expressed as T_1/2).It will be appreciated that the affinity of TCR is double will to cause K_DHalve.T_1/2It is calculated as In2 divided by dissociation rate (K_off).Therefore, T_1/2It is double to cause K_offHalve.It is preferred that the binding affinity using the identical given TCR of testing program detection Or combine half-life period for several times, take the average value of result such as 3 times or more.In a preferred embodiment, using implementing herein Surface plasmon resonance (BIAcore) method in example carries out these detections.This method detects TCR pairs of reference The Dissociation equilibrium constant K of SLLMWITQC-HLA A2 compounds_DFor 18 μM, TCR pairs of wild type is thought in the present invention The Dissociation equilibrium constant K of SLLMWITQC-HLA A2 compounds_DAlso it is 18 μM.It will cause K since the affinity of TCR is double_DSubtract Half, if so detecting Dissociation equilibrium constant Ks of the high-affinity TCR to SLLMWITQC-HLA A2 compounds_DFor 1.8 μM, then It is wild type TCR to SLLMWITQC-HLA to the affinity of SLLMWITQC-HLA A2 compounds to illustrate high-affinity TCR 10 times of the affinity of A2 compounds.The known K of those skilled in the art_DThe conversion relation being worth between unit, i.e. 1 μM=1000nM, 1nM=1000pM.

In the preference of the present invention, the affinity of the TCR and SLLMWITQC-HLA A2 compounds is wild At least 2 times of type TCR；Preferably, at least 5 times；It is highly preferred that at least 10 times.

Specifically, the TCR is to the Dissociation equilibrium constant K of SLLMWITQC-HLA A2 compounds_D≤9μM；

Any suitable method can be used to be mutated, include but not limited to foundation PCR (PCR) that A bit, according to Restriction Enzyme clone or do not depend on clone (LIC) method of connection.Many standard molecular biology teaching materials detail These methods.The more details of PCR (PCR) mutagenesis and the clone according to Restriction Enzyme can be found in Sambrook And Russell, (2001) Molecular Cloning-A Laboratory handbook (Molecular Cloning-A Laboratory Manual) (the Three editions) CSHL publishing houses.Visible (Rashtchian, (1995) Curr Opin Biotechnol 6 of more information of LIC methods (1):30-6)。

Generate the diversity for the phage particle that the method for the TCR of the present invention can be but not limited to from the such TCR of displaying The TCR that there is high-affinity to SLLMWITQC-HLA-A2 compounds is filtered out in library, such as document (Li, et al (2005) Nature Biotech 23(3):Described in 349-354).

It is to be understood that the wild type that the gene of expression wild type TCR α and β chain variable domain amino acids or expression are slightly modified The gene of the α and β chain variable domain amino acids of TCR can be adopted to prepare template TCR.Then in the variable domain of coding template TCR DNA in introduce generate the present invention high-affinity TCR needed for change.

In some preferred embodiments of the present invention, TCR of the present invention is in SEQ ID NO:α chain variable domain ammonia shown in 40 In base acid residue 52K, 53A, 54D, 55D, 56K, 92L, 97A, 98G, 99N, 100M and 102T there are one or multiple undergo mutation (use SEQ ID NO:Number shown in 40) and/or in SEQ ID NO:β chain variable domain amino acid residues 27S shown in 41, There are one in 28N, 29H, 30L, 31Y, 96D, 97P, 98R, 99A, 100G and 101T or multiple undergo mutation (uses SEQ ID NO:Number shown in 41).For example, the TCR α chains variable domain after mutation includes one or more amino acid residues selected from the group below： 52A, 52R, 52M or 52T；53P；54G；55Q or 55E；56T, 56V, 56R or 56I；92R；97S；98A；99S or 99G；100S、 100P, 100H or 100F；102I；And/or the TCR β chains variable domain after mutation includes one or more amino acid selected from the group below Residue 27S, 28N, 29H, 30L, 31Y, 96D, 97P, 98R, 99A, 100G and 101T.More specifically, it dashes forward described in α chain variable domains The concrete form of change include K52A/R/M/T, A53P, D54G, D55Q/E, K56T/V/R/I, L92R, A97S, G98A, N99S/G, One group in M100S/P/H/F or T102I or several groups；The concrete form being mutated described in β chain variable domains include S27P/H/L, One group or several in N28G, H29S/A, L30I/M/P, Y31A/G, D96G, P97F/I, R98L, A99H/P, G100I or T101Y Group.

The high-affinity TCR of the present invention includes α chain variable domain amino acid sequence SEQ ID NO:44、45、46、47、48、 49th, 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,96,97,98,99,100, one of 101 and/ Or β chain variable domain amino acid sequence SEQ ID NO:66th, 67,68,69,70,71,72,73,74,75,76,77, one of 78.Cause This, α chains variable domain amino acid sequence (the SEQ ID NO containing wild type TCR:40) TCR α chains can be with including SEQ ID NO: 66th, 67,68,69,70,71,72,73,74,75,76,77, one of 78 TCR β chains combine to form heterogeneous dimerization TCR or single-stranded TCR molecules.Alternatively, β variable domain amino acid sequences (the SEQ ID NO containing wild type TCR:41) TCR β chains can with comprising SEQ ID NO:44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、 96th, 97,98,99,100, one of 101 TCR α chains combine to form heterogeneous dimerization TCR or single chain TCR molecules.Or comprising TCR α chain variable domain amino acid sequence SEQ ID NO:44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、 59th, 60,61,62,63,64,65,96,97,98,99,100, one of 101 TCR α chains can be with including TCR β chain variable domain amino Acid sequence SEQ ID NO:66th, 67,68,69,70,71,72,73,74,75,76,77, one of 78 TCR β chains combine to be formed it is different Matter dimerization TCR or single chain TCR molecules.In the present invention, the α chains variable domain of heterogeneous dimerization TCR molecules and the ammonia of β chain variable domains are formed Base acid sequence preferably is selected from the following table 1：

Table 1

Based on the purpose of the present invention, TCR of the present invention is the part at least one TCR α and/or TCR β chain variable domains. They are usually simultaneously comprising TCR α chains variable domains and TCR β chain variable domains.They can be α β heterodimers or single stranded form Or other any forms that can be stabilized.It, can be by the overall length chain of α β heterodimerics TCR in adoptive immunotherapy (including kytoplasm and transmembrane domain) is transfected.TCR of the present invention can be used as therapeutic agent being delivered to the target of antigen presenting cell It is combined to agent or with other molecules and prepares bifunctional polypeptides and carry out directionality effect cell, TCR is preferably soluble form at this time.

For stability, disclose introduce artificial interchain two between α the and β chain constant domains of TCR in the prior art Sulfide linkage can obtain TCR molecules that are solvable and stablizing, as described in patent document 201410629321.8.Therefore, TCR of the present invention Can be the TCR that artificial interchain disulfide bond is introduced between the residue of itself α and β chain constant domain.Cysteine residues are in the TCR α and β chain constant domains between form artificial interchain disulfide bond.Cysteine residues can be substituted in appropriate site in natural TCR Other amino acid residues are to form artificial interchain disulfide bond.For example, the Thr48 of substitution TRAC*01 exons 1s and substitution TRBC1* The Ser57 of 01 or TRBC2*01 exons 1s forms disulfide bond.Cysteine residues are introduced to form other sites of disulfide bond It can also be：The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；It is aobvious outside TRAC*01 The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of son 1；The Thr45 and TRBC1*01 of TRAC*01 exons 1s Or the Asp59 of TRBC2*01 exons 1s；Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s Glu15；The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 exons 1s Pro89 and TRBC1*01 or TRBC2*01 exons 1s Ala19；Or TRAC*01 exons 1s Tyr10 and TRBC1*01 or The Glu20 of TRBC2*01 exons 1s.I.e. cysteine residues are instead of any group of site in above-mentioned α and β chain constant domains.It can be One or more C-terminals of TCR constant domains of the present invention truncate the amino of most 15 or most 10 or most 8 or less Acid, so that it does not achieve the purpose that lack native interchain disulfide bond including cysteine residues, it also can be natural by that will be formed The cysteine residues of interchain disulfide bond sport another amino acid to reach above-mentioned purpose.

As described above, the TCR of the present invention may be embodied in the artificial interchain two introduced between the residue of itself α and β chain constant domain Sulfide linkage.It should be noted that the artificial disulfide bond with or without introducing described above, TCR of the invention can contain between constant domain TRAC constant domains sequence and TRBC1 or TRBC2 constant domain sequences.The TRAC constant domains sequence and TRBC1 or TRBC2 of TCR is constant Domain sequence can be connected by the native interchain disulfide bond being present in TCR.

In addition, for stability, patent document 201510260322.4 is also disclosed in the α chains variable region of TCR and β Artificial interchain disulfide bond is introduced between chain constant region can significantly improve the stability of TCR.Therefore, high-affinity of the invention Artificial interchain disulfide bond can also be contained between the α chains variable region of TCR and β chain constant regions.It specifically, can in the α chains of the TCR Become between area and β chain constant regions formed the cysteine residues of artificial interchain disulfide bond instead of：The 46th amino acids of TRAV With the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；The 47th amino acids and TRBC1*01 of TRAV or 61 amino acids of TRBC2*01 exons 1s；The 46th amino acids of TRAV and the of TRBC1*01 or TRBC2*01 exons 1s 61 amino acids；Or the 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s.It is preferred that Ground, such TCR can include all or part of TCR α chains and (II) of (I) in addition to its transmembrane domain and remove its cross-film knot All or part of TCR β chains beyond structure domain, wherein (I) and (II) variable domain comprising TCR chains and at least a portion is constant Domain, α chains form heterodimer with β chains.It is highly preferred that such TCR can include α chains variable domain and β chains variable domain and All or part of β chains constant domain in addition to transmembrane domain, but it does not contain α chain constant domains, the α chain variable domains of the TCR Heterodimer is formed with β chains.

For stability, on the other hand, TCR of the present invention is additionally included in the TCR that its hydrophobic core region is undergone mutation, this The mutation of a little hydrophobic core regions is preferably capable the stability-enhanced mutation for making TCR of the present invention, such as in Publication No. WO2014/ Described in 206304 patent document.Such TCR can undergo mutation in its following hydrophobic core position of variable domain：(α and/or β Chain) variable region amino acid the 11st, 13,19,21,53,76,89,91,94 and/or α chain J genes (TRAJ) small peptide amino acid position It is 2nd, 4,6 reciprocal to put the 3rd, 5,7 and/or β chain J gene (TRBJ) small peptides amino acid position reciprocal, wherein amino acid sequence Position Number press the Position Number listed in international immunogenetics information system (IMGT).On those skilled in the art know International immunogenetics information system is stated, and position of the amino acid residue of different TCR in IMGT can be obtained according to the database Put number.

More specifically, the TCR that hydrophobic core region is undergone mutation in the present invention can be by the α of a flexible peptide chain link TCR The single-stranded TCR of high stability formed with the variable domains of β chains.The CDR region of TCR variable regions determines itself and small peptide-HLA compounds Between affinity, the mutation of hydrophobic core can be such that TCR more stablizes, but can't influence it between small peptide-HLA compounds Affinity.It should be noted that flexible peptide chain can be any suitable connection TCR α and the peptide chain of β chain variable domains in the present invention.This hair It is the above-mentioned high stability containing the mutation of hydrophobic core for screening the template strand of high-affinity TCR to be built in bright embodiment 1 Single-stranded TCR.Using the TCR of high stability, can more easily assess between TCR and SLLMWITQC-HLA-A2 compounds Affinity.

The α chains variable domain of single-stranded template TCR and the CDR region of β chain variable domains are identical with the CDR region of wild type TCR. That is 3 CDR of α chains variable domain are respectively CDR1 α：ATGYPS, CDR2 α：ATKADDK, CDR3 α：ALTLNNAGNMLT and β chains can 3 CDR of variable domain are respectively CDR1 β：SNHLY, CDR2 β：FYNNEI, CDR3 β：ASLDPRAGTDTQY.Single-stranded template TCR Amino acid sequence (SEQ ID NO:And nucleotide sequence (SEQ ID NO 1):79) respectively as seen in figure la and lb.It is sieved with this Select to SLLMWITQC-HLA A2 compounds have high-affinity be made of α chains variable domain and β chain variable domains it is single-stranded TCR。

Single-stranded template TCR α chains variable domain SEQ ID NO in the present invention：23 CDR, that is, CDR1, CDR2 and CDR3 difference Positioned at SEQ ID NO：27-32,50-56 and 91-102 of 2.Accordingly, numbering amino acid residues use SEQ ID NO:Number shown in 2,52K are the 3rd K of CDR2 α, and 53A is the 4th A of CDR2 α, and 54D is the 5 of CDR2 α Position D, 55D is the 6th D of CDR2 α, and 56K is the 7th K of CDR2 α, and 92L is the 2nd L of CDR3 α, and 97A is The 7th A of CDR3 α, 98G are the 8th G of CDR3 α, and 99N is the 9th N of CDR3 α, and 100M is the 10th of CDR3 α M, 102T are the 12nd T of CDR3 α.

Similarly, single-stranded template TCR β chain variable domain SEQ ID NO in the present invention：33 CDR, that is, CDR1, CDR2 and CDR3 It is located at SEQ ID NO respectively：27-31,49-54 and 93-105 of 3.Therefore, numbering amino acid residues use SEQ ID NO:Number shown in 3,27S are the 1st S of CDR1 β, and 28N is the 2nd N of CDR1 β, and 29H is CDR1 β The 3rd H, 30L is the 4th L of CDR1 β, and 31Y is the 5th Y of CDR1 β, and 96D is the 4th D of CDR3 β, and 97P is For the 5th P of CDR3 β, 98R is the 6th R of CDR3 β, and 99A is the 7th A of CDR3 β, and 100G is the 8 of CDR3 β Position G, 101T is the 9th T of CDR3 β.

The acquisition of the α β heterodimers of high-affinity that has to SLLMWITQC-HLA-A2 compounds of the present invention is logical The CDR region for crossing α and β the chain variable domain of the single-stranded TCR of the high-affinity that will be filtered out is transferred to wild type TCR α chain variable domains (SEQ ID NO:40) with β chains variable domain (SEQ ID NO:41) corresponding position and obtain.

In some embodiments of the invention, using SEQ ID NO:Number shown in 40, the α chains of TCR of the present invention are variable The hydrophobic core amino acid residue 11V in domain (i.e. the α chains variable region listed in IMGT the 11st), (i.e. the α chains listed in IMGT can by 13L Become the 13rd, area), 19L (i.e. the α chains variable region listed in IMGT the 19th), 77K (the i.e. α chains variable regions listed in IMGT the 91) and 80V (i.e. the α chains variable region listed in IMGT the 94th) in there are one or multiple undergo mutation and/or using SEQ ID NO:Number shown in 41, (i.e. the β chains listed in IMGT are variable by the hydrophobic core amino acid residue 11Q of TCR β chain variable domains The 11st, area), 13T (i.e. the β chains variable region listed in IMGT the 13rd) and 82T (the i.e. β chains variable regions the 94th listed in IMGT Position) in there are one or multiple undergo mutation.

More specifically, in some currently preferred embodiments of the present invention, using SEQ ID NO:Number shown in 40, the present invention The hydrophobic core of α chain variable domains includes one or more of amino acid residue 11L, 13V, 19V, 77I or 80I and/or using SEQ ID NO:Number shown in 41, the hydrophobic core of TCR β variable domains include one or more in amino acid residue 11L, 13V or 82V It is a.More specifically, the mutant form of the hydrophobic core of TCR α variable domains includes one in V11L, L13V, L19V, K77I or V80I Group or several groups；The mutant form of the hydrophobic core of TCR β variable domains includes one group or several groups in Q11L, T13V and T82V.

The high-affinity TCR of the present invention also includes α chain variable domain amino acid sequence SEQ ID NO:5、6、7、8、9、10、 11st, one of 12,13,14,15,16,17,18,19,20,21,22,23,24,25 and 26 and/or β chain variable domain amino acid sequences SEQ ID NO:27th, one of 28,29,30,31,32,33,34,35,36,37,38 and 39.Therefore, the above-mentioned height as template strand The single-stranded TCR α chains variable domain of stability (SEQ ID NO:2) can be SEQ ID NO with amino acid sequence:27、28、29、30、31、 32nd, 33,34,35,36,37,38 or 39 TCR β chain variable domains combine to form the single chain TCR molecules.Alternatively, above-mentioned be used as mould The single-stranded TCR β chains variable domain of high stability (the SEQ ID NO of plate chain:3) can be SEQ ID NO with amino acid sequence:5、6、7、8、 9th, 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or 26 TCR α chain variable domains combine to form institute State single chain TCR molecules.Or TCR α chain variable domain SEQ ID NO：5、6、7、8、9、10、11、12、13、14、15、16、17、 18th, one of 19,20,21,22,23,24,25 or 26 with TCR β chain variable domain SEQ ID NO:27、28、29、30、31、32、33、 34th, one of 35,36,37,38 or 39 combinations form the single chain TCR molecules.In the present invention, the α of high-affinity single chain TCR molecules Chain variable domain and the amino acid sequence of β chain variable domains preferably are selected from the following table 2：

Table 2

The present invention TCR can also multivalence complex form provide.The present invention multivalent TCR complex include two, Three, the four or more TCR of the present invention polymers that are combined and are formed, can such as be generated with four dimerization domains of p53 The tetramer or multiple TCR of the present invention and another molecule with reference to and the compound that is formed.The TCR compounds of the present invention can be used for body Outer or tracking in vivo or targeting present the cell of specific antigen, it can also be used to generate other multivalence TCR with such application and answer Close the intermediate of object.

The TCR of the present invention can be used alone, and can also be combined with conjugate with covalent or other modes, preferably with covalently side Formula combines.The conjugate includes detectable and (for diagnostic purpose, is presented wherein the TCR is used to detect The presence of the cell of SLLMWITQC-HLA-A2 compounds), therapeutic agent, PK (protein kinase) modified parts or any the above The combination of substance combines or coupling.

Detectable for diagnostic purposes includes but not limited to：Fluorescence or luminous marker, radioactively labelled substance, MRI (magnetic resonance imaging) or CT (x-ray tomography of electronic computer) contrast agent can generate detectable product Enzyme.

The therapeutic agent that can be combined or be coupled with TCR of the present invention includes but not limited to：1. radionuclide (Koppe etc., 2005, metastasis of cancer comment (Cancer metastasis reviews) 24,539)；2. biology poison (Chaudhary etc., 1989, Natural (Nature) 339,394；Epel etc., 2002, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 51,565)；3. cell factor such as IL-2 etc. (Gillies etc., 1992, National Academy of Sciences proceeding (PNAS) 89,1428；Card etc., 2004, Cancer Immunol and immunization therapy (Cancer Immunology and Immunotherapy) 53,345；Halin etc., 2003, cancer research (Cancer Research) 63,3202)；4. antibody Fc Segment (Mosquera etc., 2005, Journal of Immunology (The Journal Of Immunology) 174,4381)；5. antibody ScFv segments (Zhu etc., 1995, cancer International Periodicals (International Journal of Cancer) 62,319)；6. gold medal Nano particle/nanometer rods (Lapotko etc., 2005, cancer communication (Cancer letters) 239,36；Huang etc., 2006, it is beautiful Chemical Society of state magazine (Journal of the American Chemical Society) 128,2115)；7. virion (Peng etc., 2004, gene therapy (Gene therapy) 11,1234)；8. liposome (Mamot etc., 2005, cancer research (Cancer research) 65,11631)；9. magnetic nanosphere；10. pro-drug activation enzymes are (for example, DT- diaphorases (DTD) or connection Phenyl hydrolase-sample protein (BPHL))；11. chemotherapeutics (for example, cis-platinum) or any type of nano particle etc..

Include anti-T cell with the TCR of the present invention antibody combined or its segment or NK- cells determine antibody, such as anti-CD3 or Anti- CD28 or anti-CD16 antibody, the combination of above-mentioned antibody or its segment and TCR can pairing effect cell be oriented come it is more preferable Ground targets target cell.One preferred embodiment is the function of TCR of the present invention and anti-CD 3 antibodies or the anti-CD 3 antibodies Segment or variant combine.Specifically, the fusion molecule of TCR of the invention and AntiCD3 McAb single-chain antibody include TCR α selected from the group below Chain variable domain amino acid sequence SEQ ID NO:5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、 23、24、25、26、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65 With TCR β chains variable domain amino acid sequence SEQ ID NO selected from the group below:27、28、29、30、31、32、33、34、35、36、37、 38、39、66、67、68、69、70、71、72、73、74、75、76、77、78.Preferably, high-affinity single chain TCR molecules of the present invention SEQ ID NO can be selected from the amino acid sequence of anti-CD3 single chain antibody fusions molecule:83、84、85、86、87、88、89、 90、91、92、93。

The invention further relates to the nucleic acid molecules for encoding TCR of the present invention.The present invention nucleic acid molecules can be DNA form or Rna form.DNA can be coding strand or noncoding strand.For example, the nucleotide sequence of coding TCR of the present invention can be attached with the present invention Nucleotide sequence shown in figure is identical or the variant of degeneracy.The meaning of " variant of degeneracy " is illustrated, such as this paper institutes With " variant of degeneracy " refers to that coding has SEQ ID NO in the present invention:2 protein sequence, but with SEQ ID NO:80 The differentiated nucleotide sequence of sequence.

The present invention nucleic acid molecules full length sequence or its segment usually can with but be not limited to PCR amplification method, recombination method or Artificial synthesized method obtains.At present, it is already possible to completely by chemical synthesis come obtain encoding TCR of the present invention (or its segment, Or derivatives thereof) DNA sequence dna.Then the DNA sequence dna can be introduced various existing DNA moleculars as known in the art (or Such as carrier) and cell in.

Carrier the present invention also relates to the nucleic acid molecules comprising the present invention and the carrier or coded sequence with the present invention pass through The host cell that genetic engineering generates.

Present invention additionally comprises the separation cells for expressing TCR of the present invention, particularly T cell.There are many methods to be suitable for volume The DNA or RNA of the high-affinity TCR of code book invention carries out T cell transfection (e.g., the such as Robbins, (2008) J.Immunol.180:6116-6131).The T cell of expression high-affinity TCR of the present invention can be used for adoptive immunotherapy.This Field technology personnel understand that many appropriate methods (e.g., the such as Rosenberg, (2008) Nat for carrying out adoptive treatment Rev Cancer8(4)：299-308).

The present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition contains pharmaceutically acceptable carrier and sheet It invents TCR or of the present invention TCR compounds or presents the cell of TCR of the present invention.

The present invention also provides a kind of method for treating disease, including object in need for the treatment of is given to apply suitable present invention TCR or of the present invention TCR compounds present the cell of TCR of the present invention or the pharmaceutical composition of the present invention.

It is to be understood that amino acid name is identified using international single English alphabet herein, amino corresponding thereto Sour three English alphabet of title is write a Chinese character in simplified form：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、 Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、 Tyr(Y)、Val(V)；In the present invention, Pro60 or 60P represent the 60th proline.In addition, heretofore described mutation The K that the form of presentation of concrete form such as " K52A " represents the 52nd is substituted by A, and similarly, " K52A/R/M/T " represents the K of the 52nd Substituted or substituted or substituted or substituted by T by M by R by A.Other and so on.

In the art, when being substituted with similar nature or similar amino acid, the work(of protein will not usually be changed Energy.The 26S Proteasome Structure and Function of protein will not generally also be changed by adding one or several amino acid in C-terminal and/or N-terminal.Cause This, TCR of the present invention further includes at most 5 of TCR of the present invention, preferably at most 3, more preferably at most 2, most preferably 1 ammonia Base acid (amino acid especially outside CDR region), it is similar or similar amino acid is replaced by property, and still be able to keep Its functional TCR.

Present invention additionally comprises the TCR after slightly modifying TCR of the present invention.Modify (not changing primary structure usually) form bag It includes：Chemically derived the form such as acetylation or carboxylated of TCR of the present invention.Modification further includes glycosylation, as those are in TCR of the present invention Synthesis and processing in or further processing step in carry out it is glycosylation modified and generate TCR.This modification can pass through by TCR is completed exposed to the glycosylated enzyme (glycosylase or deglycosylating enzyme of such as mammal) of progress.Modified forms also wrap Include the sequence with phosphorylated amino acid residue (such as phosphotyrosine, phosphoserine, phosphothreonine).It further includes and is modified So as to improve its anti-proteolysis performance or optimize the TCR of solubility property.

TCR, TCR compound of the present invention or the T cell of TCR of the present invention transfections can be together with pharmaceutically acceptable carriers It is provided in pharmaceutical composition.TCR, multivalent TCR complex or the cell of the present invention is usually as the one of sterile pharmaceutical composition Part provides, and the composition generally includes pharmaceutically acceptable carrier.The pharmaceutical composition can be any suitable shape Formula (the required method for depending on giving patient).Unit dosage forms offer can be used in it, usually provides, can make in the container of sealing It is provided for a part for kit.Such kit (but nonessential) includes the use of specification.It may include multiple units Dosage form.

In addition, the TCR of the present invention can be applied alone, use also can be combined or is coupled together with other therapeutic agents (as prepared In same pharmaceutical composition).

Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to control Treat the carrier of agent administration.The term refers to some such medicament carriers：Themselves do not induce generated to receiving said composition The antibody that body is harmful to, and there is no undue toxicity after being administered.These carriers are well known to those of ordinary skill in the art.In thunder It can in bright pharmaceutical science (Remington's Pharmaceutical Sciences (Mack Pub.Co., N.J.1991)) Find discussing fully on pharmaceutically acceptable excipient.This kind of carrier includes (but being not limited to)：Brine, buffer solution, Glucose, water, glycerine, ethyl alcohol, adjuvant, and combinations thereof.

Acceptable carrier can contain liquid in therapeutic composition Chinese pharmacology, such as water, brine, glycerine and ethyl alcohol.In addition, There is likely to be complementary substances, such as wetting agent or emulsifier, pH buffer substance in these carriers.

In general, therapeutic composition can be made to injectable agent, such as liquid solution or suspension；It may also be fabricated which before the injection It is suitble in supplying solution or suspension, the solid form of liquid-carrier.

Once being made into the composition of the present invention, it can be administered by conventional route, including (but it is and unlimited In)：Intraocular, intramuscular, intravenous, subcutaneous, intracutaneous or local administration are preferably parenteral including subcutaneous, intramuscular or vein It is interior.The object waited to prevent or treated can be animal；Especially people.

When the pharmaceutical composition of the present invention is used for actual therapeutic, can various different dosage forms be used according to service condition Pharmaceutical composition.It is preferred that can be enumerated has injection, oral agents etc..

These pharmaceutical compositions can be prepared by mixing, diluting or dissolve according to conventional methods, and be added once in a while Add suitable medicated premix, such as excipient, disintegrant, adhesive, lubricant, diluent, buffer, isotonic agent (isotonicities), preservative, wetting agent, emulsifier, dispersant, stabilizer and cosolvent, and the process for preparation can root It is carried out according to dosage form with usual way.

The pharmaceutical composition of the present invention can be administered with sustained release formulation.For example, TCR of the present invention can be impregnated in gather to be sustained Object is closed as in the pill or micro-capsule of carrier, the pill or micro-capsule then are implanted into tissue to be treated by performing the operation.As sustained release The example of polymer, what can be enumerated has ethylene-vinylacetate copolymer, poly- hydroxyl-metacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid- Ethanol copolymer etc., what can preferably be enumerated is that biodegradable polymer such as lactic acid polymer and lactic acid-ethanol are total to Polymers.

When the pharmaceutical composition of the present invention is used for actual therapeutic, TCR of the present invention or TCR as active component is compound Object or the cell for presenting TCR of the present invention, can according to the weight of each patient to be treated, the age, gender, symptom degree and it is reasonable Ground is determined, finally determines rational dosage by doctor.

Main advantages of the present invention are：

(1) present invention is had selected as stencil screen to the SLLMWITQC-HLA- using the single chain TCR molecules of hydrophobic core mutation A2 compounds have the TCR of high-affinity.

(2) TCR of the invention is wild to the affinity of the SLLMWITQC-HLA-A2 compounds and/or with reference to half-life period At least 10 times of raw type TCR.

(3) affinity and/or combination of the TCR of high-affinity of the invention to the SLLMWITQC-HLA-A2 compounds Half-life period can reach the 10 of wild type TCR²-10⁵Times.

Following specific embodiment, the present invention is further explained.It is to be understood that these embodiments be merely to illustrate the present invention and It is not used in and limits the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to normal condition, Such as (Sambrook and Russell et al., molecular cloning：Laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL publishing houses) described in condition or according to the condition proposed by manufacturer.Unless In addition illustrate, otherwise percentage and number are calculated by weight.

Material and method

Experiment material used can obtain unless otherwise specified from commercially available channel in the embodiment of the present invention, wherein, E.coli DH5 α are purchased from purchased from Tiangen, E.coli BL21 (DE3) purchased from Tiangen, E.coli Tuner (DE3) Novagen, plasmid pET28a are purchased from Novagen.

The generation of the single-stranded TCR template strands of stability of 1 hydrophobic core of embodiment mutation

The method that the present invention utilizes rite-directed mutagenesis, according to patent document WO2014/206304, constructs with one The stability single chain TCR molecules that flexible small peptide (linker) connects TCR α and β chains variable domain and forms, amino acid and DNA sequences Row are respectively SEQ ID NO:1 and SEQ ID NO:79, as illustrated in figs. 1A and ib.And using the single chain TCR molecules as template into The screening of row high-affinity TCR molecules.α variable domains (the SEQ ID NO of the template strand:And β variable domains (SEQ ID NO 2):3) Amino acid sequence as shown in figures 2 a and 2b；Its corresponding DNA sequence dna is respectively SEQ ID NO:80 and 81, such as Fig. 3 a and 3b institutes Show；The amino acid sequence and DNA sequence dna of flexible small peptide (linker) are respectively SEQ ID NO:4 and 82, it is as shown in Figs. 4a and 4b.

The target gene of template strand will be carried through I double digestion of Nco I and Not, and by I double digestion of Nco I and Not PET28a carriers connect.Connection product converts and to E.coli DH5 α, is coated with the LB tablets containing kanamycins, and 37 DEG C are inverted culture Overnight, picking positive colony carries out PCR screenings, and positive recombinant is sequenced, determines that sequence correctly extracts recombinant plasmid afterwards Conversion is to E.coli BL21 (DE3), for expressing.

Expression, renaturation and the purifying of the single-stranded TCR of stability built in 2 embodiment 1 of embodiment

BL21 (DE 3) bacterium colony containing recombinant plasmid pET28a- template strands prepared in embodiment 1 is all inoculated in In LB culture mediums containing kanamycins, 37 DEG C of cultures to OD₆₀₀For 0.6-0.8, IPTG to final concentration of 0.5mM is added in, 37 DEG C Continue to cultivate 4h.5000rpm centrifugation 15min harvest cell pellets, are cracked thin with Bugbuster Master Mix (Merck) Born of the same parents' sediment, 6000rpm centrifugation 15min recycling inclusion bodys, then with Bugbuster (Merck) washed to remove cell broken Piece and membrane component, 6000rpm centrifugation 15min, collect inclusion body.By solubilization of inclusion bodies in buffer solution (20mM Tris-HCl pH 8.0,8M urea) in, high speed centrifugation removal insoluble matter, supernatant is saved backup with being dispensed after BCA standard measures in -80 DEG C.

In the single-stranded TCR inclusion body proteins dissolved to 5mg, 2.5mL buffer solutions (6M Gua-HCl, 50mM Tris- is added in HCl pH 8.1,100mM NaCl, 10mM EDTA), add DTT to final concentration of 10mM, 37 DEG C of processing 30min.With injection Device is to 125mL renaturation buffers (100mM Tris-HCl pH 8.1,0.4M L-arginines, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) in treated single-stranded TCR, 4 DEG C of stirrings are added dropwise Then renaturation solution is packed into the cellulose membrane bag filter that interception is 4kDa by 10min, bag filter is placed in the water of 1L precoolings, 4 DEG C It is slowly stirred overnight.17 it is small when after, dialyzate changes into the buffer solutions (20mM Tris-HCl pH 8.0) of 1L precoolings, 4 DEG C after Then continuous dialysis 8h changes dialyzate into identical fresh buffer and continues dialysed overnight.17 it is small when after, sample through 0.45 μm filter Membrane filtration, by anion-exchange column (HiTrap Q HP, GE Healthcare) after vacuum outgas, with 20mM Tris-HCl The 0-1M NaCl linear gradient elution liquid purifying proteins that pH 8.0 is prepared, the elution fraction of collection carry out SDS-PAGE analyses, bag It is further carried out after component concentration containing single-stranded TCR with solvent resistant column (Superdex 75 10/300, GE Healthcare) Purifying, target components also carry out SDS-PAGE analyses.

Elution fraction for BIAcore analyses further tests its purity using gel filtration.Condition is：Chromatographic column Agilent Bio SEC-3 (7.8 × 300mm of 300A, φ), mobile phase be 150mM phosphate buffers, flow velocity 0.5mL/min, 25 DEG C of column temperature, ultraviolet detection wavelength 214nm.

Embodiment 3 combines characterization

BIAcore is analyzed

The combination of TCR molecules and SLLMWITQC-HLA-A2 compounds is detected using BIAcore T200 real-time analyzers Activity.The antibody (GenScript) of anti-Streptavidin is added in into coupling buffer (10mM sodium-acetate buffers, pH 4.77), Then antibody is flowed through to the CM5 chips activated in advance with EDC and NHS, antibody is made to be fixed on chip surface, finally uses ethanolamine Hydrochloric acid solution close unreacted activating surface, complete coupling process, coupling horizontal about 15,000RU.

The Streptavidin of low concentration is made to flow through the chip surface of coated antibody, then answers SLLMWITQC-HLA-A2 It closes logistics and crosses sense channel, another passage is flowed through as reference channel, then by the biotin of 0.05mM with the flow velocity of 10 μ L/min Chip 2min closes the remaining binding site of Streptavidin.Its affinity is measured using single cycle dynamic analysis method, it will TCR is diluted to several with HEPES-EP buffer solutions (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005%P20, pH 7.4) A different concentration with the flow velocity of 30 μ L/min, flows successively through chip surface, the binding time of each sample introduction is 120s, finally It is allowed to dissociate 600s after single injected sampling.Each round uses the 10mM Gly-HCl regeneration chips of pH 1.75 after measuring.Profit With BIAcore Evaluation software computational dynamics parameters.

The preparation process of above-mentioned SLLMWITQC-HLA-A2 compounds is as follows：

A. purify

The E.coli bacterium solutions of 100ml induced expressions heavy chain or light chain are collected, 10ml is used after 4 DEG C of 8000g centrifuge 10min PBS washing thallines are once, violent with 5ml BugBuster Master Mix Extraction Reagents (Merck) afterwards Thalline is resuspended in concussion, and is rotated in room temperature and be incubated 20min, after 4 DEG C, 6000g centrifugation 15min, supernatant discarding, collection forgives Body.

Above-mentioned inclusion body is resuspended in 5ml BugBuster Master Mix, room temperature rotation is incubated 5min；Add 30ml The BugBuster of 10 times of dilution, mixing, 4 DEG C of 6000g centrifuge 15min；Supernatant discarding, the BugBuster that 30ml is added to dilute 10 times Inclusion body, mixing is resuspended, 4 DEG C of 6000g centrifuge 15min, are repeated twice, and add 30ml 20mM Tris-HCl pH 8.0 that bag is resuspended Contain body, mixing, 4 DEG C of 6000g centrifuge 15min, finally dissolve inclusion body, SDS-PAGE detections with 20mM Tris-HCl 8M urea Inclusion body purity, BCA kits survey concentration.

B. renaturation

The small peptide SLLMWITQC (Beijing SBS Genetech gene technology Co., Ltd) of synthesis is dissolved in DMSO to 20mg/ml Concentration.The inclusion body of light chain and heavy chain is dissolved with 8M urea, 20mM Tris pH 8.0,10mM DTT, is added in before renaturation 3M guanidine hydrochlorides, 10mM sodium acetates, 10mM EDTA are further denatured.SLLMWITQC peptides are added in into renaturation with 25mg/L (final concentration) Buffer solution (0.4M L-arginines, 100mM Tris pH 8.3,2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced forms Glutathione, 0.2mM PMSF, are cooled to 4 DEG C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, Heavy chain adds in three times, 8h/ times), renaturation carries out at least 3 days at 4 DEG C to completion, and can SDS-PAGE detections renaturation success.

C. purified after renaturation

Make dialysis with the 20mM Tris pH 8.0 of 10 volumes to replace renaturation buffer, at least replace buffer solution and come twice Fully reduce the ionic strength of solution.With 0.45 μm of cellulose acetate sheets filtration protein solution after dialysis, it is then loaded into On HiTrap Q HP (GE General Electric Co. Limited) anion-exchange column (5ml bed volumes).Instrument (the general electricity of GE is purified using Akta Gas company), the 0-400mM NaCl linear gradients liquid elution albumen that 20mM Tris pH 8.0 are prepared, pMHC is about in 250mM It is eluted at NaCl, collects all peak components, SDS-PAGE detection purity.

D. biotinylation

With Millipore super filter tubes by the pMHC molecular concentrations of purifying, while it is 20mM Tris pH by buffer exchange 8.0, then add in biotinylation reagent 0.05M Bicine pH 8.3,10mM ATP, 10mM MgOAc, 50 μM of D- Biotin, 100 μ g/ml BirA enzymes (GST-BirA), incubation at room temperature mixture are stayed overnight, and whether SDS-PAGE detections biotinylation Completely.

E. the compound after purifying biological elementization

PMHC molecular concentrations after biotinylation is marked with Millipore super filter tubes are to 1ml, using gel permeation chromatography The pMHC of purifying biological elementization, using Akta purifying instrument (GE General Electric Co. Limited), with filtered PBS pre-equilibrations HiPrep^TM 16/60S200HR columns (GE General Electric Co. Limited), biotinylation pMHC molecules concentrated loading 1ml, then with PBS with 1ml/ Min flow velocitys elute.Biotinylated pMHC molecules occur in about 55ml as unimodal elution.Merge the group containing protein Point, it is concentrated with Millipore super filter tubes, BCA methods (Thermo) measure protein concentration, add in protease inhibitors cocktail (Roche) packing of biotinylated pMHC molecules is stored in -80 DEG C.

The generation of the single-stranded TCR of 4 high-affinity of embodiment

Display technique of bacteriophage is to generate TCR high-affinities Mutant libraries to screen a kind of means of high-affinity variant. By Li et al. ((2005) Nature Biotech 23 (3):349-354) the TCR phage displays and screening technique of description are applied to Single-stranded TCR templates in embodiment 1.It establishes the library of high-affinity TCR by being mutated the CDR region of the template strand and is washed in a pan Choosing.Those skilled in the art can be obtained by reading above-mentioned document and described build storehouse and screening technique.I.e. by using with institute The primer of the one or more codons variation needed is realized to as the plasmid containing related DNA of template.By a few wheel elutriations Phage library afterwards is and corresponding antigens have specific binding, therefrom picking monoclonal, and carry out sequence analysis.

Using the phase interaction of the analysis of BIAcore methods TCR molecules and SLLMWITQC-HLA-A2 compounds in embodiment 3 With, filtered out affinity and/or with reference to half-life period be wild type TCR at least 2 times of high-affinity TCR, that is, filter out The Dissociation equilibrium constant K of high-affinity TCR combination SLLMWITQC-HLA-A2 compounds_DIt is combined less than or equal to wild type TCR The Dissociation equilibrium constant K of SLLMWITQC-HLA-A2 compounds_DHalf, it is as a result as shown in table 3 below.Utilize the above method Detect reference TCR and the K of SLLMWITQC-HLA-A2 compounds interaction_DIt is worth for 18 μM, Curves of Interaction is as schemed Shown in 14, i.e. wild type TCR and the K of SLLMWITQC-HLA-A2 compounds interaction_DValue is also 18 μM.

Specifically, using SEQ ID NO:Number shown in 40, the α chain variable domains of these high-affinities TCR mutant Amino acid in following one or more sites is undergone mutation：52K、53A、54D、55D、56K、92L、97A、98G、99N、 100M, 102T and/or use SEQ ID NO:Number shown in 41, the β chain variable domains of these high-affinities TCR mutant exist Amino acid 27S, 28N, 29H, 30L, 31Y, 96D, 97P, 98R, 99A, 100G, the 101T in following one or more site occur prominent Become.

More specifically, using SEQ ID NO:Number shown in 40, the α chains variable domain of these high-affinities TCR include choosing From one or more amino acid residue 52A, 52R, 52M or 52T of the following group；53P；54G；55Q or 55E；56T, 56V, 56R or 56I；92R；97S；98A；99S or 99G；100S, 100P, 100H or 100F；102I；And/or using SEQ ID NO:Shown in 41 Number, the β chains variable domain of these high-affinities TCR include one or more amino acid residue 27P, 27H selected from the group below or 27L；28G；29S or 29A；30I, 30M or 30P；31A or 31G；96G；97F or 97I；98L；99H or 99P；100I；101Y.

α chains variable domain (the SEQ ID NO of the single-stranded TCR of high-affinity:5、6、7、8、9、10、11、12、13、14、15、16、 26) and β chains variable domain (SEQ ID NO 17,18,19,20,21,22,23,24,25 and:27、28、29、30、31、32、33、34、 35th, 36,37,38 and specific amino acid sequence 39) respectively as shown in Fig. 5 a-v and Fig. 6 a-m.

Table 3

After testing, the affinity and/or combination half-life period of TCR α chains variable domain and TCR β chain variable domains reach in upper table At least 2 times of wild type TCR.In addition, the α chain variable domain sequences of the single-stranded TCR of high-affinity are respectively SEQ ID NO.:6、7、8、 9 or 18；And when β chain variable domain sequences are respectively 28,29 or 30, K_DAlso between 250pM~700nM.

The generation of the 5 heterogeneous dimerization TCR of high-affinity α β of embodiment

The CDR region mutation of the single-stranded TCR of the high-affinity screened in embodiment 4 is introduced into the heterogeneous dimerization TCR's of α β In the corresponding site of variable domain, and its affinity with SLLMWITQC-HLA-A2 compounds is detected by BIAcore.It is above-mentioned The method that the introducing of CDR region high-affinity catastrophe point uses rite-directed mutagenesis well known to those skilled in the art.Above-mentioned wild type TCR α chains and β chains variable domain amino acid sequence respectively such as Fig. 7 a (SEQ ID NO:And 7b (SEQ ID NO 40):41) shown in.

It should be noted that obtain more stable sTCR, more easily to assess TCR and SLLMWITQC-HLA Binding affinity between A2 compounds and/or with reference to half-life period, the heterogeneous dimerization TCR of α β can be in the constant region of α and β chains A cysteine residues are introduced respectively to form the TCR of artificial interchain disulfide bond, and it is residual to introduce cysteine in the present embodiment The amino acid sequence of TCR α and β chains is respectively such as Fig. 8 a (SEQ ID NO after base:42) and 8b shown in (SEQ ID NO:43), introduce Cysteine residues with overstriking letter represent.

In addition, to obtain more stable sTCR, more easily to assess TCR and SLLMWITQC-HLA A2 Binding affinity between compound and/or with reference to half-life period, the heterogeneous dimerization TCR of α β can also be in the variable region of α chains and β chains Constant region in introduce a cysteine residues respectively to form the TCR of artificial interchain disulfide bond, such heterogeneous dimerization TCR can be with or without α chain constant regions.TCR α chains variable domains and β chain amino after cysteine residues are introduced in the present embodiment Acid sequence is respectively such as Figure 12 a (SEQ ID NO:And 12b (SEQ ID NO 94):95) shown in, the cysteine residues of introducing are to add Thick letter represents.

Pass through《Molecular Cloning: A Laboratory room handbook》(Molecular Cloning a Laboratory Manual) the (the 3rd Version, Sambrook and Russell) described in standard method by the extracellular sequence gene of TCR α and β chains to be expressed through synthesis After be inserted respectively into expression vector pET28a+ (Novagene), the cloning site of upstream and downstream is NcoI and NotI respectively.CDR region Mutation pass through over-lap PCR well known to those skilled in the art (overlap PCR) introduce.Insert Fragment confirms nothing by sequencing By mistake.

Expression, renaturation and the purifying of the heterogeneous dimerization TCR of 6 α β of embodiment

The expression vector of TCR α and β chains is converted by chemical transformation into expression bacterium BL21 (DE3), bacterium respectively It is grown with LB culture solutions, in OD₆₀₀It is induced when=0.6 with final concentration 0.5mM IPTG, the bag formed after α the and β chains expression of TCR Contain body to extract by BugBuster Mix (Novagene), and through the repeated multiple times washing of BugBuster solution, forgive Body is finally dissolved in 6M guanidine hydrochlorides, 10mM dithiothreitol (DTT)s (DTT), 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris (pH 8.1) in.

Dissolved TCR α and β chains are with 1：1 mass ratio is quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), in 3.7mM cystamine, 6.6mM β-mercapoethylamine (4 DEG C), final concentration of 60mg/mL.Mixing Solution is placed in the deionized water of 10 times of volumes dialysis (4 DEG C) afterwards, 12 it is small when after change deionized water into buffer solution (20mM Tris, pH 8.0) continue at 4 DEG C dialysis 12 it is small when.Solution after the completion of dialysis after 0.45 μM of membrane filtration, by the moon from Sub- exchange column (HiTrap Q HP, 5ml, GE Healthcare) purifying.Eluting peak contains the successful α and β dimers of renaturation TCR is confirmed by SDS-PAGE glue.TCR then by gel permeation chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare) it is further purified.TCR purity after purification is measured by SDS-PAGE is more than 90%, and concentration is by BCA methods It determines.

7 BIAcore analysis results of embodiment

The heterogeneous dimerization TCR and SLLMWITQC- of α β for introducing high-affinity CDR region are detected using method described in embodiment 3 The affinity of HLA-A2 compounds.

The CDR region filtered out in the single-stranded TCR α of high-affinity and β chains is transferred to wild type TCR α chain variable domains SEQ respectively ID NO:40 and β chain variable domain SEQ ID NO:41 corresponding position forms the heterogeneous dimerization TCR of α β.Obtained new TCR α and β Chain variable domain amino acid sequence, respectively as shown in Fig. 9 a-v and Figure 10 a-m.In addition, the high-affinity that the present invention will also filter out CDR region is transferred to described in embodiment 5 in α variable domains (SEQ ID NO:94) with β chains constant domain (SEQ ID NO:95) in, shape α chain constant regions are free of into the heterogeneous dimerization TCR of α β, such TCR.Since the CDR region of TCR molecules determines itself and corresponding pMHC The affinity of compound, so those skilled in the art are it is contemplated that introduce the heterogeneous dimerization TCR of α β of high-affinity catastrophe point With the high-affinity to SLLMWITQC-HLA-A2 compounds.Using method construction of expression vector described in embodiment 5, utilize The heterogeneous dimerization TCR of α β that method described in embodiment 6 is mutated above-mentioned introducing high-affinity are expressed, renaturation and purifying, so Its affinity with SLLMWITQC-HLA-A2 compounds is measured using BIAcore T200 afterwards, it is as shown in table 4 below.

Table 4

From upper table 4, the heterogeneous dimerization TCR of α β of introducing CDR region catastrophe point, which are maintained, answers SLLMWITQC-HLA-A2 Close the high-affinity of object.The affinity of the heterogeneous dimerization TCR is parents of the wild type TCR to SLLMWITQC-HLA-A2 compounds With at least 2 times of power.

8 anti-CD 3 antibodies of embodiment and expression, renaturation and the purifying of the fusion of the single-stranded TCR of high-affinity

The high-affinity single chain TCR molecules of the present invention with the single chain molecule (scFv) of anti-cd 3 antibodies are merged, are built Fusion molecule.Method by being overlapped (overlap) PCR, designs primer, connects anti-CD 3 antibodies and the single-stranded TCR of high-affinity The gene of molecule designs intermediate connection small peptide (linker) as GGGGS, and makes to limit on the genetic fragment band of fusion molecule Property restriction enzyme site Nco I and Not I.By pcr amplification product through I double digestion of Nco I and Not, with passing through I double digestion of Nco I and Not PET28a carriers connection.Connection product is converted to E.coli DH5 α competent cells, is coated with the LB tablets containing kanamycins, 37 DEG C of inversion overnight incubations, picking positive colony carry out PCR screenings, positive recombinant are sequenced, after determining that sequence is correct Recombinant plasmid transformed is extracted to E.coli BL21 (DE3) competent cell, for expressing.

The expression of fusion protein

Expression plasmid containing target gene is transformed into coli strain BL21 (DE3), coating LB tablets (block that 50 μ g/ml of mycin) it is placed in 37 DEG C of overnight incubations.Next day chooses clone and is seeded to 10ml LB fluid nutrient mediums (50 μ g/ of kanamycins Ml 2-3h) is cultivated, by volume 1:100 are seeded in 1L LB culture mediums (50 μ g/ml of kanamycins), continue culture to OD₆₀₀ For 0.5-0.8, then the expression of destination protein is induced using the IPTG of final concentration of 0.5mM.Induce 4 it is small when after, with 6000rpm centrifugation 10min harvest cells.PBS buffer solution washing thalline once, and dispenses thalline, takes and is equivalent to the thin of 200ml The thalline of bacterium culture cracks bacterium with 5ml BugBuster Master Mix (Novagen), is received with 6000g centrifugations 15min Collect inclusion body.Then 4 detergent washings are carried out to remove cell fragment and membrane component.Then, bag is washed with buffer solution such as PBS Contain body to remove detergent and salt.Finally, the inclusion body Tris buffer solutions of the urea containing 8M are dissolved, and it is dense to measure inclusion body Degree, is placed in after packing -80 DEG C of freezen protectives.

The refolding of fusion protein

The defrosting of about 10mg inclusion bodys is taken out from -80 DEG C of ultra low temperature freezers, adds dithiothreitol (DTT) (DTT) extremely final concentration of 10mM, incubated in 37 DEG C 30min to 1 it is small when to ensure that disulfide bond is opened completely.Then inclusion body sample solution is dripped respectively Enter 4 DEG C of precooling refolding buffers of 200ml (100mM Tris pH8.1,400mM L-arginines, 2mM EDTA, 5M urea, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine), 4 DEG C are slowly stirred about 30 minutes.Renaturation solution is with 8 The H of times volume precooling₂When O dialysis 16-20 is small.It is dialysed twice with the 10mM Tris pH 8.0 of 8 times of volumes again, 4 DEG C are continued thoroughly When analysis about 8 is small, following purifying is carried out after dialysis after sample filtering.

The first step purifying of fusion protein

Refolding object (in 10mM Tris pH 8.0) by dialysis is pre- using POROS HQ/20 anion-exchange chromatographies Column (Applied Biosystems) is filled, carrying out gradient with 0-600mM NaCl in AKTA purifying instrument (GE Healthcare) washes It is de-.Each component is analyzed by the SDS-PAGE of coomassie brilliant blue staining, is then combined with.

The second step purifying of fusion protein

The sample solution that first step purifying merges is concentrated for this step purifying, utilizes the pre-equilibration in PBS buffer solution Superdex 75 10/300GL gel permeation chromatographies prepacked column (GE Healthcare) purified fusion protein, Coomassie brilliant blue The component of the SDS-PAGE analysis appearances of dyeing, is then combined with.

9 anti-CD 3 antibodies of embodiment and expression, renaturation and the purifying of the fusion of the heterogeneous dimerization TCR of high-affinity α β

The single-chain antibody (scFv) of anti-CD3 with the heterogeneous dimerization TCR of α β is merged, prepares fusion molecule.The scFv of anti-CD3 It is merged with the β chains of TCR, which can include the β chain variable domains of any above-mentioned heterogeneous dimerization TCR of high-affinity α β, fusion The TCR α chains of molecule can include the α chain variable domains of any above-mentioned heterogeneous dimerization TCR of high-affinity α β.

The structure of fusion molecule expression vector

1. the structure of α chain expression vectors

The target gene of the α chains of the heterogeneous dimerization TCR of α β will be carried through I double digestion of Nco I and Not, with passing through Nco I and Not I The pET28a carriers connection of double digestion.Connection product converts and to E.coli DH5 α, is coated on the LB tablets containing kanamycins, and 37 DEG C be inverted overnight incubation, picking positive colony carry out PCR screenings, positive recombinant is sequenced, determines that sequence is correctly taken out afterwards Recombinant plasmid transformed is put forward to E.coli Tuner (DE3), for expressing.

2. the structure of anti-CD3 (scFv)-β chain expression vectors

Method by being overlapped (overlap) PCR designs primer by anti-CD3scFv and the heterogeneous dimerization TCR β of high-affinity Chain gene connects, and intermediate connection small peptide (linker) is GGGGS, and makes the scFv of anti-CD3 and high-affinity heterogeneous Restriction endonuclease sites Nco I (CCATGG) and Not I on the genetic fragment band of the fusion protein of dimerization TCR β chains (GCGGCCGC).By pcr amplification product through I double digestion of Nco I and Not, with the pET28a carriers by I double digestion of Nco I and Not Connection.Connection product is converted to E.coli DH5 α competent cells, is coated with the LB tablets containing kanamycins, and 37 DEG C are inverted culture Overnight, picking positive colony carries out PCR screenings, and positive recombinant is sequenced, determines that sequence correctly extracts recombinant plasmid afterwards Conversion is to E.coli Tuner (DE3) competent cell, for expressing.

Expression, renaturation and the purifying of fusion protein

Expression plasmid is converted respectively into E.coli Tuner (DE3) competent cell, be coated with LB tablet (kanamycins 50 μ g/mL) it is placed in 37 DEG C of overnight incubations.Next day chooses clone and is seeded to 10mL LB fluid nutrient mediums (50 μ g/mL of kanamycins) Cultivate 2-3h, by volume 1:100 are seeded in 1L LB culture mediums, and it is 0.5-0.8 to continue culture to OD600, adds in final concentration The expression of destination protein is induced for 1mM IPTG.Induce 4 it is small when after, with 6000rpm centrifugation 10min harvest cell.PBS is buffered Liquid washing thalline once, and dispenses thalline, takes the thalline 5mL BugBuster for the bacterial cultures for being equivalent to 200mL Master Mix (Merck) crack bacterium, and inclusion body is collected with 6000g centrifugations 15min.Then carry out the washing of 4 detergent with Remove cell fragment and membrane component.Then, inclusion body is washed with buffer solution such as PBS to remove detergent and salt.Finally, will forgive Body guanidine hydrochloride containing 6M, 10mM dithiothreitol (DTT)s (DTT), 10mM ethylenediamine tetra-acetic acids (EDTA), 20mM Tris, pH 8.1 are slow Solution dissolving is rushed, and measures and forgives bulk concentration, is placed in after packing -80 DEG C of freezen protectives.

Dissolved TCR α chains and anti-CD3 (scFv)-β chains are with 2：5 mass ratio is quickly mixed in 5M urea (urea), 0.4M L-arginines (L-arginine), 20mM Tris pH 8.1,3.7mM cystamine, 6.6mM β- Mercapoethylamine (4 DEG C), final concentration α chains and anti-CD3 (scFv)-β chains are respectively 0.1mg/mL, 0.25mg/mL.

Solution is placed in the deionized water of 10 times of volumes dialysis (4 DEG C) after mixing, 12 it is small when after deionized water is changed into Buffer solution (10mM Tris, pH 8.0) continue at 4 DEG C dialysis 12 it is small when.Solution after the completion of dialysis is through 0.45 μM of filter membrane mistake After filter, purified by anion-exchange column (HiTrap Q HP 5ml, GE healthcare).It is successful that eluting peak contains renaturation TCR α chains and the TCR of anti-CD3 (scFv)-β chain homodimers are confirmed by SDS-PAGE glue.TCR fusion molecules then pass through size Exclusion chromatography (S-100 16/60, GE healthcare) is further purified and anion-exchange column (HiTrap Q HP 5ml, GE healthcare) it purifies again.TCR fusion molecules purity after purification is measured by SDS-PAGE more than 90%, dense Degree is measured by BCA methods.

In addition, the scFv of anti-CD3 can also be merged with the α chain variable domains of the heterogeneous dimerization TCR of the α β not comprising α chain constant regions, The α chains variable domain of the heterogeneous dimerization TCR can be SEQ ID NO:97, β chain variable domains can be SEQ ID NO:41；It is described The α chains variable domain of heterogeneous dimerization TCR can be SEQ ID NO:97, β chain variable domains can be SEQ ID NO:66；It is described heterogeneous The α chains variable domain of dimerization TCR can be SEQ ID NO:100, β chain variable domains can be SEQ ID NO:71；Described heterogeneous two The α chains variable domain of poly- TCR can be SEQ ID NO:101, β chain variable domains can be SEQ ID NO:66；The heterogeneous dimerization The α chains variable domain of TCR can be SEQ ID NO:99, β chain variable domains can be SEQ ID NO:72；The heterogeneous dimerization TCR α chains variable domain can be SEQ ID NO:98, β chain variable domains can be SEQ ID NO:72；The α of the heterogeneous dimerization TCR Chain variable domain can be SEQ ID NO:96, β chain variable domains can be SEQ ID NO:66.Carrier construction as stated above, into Row expression, renaturation and purifying.

Embodiment 10 transfects the functional experiment of the effector cell of high-affinity TCR of the present invention

The present embodiment, which demonstrates the effector cell for transfecting high-affinity TCR of the present invention, has target cell good specificity to swash Effect living.

Functions and specificity of the high-affinity TCR of the present invention in cell are detected by ELISPOT experiments.Art technology The known method using ELISPOT experiment detection cell functions of personnel.The present embodiment IFN-γ ELISPOT experiments are with from health The PBL cells being separated in the blood of volunteer through slow-virus transfection TCR as effector cell (respectively label TCR1, TCR2, TCR3, TCR4), control group effector cell is marked as NC (untransfected TCR) and GFP (Transfection of GFP).Target cell system is IM9 (89), U266B1 (6940), 293T (1) cell.Wherein, target cell system IM9 (89) and U266B1 (6940) expression related antigen.

Prepare ELISPOT tablets first.ELISPOT tablets Ethanol activation is coated with, and 4 DEG C overnight.It tests the 1st day, goes to exchange By liquid, washing closing is incubated two hours, removes confining liquid, in the following order adds in each component of experiment at room temperature ELISPOT tablets：Culture medium adjusts effector cell to 1X10⁵A cells/ml, culture medium adjust each target cell system to 2X10⁵It is a Cells/ml.100 2X105 cells/mls of μ L target cell systems (i.e. 20,000 cells/well), 100 μ L are taken after mixing Effector cell 1X10⁵A cells/ml (i.e. 10,000 cells/well) is added in corresponding aperture, and sets two multiple holes.It incubated Night (37 DEG C, 5%CO₂).It tests the 2nd day, washing flat board simultaneously carries out secondary detection and colour developing, and dry tablet recycles immune spot Point plate reader (ELISPOT READER system；AID20 companies) count the spot formed on film.Experimental result is as schemed Shown in 16, transfecting the effector cell of high-affinity TCR of the present invention has target cell good specific activation to act on.

The functional experiment of the fusion protein of 11 high-affinity TCR of the present invention of embodiment and anti-CD 3 antibodies

The present embodiment demonstrates the fusion protein of high-affinity TCR and anti-CD 3 antibodies of the present invention, and can to redirect effect thin Born of the same parents, and with good activation.

The known method using ELISPOT experiment detection cell functions of those skilled in the art.The present embodiment IFN-γ Effector cell used is the CD8+T cells being separated to from the blood of healthy volunteer in ELISPOT experiments, and target cell system is Loading peptides pNY (A0201NY-ESO-1：SLLMWITQC T2 cells), control group are Loading peptides pGP100 (A0201gp100：YLEPGPVTA T2 cells).

Prepare ELISPOT tablets first.ELISPOT tablets Ethanol activation is coated with, and 4 DEG C overnight.It tests the 1st day, goes to exchange By liquid, washing closing is incubated two hours, removes confining liquid, in the following order adds in each component of experiment at room temperature ELISPOT tablets：Culture medium adjusts CD8+T cells to 2X10⁴A cells/ml, culture medium adjust each target cell system to 2X10⁵ Albumen is diluted to concentration as 0.04 μM by a cells/ml, culture medium, one by one 10 times of gradient dilutions, totally 6 concentration gradients.It is mixed 50 μ L diluted protein solutions, 50 μ L target cell systems 2X10 are taken after closing uniformly⁵A cells/ml (i.e. 10,000 cells/well), 100 μ L effector cell 2X10⁴A cells/ml (i.e. 2,000 effector cell/hole) is added in corresponding aperture, and sets two multiple holes.Temperature Educate overnight (37 DEG C, 5%CO₂).It tests the 2nd day, washing flat board simultaneously carries out secondary detection and colour developing, and dry tablet, recycling is exempted from Epidemic disease patch plate reader (ELISPOT READER system；AID20 companies) count the spot formed on film.Experimental result As shown in figure 17, the fusion protein of high-affinity TCR and anti-CD 3 antibodies of the present invention can redirect effector cell, and with very Good activation.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited It encloses.

Claims

1. a kind of T cell receptor (TCR), which is characterized in that it has the activity with reference to SLLMWITQC-HLA A2 compounds, and And the T cell receptor includes TCR α chains variable domains and TCR β chain variable domains, the TCR α chains variable domain includes 3 CDR regions, institute It is as follows to state the consensus sequence of 3 CDR regions of TCR α chain variable domains,

CDR1α：ATGYPS

CDR2α：ATKADDK

CDR3α：ALTLNNAGNMLT, and contain at least one following mutation：

And/or the TCR β chains variable domain includes 3 CDR regions, the consensus sequence of 3 CDR regions of the TCR β chain variable domains is such as Under,

CDR1β：SNHLY

CDR2β：FYNNEI

CDR3β：ASLDPRAGTDTQY, and contain at least one following mutation：

Also, the T cell receptor (TCR), comprising TCR α chains variable domains and TCR β chain variable domains, the TCR α chains variable domain bag Include CDR1 α, CDR2 α and CDR3 α；Also, the CDR1 α include sequence:ATGYPS；

The TCR include TCR α chains variable domains and TCR β chain variable domains, the TCR β chains variable domain include CDR1 β, CDR2 β and CDR3 β, wherein the CDR2 β include sequence：FYNNEI；

Also, the TCR has any one group in following s-1 to s-42 of CDR：

2. TCR as described in claim 1, which is characterized in that any one group in following 1 to 18 of the TCR：

And/or any one group in following s-1 to s-42 of the TCR：

。

3. TCR as described in claim 1, which is characterized in that the TCR is the heterogeneous dimerization TCR of α β.

4. TCR as described in claim 1, which is characterized in that the TCR includes the whole (I) in addition to its transmembrane domain Or all or part of TCR β chains of part TCR α chains and (II) in addition to its transmembrane domain, wherein (I) and (II) includes The variable domain of TCR chains and at least a portion constant domain.

5. TCR as described in claim 1, which is characterized in that the TCR is the heterogeneous dimerization TCR of α β, and the α chains of the TCR are variable Contain artificial interchain disulfide bond between area and β chain constant regions.

6. TCR as claimed in claim 5, which is characterized in that formed between the α chains variable region of the TCR and β chain constant regions The cysteine residues of artificial interchain disulfide bond are instead of selected from following one or more groups of sites：

The 47th amino acids of TRAV and the 60th amino acids of TRBC1*01 or TRBC2*01 exons 1s；

Wherein, the Position Number of the amino acid sequence of TRAV is according to the Position Number listed in IMGT；

The Position Number of TRBC1*01 or TRBC2*01 amino acid sequences carries out Position Number by the order from N-terminal to C-terminal successively.

7. TCR as claimed in claim 5, which is characterized in that contain artificial interchain two between α chains variable region and β chain constant regions The TCR of sulfide linkage includes α chains variable domain and β chains variable domain and all or part of β chains constant domain in addition to transmembrane domain, but It does not include α chain constant domains, and α chains variable domain and the β chains of the TCR form heterodimer.

8. TCR as claimed in claim 5, which is characterized in that contain artificial interchain two between α chains variable region and β chain constant regions The TCR of sulfide linkage includes all or part of TCR α chains and (II) of (I) in addition to the TCR transmembrane domains and removes the TCR cross-films All or part of TCR β chains beyond structural domain, wherein (I) and (II) variable domain comprising TCR chains and at least a portion is constant Domain.

9. TCR as claimed in claim 5, which is characterized in that the TCR is the heterogeneous dimerization TCR of α β, it includes (I) except described The all or part of all or part of TCR α chains and (II) in addition to the TCR transmembrane domains beyond TCR transmembrane domains TCR β chains, wherein (I) and (II), comprising the variable domain of TCR chains and at least a portion constant domain, α chains constant region and β chains are constant Contain artificial interchain disulfide bond between area.

10. TCR as described in claim 1, which is characterized in that form artificial chain between the TCR α and the constant region of β chains Between disulfide bond cysteine residues instead of selected from following one or more groups of sites：

The Ser57 of Thr48 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser77 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser17 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Asp59 of Thr45 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Glu15 of Ser15 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

The Ser54 of Arg53 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；TRAC*01 exons 1s The Ala19 of Pro89 and TRBC1*01 or TRBC2*01 exons 1s；With

The Glu20 of Tyr10 and TRBC1*01 or the TRBC2*01 exons 1 of TRAC*01 exons 1s；

Wherein, the Position Number of TRAC*01 and TRBC1*01 or TRBC2*01 amino acid sequences is by successively suitable from N-terminal to C-terminal Sequence carries out Position Number.

11. TCR as described in claim 1, which is characterized in that the TCR is single-stranded TCR.

12. TCR as claimed in claim 11, which is characterized in that the TCR is made of α chains variable domain and β chain variable domains Single-stranded TCR, the α chains variable domain and β chains variable domain are connected by a flexible short peptide sequence.

13. TCR as described in claim 1, which is characterized in that the hydrophobic core of the TCR is undergone mutation.

14. TCR as described in claim 1, which is characterized in that the hydrophobic core of the TCR α chains variable domain and/or β chain variable domains It undergos mutation.

15. TCR as claimed in claim 13, which is characterized in that the TCR that the hydrophobic core is undergone mutation is by α chain variable domains With the single-stranded TCR of β chains variable domain composition, the α chains variable domain is connected with β chains variable domain by a flexible short peptide sequence.

16. TCR as claimed in claim 13, which is characterized in that the hydrophobic core mutation of the TCR is happened at SEQ ID NO:40 One or more acid residues sites selected from the group below of shown α chains variable domain：11V, 13L, 19L, 77K and 80V；Wherein, Numbering amino acid residues use SEQ ID NO:Number shown in 40；And/or

The hydrophobic core mutation is happened at SEQ ID NO:One or more amino acid selected from the group below of the variable domains of β chains shown in 41 Residue positions：11Q, 13T and 82T, wherein, numbering amino acid residues use SEQ ID NO:Number shown in 41.

17. TCR as claimed in claim 16, which is characterized in that the α chain variable domains of the TCR after hydrophobic core mutation include One or more amino acid residue selected from the group below：11L, 13V, 19V, 77I and 80I；It is and/or described after hydrophobic core mutation The β chains variable domain of TCR includes one or more amino acid residues selected from the group below：11L, 13V and 82V.

18. TCR as described in claim 1, which is characterized in that the TCR is soluble.

19. TCR as described in claim 1, which is characterized in that the α chains of the TCR and/or C- the or N- ends of β chains are combined with Conjugate.

20. TCR as claimed in claim 19, which is characterized in that the conjugate combined with the TCR for detectable, Therapeutic agent, PK modified parts or combination thereof.

21. TCR as claimed in claim 20, which is characterized in that the therapeutic agent combined with the TCR is to be connected to the TCR α or β chains C- or N- ends anti-CD 3 antibodies.

22. TCR as claimed in claim 21, which is characterized in that the α chain variable domains of the TCR combined with anti-CD 3 antibodies The β chains variable domain amino acid sequence of amino acid sequence and the TCR combined with anti-CD 3 antibodies are selected from following combination：

。

23. TCR as claimed in claim 22, which is characterized in that the TCR and the amino acid sequence of anti-CD 3 antibodies fusion molecule Row preferably are selected from：SEQ ID NO:83-93.

24. a kind of multivalent TCR complex, which is characterized in that comprising at least two TCR molecules, and at least one TCR therein TCR of the molecule any one of the claims.

25. a kind of nucleic acid molecules, which is characterized in that the nucleic acid molecules include any TCR of coding claim 1-23 Nucleotide sequence or its complementary series.

26. a kind of carrier, which is characterized in that the carrier contains the nucleic acid molecules described in claim 25.

27. a kind of host cell, which is characterized in that contain the carrier or dye described in claim 26 in the host cell The nucleic acid molecules described in the claim 25 of external source are integrated in colour solid.

A kind of 28. separated cell, which is characterized in that the TCR any one of the cell expression claim 1-23.

29. a kind of pharmaceutical composition, which is characterized in that the composition contains pharmaceutically acceptable carrier and claim The TCR compounds described in TCR or claim 24 any one of 1-23 or the cell described in claim 28.

30. in the TCR compounds or claim 28 described in claim 1-23 any one of them TCR, claim 24 The purposes of the cell, which is characterized in that be used to prepare the drug for the treatment of tumour.

A kind of 31. method for preparing any TCR in claim 1-23, which is characterized in that including step：

(i) host cell described in claim 27 is cultivated, so as to express any TCR in claim 1-23；

(ii) isolated or purified goes out the TCR.