CN101061138A - T-cell receptors containing a non-native disulfide interchain bond linked to therapeutic agents - Google Patents

T-cell receptors containing a non-native disulfide interchain bond linked to therapeutic agents Download PDF

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CN101061138A
CN101061138A CNA2005800393813A CN200580039381A CN101061138A CN 101061138 A CN101061138 A CN 101061138A CN A2005800393813 A CNA2005800393813 A CN A2005800393813A CN 200580039381 A CN200580039381 A CN 200580039381A CN 101061138 A CN101061138 A CN 101061138A
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tcr
antibody
therapeutic substance
sequence
gly
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B·K·雅各布森
T·B·安德森
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Avidex Ltd
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract

The present invention provides a dimeric TCR (dTCR) or single-chain TCR (scTCR) associated with selected therapeutic agents, wherein said TCR comprises a first segment constituted by an amino acid sequence corresponding to a TCR a chain variable domain sequence fused to the N terminus of an amino acid sequence corresponding to a TCR a chain constant domain extracellular sequence, a second segment constituted by an amino acid sequence corresponding to a TCR ss chain variable domain fused to the N terminus of an amino acid sequence corresponding to TCR ss chain constant domain extracellular sequence, a disulfide bond between the first and second chains, said disulfide bond being one which has no equivalent in native ass T cell receptors, and in the case of said scTCRs further comprising a linker sequence linking the C terminus of the first segment to the N terminus of the second segment, or vice versa, the length of the linker sequence and the position of the disulfide bond being such that the variable domain sequences of the first and second segments are mutually orientated substantially as in native ass T cell receptors.

Description

The TXi Baoshouti that contains the non-natural interchain disulfide bond that is connected with therapeutic substance
The present invention relates to contain TXi Baoshouti (TCR) with therapeutic substance bonded non-natural interchain disulfide bond.
Background of invention
Novel TCR therapeutical agent combination disclosed herein can be used for treating autoimmune disease, organ rejection, graft versus host disease (GVH disease) (GVHD) and cancer.TCR in the TCR therapeutical agent combination disclosed herein partly is a targeting moiety.
The invention summary
The present invention has prepared available and therapeutic substance bonded dimer TCR (dTCR) or strand TCR (scTCR) first, and described material is selected from: IL-1, IL-1 α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-β, IFN-γ, lymphotoxin, TNF α, anti--CD2 antibody, anti-CD 3 antibodies, anti-CD 4 antibodies, anti--CD8 antibody, anti--CD44 antibody, anti--CD45RA antibody, anti--CD45RB antibody, anti--CD45RO antibody, anti--Thy1.2 antibody, antilymphocyte globulin (ALG), anti--α β TCR antibody, anti--gamma delta T CR antibody, anti--CD49a antibody, anti--CD49b antibody, anti--CD49c antibody, anti--CD49d antibody, anti--CD49e antibody, anti--CD49f antibody, anti--TCR V β 8 antibody, anti--CD16 antibody, anti--CD28 antibody, CTLA-4-Ig, anti--B7.2 antibody, anti-CD 40 L antibody, anti--ICAM-1 antibody, ICAM-I, anti--Mac antibody, anti--LFA-1 antibody, anti--the IFN-gamma antibodies, IFN-γ, IFN-γ R/IgG1 syzygy, anti--IL-2R antibody, IL-2R antibody, IL-2 diphtheria (Diptheria)-toxin protein, anti--IL-12 antibody, IL-12 antagonist (p40), anti--IL-1 antibody, the IL-1 antagonist, L-Glutamic decarboxylase (GAD), anti--GAD antibody, viral protein and peptide, bacterioprotein or peptide, A-galactosyl-ceramide, thyrocalcitonin, niacinamide, anti--oxygenant (vitamin-E, the probucol analogue, probucol+deflazacort (deflazacoert) or aminoguanidine), anti--inflammatory drugs (pentoxifylline or rolipram (Rolipram)), immunomodulator (Roquinimex, Ling-zhi-8, the D-dextran, multifunctional protein 14, Ciamexon, cholera toxin B, vanadate or vitamin D 3 analogs, small molecules CD80 inhibitor, male sex hormone, IGF-1, thing (Immunomanipulation) (natural antibody) is controlled in immunity, the lupus idiotype, lipopolysaccharides), sulfatide, bee venom, the Kampo preparation, silica, Sphingolipids,sialo, anti-asialo (Antiasialo) GM-1 antibody, Unidasa, concanavalin A, anti--I class MHC antibody, or anti--II class MHC antibody, S-Neoral, FK-506, azathioprine, rapamycin or Gusperimus (Deoxyspergualin), the functional variant or the fragment of PE38 Pseudomonas exotoxin or above-mentioned any material; Wherein said TCR comprises: by first section that the aminoacid sequence corresponding to TCR α chain variable region sequence constitutes, this aminoacid sequence merges with aminoacid sequence N-terminal corresponding to the outer sequence of TCR α chain constant region born of the same parents; By second section that the aminoacid sequence corresponding to TCR β chain variable region sequence constitutes, this aminoacid sequence merges with aminoacid sequence N-terminal corresponding to the outer sequence of TCR β chain constant region born of the same parents; A disulfide linkage is arranged between this first and second chain, and described disulfide linkage does not have Equivalent in natural α β TXi Baoshouti; Be example with described scTCR, it also contains the joint sequence that connects this first section C-terminal and this second section N-terminal, or vice versa, and the length of this joint sequence and the position of this disulfide linkage should make variable region sequences directed each other basically as natural α β TXi Baoshouti of described first and second sections.
Detailed Description Of The Invention
The invention provides and therapeutic substance bonded dimer TCR (dTCR) or strand TCR (scTCR), described material is selected from: IL-1, IL-1 α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-β, IFN-γ, lymphotoxin, TNF α, anti--CD2 antibody, anti-CD 3 antibodies, anti-CD 4 antibodies, anti--CD8 antibody, anti--CD44 antibody, anti--CD45RA antibody, anti--CD45RB antibody, anti--CD45RO antibody, anti--Thy1.2 antibody, antilymphocyte globulin (ALG), anti--α β TCR antibody, anti--gamma delta T CR antibody, anti--CD49a antibody, anti--CD49b antibody, anti--CD49c antibody, anti--CD49d antibody, anti--CD49e antibody, anti--CD49f antibody, anti--TCR V β 8 antibody, anti--CD16 antibody, anti--CD28 antibody, CTLA-4-Ig, anti--B7.2 antibody, anti-CD 40 L antibody, anti--ICAM-1 antibody, ICAM-I, anti--Mac antibody, anti--LFA-1 antibody, anti--the IFN-gamma antibodies, IFN-γ, IFN-γ R/IgG1 syzygy, anti--IL-2R antibody, IL-2R antibody, IL-2 diphtheria (Diptheria)-toxin protein, anti--IL-12 antibody, IL-12 antagonist (p40), anti--IL-1 antibody, the IL-1 antagonist, L-Glutamic decarboxylase (GAD), anti--GAD antibody, viral protein and peptide, bacterioprotein or peptide, A-galactosyl-ceramide, thyrocalcitonin, niacinamide, anti--oxygenant (vitamin-E, the probucol analogue, probucol+deflazacort or aminoguanidine), anti--inflammatory drugs (pentoxifylline or rolipram), immunomodulator (Roquinimex, Ling-zhi-8, the D-dextran, multifunctional protein 14, Ciamexon, cholera toxin B, vanadate or vitamin D 3 analogs, small molecules CD80 inhibitor, male sex hormone, IGF-1, thing (Immunomanipulation) (natural antibody) is controlled in immunity, the lupus idiotype, lipopolysaccharides), sulfatide, bee venom, the Kampo preparation, silica, Sphingolipids,sialo, anti-asialo GM-1 antibody, Unidasa, concanavalin A, anti--I class MHC antibody, or anti--II class MHC antibody, S-Neoral, FK-506, azathioprine, rapamycin or Gusperimus, the functional variant or the fragment of PE38 Pseudomonas exotoxin or above-mentioned any material; Wherein said TCR comprises: by first section that the aminoacid sequence corresponding to TCR α chain variable region sequence constitutes, this aminoacid sequence merges with aminoacid sequence N-terminal corresponding to the outer sequence of TCR α chain constant region born of the same parents; By second section that the aminoacid sequence corresponding to TCR β chain variable region sequence constitutes, this aminoacid sequence merges with aminoacid sequence N-terminal corresponding to the outer sequence of TCR β chain constant region born of the same parents; A disulfide linkage is arranged between this first and second chain, and described disulfide linkage does not have Equivalent in natural α β TXi Baoshouti; Be example with described scTCR, it also contains the joint sequence that connects described first section C-terminal and the described second section N-terminal, or vice versa, and the length of this joint sequence and the position of this disulfide linkage should make variable region sequences directed each other basically as natural α β TXi Baoshouti of described first and second sections.
Term used herein " with therapeutic substance bonded dimer TCR (dTCR) or strand TCR (scTCR) " is interpreted as representing that TCR is with the therapeutic substance covalency or otherwise be connected.Described therapeutic substance can directly link to each other with TCR, also can link to each other indirectly through shank.
Term used herein " functional variant " is interpreted as representing the analogue with identical treatment effect of disclosed therapeutic substance.For example, those skilled in the art will know that with material disclosed herein and compare, can be created in the therapeutical agent that mixes minor alteration in its chemical structure or the aminoacid sequence but can not change the therapeutic action of this material.This common variant is included in the scope of the present invention.
Functional antibodies fragment and variant
The antibody fragment and the variant/analogue that are applicable to composition described herein and method include but not limited to following.
Antibody fragment
The known fragment that may produce certain the given antibody that has kept parental generation antibody identical combination characteristic basically of those skilled in the art.This segmental details hereinafter is provided:
Small antibody (minibody)-these constructions by have amputation the antibody of Fc part form.Therefore, they have kept the complete binding domains of its antibody of deriving.
Fab fragment-these (constructions) comprise a light chain immunoglobulin that links to each other with a part of covalency of heavy chain immunoglobulin.For example, the Fab fragment comprises an antigen binding site.The Fab fragment is defined as the part of the IgG that discharges with the papoid processing.This fragment generally can produce through recombinant DNA technology.(Reeves etc., (2000), " immunology lecture notes " (Lecture Notes on Immunology), (the 4th edition), BlackwellScience publishes)
F (ab ') 2Fragment-these (constructions) comprise a kind of two antigen binding sites and hinge region of antibody.F (ab ') 2Fragment is defined as the part of the IgG that discharges with pepsin.This fragment generally can produce through recombinant DNA technology.(Reeves etc., (2000), " immunology lecture notes " (Lecture Notes on Immunology), (the 4th edition), Blackwell Science publishes)
Fv fragment-these (constructions) comprise heavy (chain) variable region of the immunoglobulin (Ig) that links to each other with immunoglobulin light (chain) variable region covalency.Many Fv designs have been produced.These (constructions) comprise by introducing disulfide linkage and strengthen associating dsFv between two structural domains.Perhaps, can utilize peptide linker that two structural domains are combined becomes a polypeptide and forms scFv.Also produced the variable region of containing heavy chain immunoglobulin or light chain, and the Fv construction that combines with the variable region or the constant region of corresponding heavy chain immunoglobulin or light chain.But Fv is multimerization and form double antibody or three antibody (Maynard etc., (2000), Annu Rev Biomed Eng, 2 339-376) also.
Nanobodies TM-these constructions of being put goods on the market by Ablynx (Belgium) comprise heavy (chain) variable region of a synthetic immunoglobulin (Ig) derived from camel (camelid) (for example, camel or yamma) antibody.
Domain antibodies-these constructions that put goods on the market by Domantis (Belgium) comprise heavy (chain) variable region of an immunoglobulin (Ig) or immunoglobulin light (chain) variable region of affinity maturation.
Antibody variants and analogue
The defined antibody function characteristic of the present invention is that their energy specificitys are in conjunction with target ligands.Those skilled in the art are known can engineered many other proteinic this binding characteristics.Be applicable to that the antibody variants of the present composition and method and the example of congener include but not limited to following.
Based on protein scaffolds in conjunction with polypeptide-this comprises the proteinic sudden change analogue that contains natural coupling collar in conjunction with construction family.Example comprise by Affibody (Sweden) put goods on the market based on triple helical motif Affibodies antibody derived from one of IgG binding domains of streptococcus aureus (Staphylococcus aureus) albumin A.Another example be by EvoGenix (Australia) put goods on the market based on having transplanted the Evibodies antibody that is similar to antibodies ring structure territory CTLA-4 ectodomain.Last example is the Cytokine Traps that the cytokine receptor structural domain is arranged by the grafting in the antibody support that RegeneronPharmaceuticals (US) puts goods on the market.(Nygren etc., (2000) Current Opinion in Structural biology, 7 463-469) utilize support through the engineered summary that adds novel binding site in protein.This summary mentions that following protein originates as support: CP1 zinc refers to (structure), amylase inhibiting peptide (Tendamistat), Z structural domain (albumin A congener), PST1, coiled coil, LACI-D1 and cytochrome b 562Other protein scaffolds research report has adopted fibronectin, green fluorescent protein (GFP) and ankyrin to repeat (sequence).
Those skilled in the art are known can produce can with certain given protein ligands different piece bonded antibody or its fragment, variant or analogue.For example, can prepare anti-CD 3 antibodies at one of polypeptide chain (that is, γ, δ, ε, ζ and η CD3 chain) that forms this (CD3) mixture.Can be the used preferred anti-CD 3 antibodies of the present composition and method with the antibody of ε CD3 chain combination.
The present invention provides and therapeutic substance bonded dTCR or scTCR on the other hand, and described material is selected from: IL-1, IL-1 α, IL-3, IL-5, IL-6, IL-7, IL-11, IL-12, TGF-β, lymphotoxin, TNF α, anti--CD2 antibody, anti-CD 4 antibodies, anti--CD8 antibody, anti--CD44 antibody, anti--CD45RA antibody, anti--CD45RB antibody, anti--CD45RO antibody, anti--Thy1.2 antibody, antilymphocyte globulin (ALG), anti--α β TCR antibody, anti--gamma delta T CR antibody, anti--CD49a antibody, anti--CD49b antibody, anti--CD49c antibody, anti--CD49d antibody, anti--CD49e antibody, anti--CD49f antibody, anti--TCR V β 8 antibody, anti--CD16 antibody, anti--CD28 antibody, CTLA-4-Ig, anti--B7.2 antibody, anti-CD 40 L antibody, anti--ICAM-1 antibody, ICAM-1, anti--Mac antibody, anti--LFA-1 antibody, anti--the IFN-gamma antibodies, IFN-γ, IFN-γ R/IgG1 syzygy, anti--IL-2R antibody, IL-2R antibody, IL-2 diphtheria (Diptheria)-toxin protein, anti--IL-12 antibody, IL-12 antagonist (p40), anti--IL-1 antibody, the IL-1 antagonist, L-Glutamic decarboxylase (GAD), anti--GAD antibody, viral protein and peptide, bacterioprotein or peptide, A-galactosyl-ceramide, thyrocalcitonin, niacinamide, anti--oxygenant (vitamin-E, the probucol analogue, probucol+deflazacort or aminoguanidine), anti--inflammatory drugs (pentoxifylline or rolipram), immunomodulator (Roquinimex, Ling-zhi-8, the D-dextran, multifunctional protein 14, Ciamexon, cholera toxin B, vanadate or vitamin D 3 analogs, small molecules CD80 inhibitor, male sex hormone, IGF-1, thing (Immunomanipulation) (natural antibody) is controlled in immunity, the lupus idiotype, lipopolysaccharides), sulfatide, bee venom, the Kampo preparation, silica, Sphingolipids,sialo, anti-asialo GM-1 antibody, Unidasa, concanavalin A, anti--I class MHC antibody, or anti--II class MHC antibody, S-Neoral, FK-506, azathioprine, rapamycin or Gusperimus, or the functional variant or the fragment of above-mentioned any material
" anti--the T cell " antibody
One group of preferred immunomodulator of the present invention be can with only by epi-position bonded antibody or its functional fragment or the variant/analogue of T cell or NK cell (NK) presented by cells.Below be the antibody of selectively targeted these cells of energy:
Anti-CD 3 antibodies, anti-CD 4 antibodies, anti--CD8 antibody, anti--α β TCR antibody, anti--CD49a antibody, anti--CD49b antibody, anti--CD49c antibody, anti--CD49d antibody, anti--CD49e antibody, anti--CD49f antibody, anti--gamma delta T CR antibody, anti--TCR V β 8 antibody and anti--CD28 antibody.
Those skilled in the art will know that the T cell and/or the specific subgroup of NK cell of the above antibody of great majority institute target.Have only all NK cells of anti-CD 3 antibodies target and T cell.
This antibody that links to each other with soluble T CR and form the difunctional composition of the present invention can cause T cell and/or NK cellular localization in the cell of expressing soluble T CR homeopeptide-MHC part.Do not think bound by theoryly, these antibody combine with T cell or NK cell can activate these cells.
The present invention provides and therapeutic substance bonded dTCR or scTCR on the other hand, and described therapeutic substance is selected from IL-10, IL-4 or IL-3 or their functional variant or fragment.
One aspect of the present invention provides tissue-specific dTCR or scTCR.In an embodiment aspect this, described dTCR or scTCR have specificity to the tissue as the autoreactive T cell target in autoimmune disease, organ rejection or graft versus host disease (GVH disease) (GVHD).In the particular aspect this, described dTCR or scTCR are that islet cells is specific.T cell clone NY8.3 (Santamaria etc., J.Immunology, (1995), 154 2494-2503 and Nagata etc., (1995), J Immunology, 1522042-2050) and G9C8 (Wong etc., J Exp Med, (1996), 183 67-76) be islet cells specificity mouse T cell clone's example.NY8.3 T cell clone is mouse H2-K dThe specificity clone of G-6-Pase catalytic subunit associated protein (IGRP) derived peptide that MHC presents, G9C8 T cell clone is mouse H2-K dThe specificity clone of the Regular Insulin derived peptide that MHC presents.
The present invention provides and therapeutic substance bonded dTCR or scTCR on the other hand, and described therapeutic substance is selected from IL-15, IL-21, IL-23, PE38 Pseudomonas exotoxin, IFN-γ or anti-CD 3 antibodies or their functional variant or fragment.
In one aspect of the invention, be dTCR with therapeutic substance bonded TCR.In others of the present invention, TCR is scTCR with the therapeutic substance bonded.
It is that TCR of the present invention combines preferred joint with therapeutic substance that two class joints are arranged.By polyalkylene glycol chain and therapeutic substance bonded TCR of the present invention is one of embodiment of this aspect.Peptide linker is another kind of TCR joint.About they application in forming the TCR polymer, hereinafter gone through this two classes joint.The embodiment 6 of this paper provides two kinds of peptide linker examples that can be used for connecting TCR and therapeutic substance.The known various peptide linkers of those skilled in the art are suitable for connecting TCR β chain and required therapeutic substance.Below be other example that can be used for the joint sequence of this purpose:
Its coding of ggcggtccg-Gly-Gly-Pro joint.
Cccggg-its coding comprises the Pro-Gly joint of Xma1 restriction enzyme site.
As mentioned above, the TCR in the TCR therapeutic substance combination described herein partly is a targeting moiety.TCR of the present invention can target TCR part, for example peptide-MHC or CD1-antigenic compound.For example, if compare with the natural TCR of the specificity of this part, higher and/or dissociation rate is an ideal to these TCR more slowly to the avidity of this TCR part.The contriver of application WO 2004/044004 of awaiting the reply has described in detail with the natural TCR of the specificity of this part and has compared, the preparation method of and/or TCR that dissociation rate slower higher to the avidity of this TCR part.This TCR is to the avidity (K of TCR part D) preferably be higher than 1 μ M and/or dissociation rate (k OFF) be preferably lower than 1 * 10 -3S -1More preferably this TCR is to the avidity (K of TCR part D) be higher than 10nM and/or dissociation rate (k OFF) be lower than 1 * 10 -4S -1Most preferably this TCR is to the avidity (K of TCR part D) be higher than 1nM and/or dissociation rate (k OFF) be lower than 1 * 10 -5S -1
Can adopt any known method to detect avidity (K D) and/or dissociation rate (k Off).Preferred method is embodiment 3 described surperficial plasmon resonance (Biacore) methods.
Just in the broadest sense, TCR of the present invention can be strand TCR (scTCR) or dimer TCR (dTCR) form as described in WO 04/033685 and WO 03/020763.
Suitable scTCR form comprises: first section that is made of the aminoacid sequence corresponding to TCR α chain variable region, by second section that the aminoacid sequence corresponding to TCR β chain variable region sequence constitutes, described aminoacid sequence merges with N-terminal corresponding to the aminoacid sequence of the outer sequence of TCR β chain constant region born of the same parents; With the joint sequence that is connected this first section C-terminal and this second section N-terminal.
Perhaps, first section can be made of the aminoacid sequence corresponding to TCR β chain variable region, second section can be made of the aminoacid sequence corresponding to TCR α chain variable region sequence, and described aminoacid sequence merges with N-terminal corresponding to the aminoacid sequence of the outer sequence of TCR α chain constant region born of the same parents.
More particularly, first section can be made of the aminoacid sequence corresponding to TCR α chain variable region sequence, and this aminoacid sequence merges with N-terminal corresponding to the aminoacid sequence of the outer sequence of TCR α chain constant region born of the same parents; Second section can be made of the aminoacid sequence corresponding to TCR β chain variable region sequence, and this aminoacid sequence merges with N-terminal corresponding to the aminoacid sequence of the outer sequence of TCR β chain constant region born of the same parents; And between first and second chains, can there be a disulfide linkage that does not have Equivalent in the natural α β TXi Baoshouti.
In above scTCR form, joint sequence can connect the first section C-terminal and the second section N-terminal, and its sequence is suc as formula-PGGG-(SGGGG) 5-P-(SEQ ID NO:1) or-PGGG-(SGGGG) 6Shown in-the P-(SEQ IDNO:2), wherein P is a proline(Pro), and G is a glycine, and S is a Serine.
The suitable dTCR form of TCR of the present invention contains first polypeptide and second polypeptide, wherein merges corresponding to the sequence of TCR α chain variable region sequence and corresponding to the sequence of N of the outer sequence of TCR α chain constant region born of the same parents is terminal in first polypeptide; Merge corresponding to the sequence of TCR β chain variable region sequence and corresponding to the sequence of N of the outer sequence of TCR β chain constant region born of the same parents is terminal in second polypeptide; This first and second polypeptide links to each other by the disulfide linkage that does not have Equivalent in natural α β TXi Baoshouti.
Described first polypeptide can contain with corresponding to the terminal TCR α chain variable region sequence that merges of the sequence of N of the outer sequence of TCR α chain constant region born of the same parents, and the sequence of second polypeptide corresponding to TCR β chain variable region sequence and with merge corresponding to the sequence of N of the outer sequence of TCR β chain constant region born of the same parents is terminal, this first and second polypeptide by replacing the TRAC*01 exons 1 Thr 48 and cysteine residues or the disulfide linkage between its inhuman Equivalent of the Ser 57 that replaces TRBC1*01 or TRBC2*01 exons 1 link to each other.(terms such as " TRAC " this paper according to " TXi Baoshouti handbook (T cell receptor Factsbook), (2001), LeFranc and LeFranc, Science Press, ISBN 0-12-441352-8 name).
The dTCR of TCR of the present invention or scTCR form can contain the aminoacid sequence corresponding to outer constant region of people α β TCR born of the same parents and variable region sequences, with the amino-acid residue that can be connected described constant region sequence and there is not the disulfide linkage of Equivalent in natural TCR.This disulfide linkage and natural TCR in corresponding its β carbon atom between cysteine residues, for example at the Thr 48 that replaces the TRAC*01 exons 1 with replace between the cysteine residues or its inhuman Equivalent of Ser 57 of TRBC1*01 or TRBC2*01 exons 1 less than the amino-acid residue of 0.6nm.For TCR α chain, other site that can introduce halfcystine formation disulfide linkage is the following residue in the TRAC*01 exons 1, for TCR β chain, is the following residue in TRBC1*01 or the TRBC2*01 exons 1:
TCR α chain TCR β chain Natural β carbon distance (nm)
Thr 45 Tyr 10 Thr 45 Ser 15 Ser 77 Ser 17 Asp 59 Glu 15 0.533 0.359 0.560 0.59
Except above-mentioned non-natural disulfide linkage, contain disulfide linkage between the residue of those residues that the dTCR of TCR of the present invention or scTCR form can connect by disulfide linkage in corresponding to natural TCR.
The sequence of the dTCR of TCR of the present invention or scTCR form does not preferably contain strides film or tenuigenin sequence corresponding to natural TCR.
One embodiment of the invention provide and therapeutic substance bonded TCR, and wherein said therapeutic substance is the PE38 extracellular toxin.
The PE38 extracellular toxin is the clipped form of Pseudomonas exotoxin.This natural polypeptides is the 66kDa protein that is made of structural domain IA, II, IB and III.The PE38 derivative is made of amino acid 380-399 and the domain II I of domain II, structural domain IB.Those skilled in the art understand that the present invention can utilize other clipped form (for example PE40) of Pseudomonas exotoxin.The preferred variants of the used PE38 of the present invention contains sudden change in its domain II I, thereby the C-end amino acid is KDEL.In the past (research) show that the terminal sudden change of these C-has improved the toxicity of Pseudomonas exotoxin.(Kreitman etc., (1995), J Biochem, 307 29-37).
In an embodiment preferred, contain aminoacid sequence shown in (SEQ ID NO:73) and (SEQ ID NO:71) with the described TCR of PE38 extracellular toxin bonded.(seeing Figure 29 b and 28b respectively)
The TCR monomer of PEGization
In one embodiment, the present invention also combines with at least one polyalkylene glycol chain with therapeutic substance bonded TCR.The known many methods of those skilled in the art can cause this combination.In an embodiment preferred, this polyalkylene glycol chain links to each other with the TCR covalency.In another embodiment, the polyalkylene glycol chain of this aspect of the present invention contains at least two polyethylene repeating units.
Multivalence TCR mixture
One aspect of the present invention provides the multivalence TCR mixture that contains with at least two TCR of therapeutic substance bonded.In an embodiment aspect this, at least two TCR molecules link to each other through shank and form the multivalence mixture.This multivalence TCR mixture can link to each other through nonpeptidic polymer chain or peptide linker sequence.This mixture is preferably water miscible, thereby should select shank like this.In addition, shank preferably can with the TCR molecule on clear and definite position link to each other, thereby reduce the structure diversity of the mixture that forms as far as possible.Polymer chain or peptide linker sequence are not being extended between each the TCR amino-acid residue in the TCR variable region sequences in the TCR mixture of the present invention that embodiment provided of this aspect.
Because mixture of the present invention can be used for medical treatment, shank should be considered their pharmacy suitability, and for example their immunogenicity is selected.
Such as the known example that meets the shank of above ideal standard in antibody fragment connection area.
Two class joints are preferred for producing multivalence TCR molecule of the present invention.Wherein TCR provides an embodiment of this aspect by the continuous TCR mixture of the present invention of polyalkylene glycol chain.
The first kind is a hydrophilic polymer, for example polyalkylene glycol.The most frequently used in this base polymer is polyoxyethylene glycol or the PEG that structure is shown below.
HOCH 2CH 2O(CH 2CH 2O) n-CH 2CH 2OH
Wherein n is greater than 2.Yet other polymkeric substance comprises the multipolymer of polypropylene glycol and ethylene glycol and propylene glycol according to other suitable, the optional polyalkylene glycol that replaces.
This polymkeric substance can be used for the PK distribution situation that processing or coupling therapeutic substance, particularly polypeptide or protein therapeutic agent change this therapeutical agent valuably.It is believed that the PEG molecular energy form " shell " around this therapeutical agent thus and spatially hinder therapeutical agent and immune system response and reduce the PK distribution situation that its proteasome degradation has improved PEG-therapeutical agent conjugate.(Casey etc., (2000), Tumor Targetting, 4 235-244).The big I of used hydrophilic polymer is used concrete the selection according to the treatment of TCR mixture expection.Therefore, for example see through when organizing, as when being used for the treatment of tumour, should utilize the low-molecular weight polymer of 5KDa magnitude when this product of needs leaves circulation (system).Existing many survey articles and books are described PEG and the application of similar molecule in pharmaceutical preparation in detail.For example, Harris and Zalipsky, (1997), " chemistry and biology of polyoxyethylene glycol is used ACS handbook (Chemistry and Biological Applications of Polyethylene Glycol ACSBooks), Washington, D.C..
Used polymkeric substance can have linear or branched configurations.Can comprise that glycerine and glycerine oligomer, tetramethylolmethane, sorbyl alcohol and Methionin induce the PEG of branch molecule or derivatives thereof by adding component.
This polymkeric substance for example in its one or both ends, thereby and/or contains the chemical reactivity group this polymkeric substance is linked to each other with target site among the TCR generally in its structure at skeleton side chain place.As follows, this chemical reactivity group can directly link to each other with hydrophilic polymer, and perhaps as follows can have one spacer groups/part between hydrophilic polymer and reactive behavior chemistry (group):
Reactive behavior chemistry (group)-hydrophilic polymer-reactive behavior chemistry (group)
Reactive behavior chemistry (group)-spacer groups-hydrophilic polymer-spacer groups-reactive behavior chemistry (group)
The spacer groups that is used to form the above-mentioned type construction can be reactionless active, chemically stable, any organic moiety of catenate.This spacer groups includes but not limited to following group:
-(CH 2) n-, n=2 to 5 wherein
-(CH 2) 3NHCO(CH 2) 2
The divalent alkyl spacer groups provides another embodiment of this aspect in the multivalence TCR mixture of the present invention between polyalkylene glycol chain and itself and the TCR tie point that has been connected therapeutic substance.
Polyalkylene glycol chain contains at least two polyoxyethylene glycol repeating units in the multivalence TCR mixture of the present invention, and another embodiment of this aspect is provided.
Can utilize various coupling chemistry (reagent) that polymer molecule is coupled to protein and peptide therapeutics.Select only coupling chemistry (reagent) to depend primarily on required coupling site.For example, following coupling chemistry (reagent) (is originated: the one or more ends that Nektar molecular engineering catalogue 2003 (Nektar Molecular Engineering Catalogue 2003)) have been used to connect the PEG molecule: N-maleimide, vinyl sulfone(Remzaol, benzotriazole carbonic ether, succinimide propionic ester (Succinimidyl proprionate), succinimide butyric ester, thioester, acetaldehyde, acrylate, vitamin H and primary amine.
As mentioned above, the non-PEG polymkeric substance multimerization that also can be TCR of the present invention provides suitable joint.For example, can utilize the part that contains the maleimide end that links to each other by aliphatic chain, for example BMH and BMOE (Pierce, production number 22330 and 22323).
Peptide linker is another kind of TCR joint.These joints are made up of amino acid chain, and its effect is to produce simple joint or the multimerization structural domain that is used to connect on the TCR molecule.Once produced with vitamin H/Streptavidin system in the past and be used for the external TCR tetramer (referring to WO/99/60119) in conjunction with research.Yet Streptavidin is microbe-derived polypeptide, therefore is used for the treatment of in the agent undesirable.
TCR is by continuous another embodiment that this aspect is provided of the peptide linker of derived from human multimerization structural domain in the TCR mixture of the present invention.There are many people's albumen that contain the multimerization structural domain to can be used for producing multivalence TCR mixture.For example, compare with monomer scFV fragment, the scFv antibody fragment tetramer that utilizes p53 four dimerization structural domains to be produced has shown that the lasting retention time of its serum increases and the speed of dissociating significantly reduces.(Willuda etc., (2001) J.Biol.Chem.276 (17) 14385-14392).Oxyphorase also has the four dimerization structural domains that possibility is used for such application.
Soluble T CR of the present invention or multivalence TCR mixture can link to each other with the enzyme that prodrug can be converted into medicine.This makes prodrug only change medicine (that is, by the sTCR target) at the position of needs.
Therapeutic is used
The present invention also provides the method that therapeutic substance is delivered to target cell, this method is included in and allows under potential target cell and TCR of the present invention or the multivalence TCR mixture bonded condition the two to be contacted, and described TCR or multivalence TCR mixture are that given peptide-MHC mixture is specific.
Specifically, can utilize soluble T CR of the present invention or multivalence TCR mixture that therapeutic substance is delivered to and to present position, concrete antigenic cell place.This can be used for many situations, when for example resisting tumour or autoimmune disease.The sending of therapeutic substance should apply its effect but not only to working with its bonded cell in the part.
Therefore, a kind of concrete scheme imagination links to each other molecules of immunization stimulus with the TCR of the present invention or the multivalence TCR mixture of specific for tumour antigen.For cancer therapy, the vicinity that is positioned tumour or transfer (tumour) can improve the effect of toxin or immunostimulant.Perhaps, can utilize soluble T CR of the present invention or multivalence TCR mixture immunosuppressor to be delivered to the position, cell place that to present the relevant specific antigen of autoimmune disease.For example, can utilize the islet cells specificity TCR immunosuppressor (as IL-10, IL-4 or IL-13 or their functional variant or fragment) to be delivered to diabetic subject's islet cells.
For vaccine delivery, vaccine antigen can be positioned near the antigen presenting cell, thereby can improve antigenic effectiveness.
Estimate to give patient's Interferon, rabbit (IFN), IFN-γ for example gives can improve with therapeutic substance bonded TCR the expression level of peptide-MHC on the target cell then and/or simultaneously.This is useful especially for the treatment cancer.
Other embodiment of the present invention provides the pharmaceutical composition that contains with therapeutic substance bonded TCR or its multivalence TCR mixture and pharmaceutically acceptable carrier.
The present invention also provides a kind of treatment method for cancer, comprise the object significant quantity of suffering from this cancer with therapeutic substance bonded TCR or its multivalence TCR mixture.In a related embodiment, the invention provides the application in the composition of preparation treatment cancer with therapeutic substance bonded TCR or its multivalence TCR mixture.IL-15, IL-21 or anti-CD 3 antibodies or their functional variant or fragment are the particularly preferred therapeutical agents that is used for the treatment of cancer.
The present invention also provides a kind of method for the treatment of autoimmune disease, organ rejection or GVHD, comprise the object significant quantity of suffering from this autoimmune disease, organ rejection or GVHD with therapeutic substance bonded TCR or its multivalence TCR mixture.In a related embodiment, the invention provides the application in the composition of preparation treatment autoimmune disease, organ rejection or GVHD with therapeutic substance bonded TCR or its multivalence TCR mixture.IL-10, IL-4 or IL-13 or their functional variant or fragment are the particularly preferred therapeutical agents that is used for the treatment of autoimmune disease, organ rejection or GVHD.In another related embodiment, dTCR of the present invention or scTCR are tissue-specific.In other relevant embodiment, described dTCR or scTCR are that the target position of autoreactive T cell is tissue-specific in autoimmune disease, organ rejection or the graft versus host disease (GVH disease) (GVHD).In a particular, the invention provides the method for treatment diabetes, wherein said dTCR or scTCR are that islet cells is specific.
Can comprise from the cancer that the inventive method benefits: leukemia, head, neck (cancer), lung (cancer), mammary gland (cancer), colon (cancer), uterine cervix (cancer), liver (cancer), pancreas (cancer), ovary (cancer) and testis (cancer).
Can comprise following from the autoimmune disease that the inventive method benefits:
Acute disseminated encephalomyelitis,
Adrenal insufficiency,
Allergic angiitis and granuloma (Allergic angiitis and granulomatosis),
Amyloidosis (Amylodosis),
Ankylosing spondylitis,
Asthma,
The autoimmunity bronzed disease,
The autoimmunity alopecia,
ACAH,
Autoimmune hemolytic anemia (Autoimmune haemolytic anaemia),
Autoimmunity neutrophil minimizing (Autoimmune Neutrogena),
Autoimmune thrombocytopenic purpura,
Behet's disease,
Cerebellar degeneration,
Chronic active hepatitis,
Chronic inflammatory demyelination polyneural root sacred disease (Chronic inflammatory demyelinatingpolyradiculoneuropathy),
Have monoclonal gammopathy chronic neuropathic,
Classical polyarteritis nodosa,
Adrenal,congenital hyperplasia,
Cryopathy (Cryopathies),
Dermatitis herpetiformis,
Diabetes,
Yi-Lang syndrome,
Encephalomyelitis,
Epidermolysis bullosa acquisita,
Erythema nodosum,
Gluten susceptibility enteropathy,
Goodpasture's syndrome,
Ge-Ba syndrome,
Hashimoto thyroiditis,
Hyperthyroidism,
Idiopathic hemochromatosis,
The special property sent out membranous glomerulonephritis,
The separation property vasculitis of central nervous system,
Mucocutaneous lymphnode syndrome,
Slight variation ephrosis,
Mix type vasculitis (Miscellaneous vasculitides),
MCTD,
Have block many focuses motor neuropathy (Multifocal motor neuropathy withconduction block),
Multiple sclerosis,
Myasthenia gravis,
Opsoclonus-myoclonic syndrome (Opsoclonus-myoclonus syndrome),
Pemphigoid (Pemphigoid),
Pemphigus (Pemphigus),
Pernicious anemia,
Polymyositis/dermatomyositis,
Infection posterior joint inflammation,
The sclerosis of primary bile duct,
Psoriatic,
Reactive arthritis,
RD,
Retinopathy,
Rheumatoid arthritis,
Sclerosing cholangitis,
Xerodermosteosis,
Stiff-man syndrome,
Subacute thyroiditis,
Systemic lupus erythematous,
Whole body shape necrotizing vasculitis,
Sjogren's syndrome disease (scleroderma),
Aortic arch syndrome,
Temporal arteritis,
Thromboangiitis obliterans,
I type and II type autoimmune polyglandular syndrome,
Ulcerative colitis,
Uveitis,
Wegner granulomatosis
The part that therapeutic composition of the present invention can be used as the Bactericidal medicine composition that comprises pharmaceutically acceptable carrier usually provides.This pharmaceutical composition can be taked any suitable form (depend on and give the patient required method it).This pharmaceutical composition can provide by unit dosage, is contained in usually in the container of sealing, and a part that can be used as test kit provides.Common (though optional) is equipped with working instructions in this test kit.This test kit can be equipped with a plurality of described unit dosage.
This pharmaceutical composition can adopt any suitable way to give, for example parenteral, transdermal or suction, preferred parenteral (comprise subcutaneous, intramuscular, or most preferably intravenously) approach.Can pass through the known any method of pharmaceutical field, for example mixed active composition and vehicle or vehicle prepare this composition under aseptic condition.
Depend on the disease of being treated or illness, individual age and the physical appearance etc. for the treatment of, the dosage range of material of the present invention is very wide, and the doctor can finally determine used suitable dose.
Others
With therapeutic substance bonded scTCR or dTCR (wherein TCR preferably is made of constant region or variable region sequences corresponding to the human sequence) can pure substantially form or provide as purifying or isolating preparation.For example, can be substantially free of other proteinic form provides.
The preferred feature of each side of the present invention is the same with other each side of having done necessary correction.The prior art file that this paper mentions is at utmost included this paper in according to law allowed.
Embodiment
Following examples have further described the present invention, but the scope that does not limit the present invention in any way.
With reference to the following drawings:
Fig. 1 a and 1b have shown the α of solubility A6 TCR and the nucleotide sequence of β chain respectively, and described nucleotide sequence has been introduced a halfcystine codon through sudden change.The halfcystine codon that shadow representation is introduced;
Fig. 2 a has shown the outer aminoacid sequence of the born of the same parents of A6 TCR α chain, and it comprises the T that is used to produce novel interchain disulfide bond 48→ C suddenly change (underscore); Fig. 2 b has shown the outer aminoacid sequence of the born of the same parents of A6 TCR β chain, and it comprises the S that is used to produce novel interchain disulfide bond 57→ C suddenly change (underscore);
Fig. 3 a has shown A6 TCR α chain-ordering, and its novel cysteine residue that comprises sudden change is to mix the BamH1 restriction site.Shadow representation is for forming the sudden change that the BamH1 restriction site is introduced;
Fig. 3 b and 3c have shown through sudden change and have contained the JM22TCR α of the extra cysteine residues that forms the non-natural disulfide linkage and the dna sequence dna of β chain;
Fig. 4 a and 4b have shown aminoacid sequence outside the JM22 TCR α of the generation of dna sequence dna shown in Fig. 3 b and the 3c and β chain born of the same parents respectively;
Fig. 5 a and 5b have shown the α of solubility AH-1.23 TCR and the dna sequence dna of β chain respectively, and this sequence has been introduced a new halfcystine codon (with shadow representation) through sudden change;
Fig. 6 a and 6b have shown aminoacid sequence outside the AH-1.23 TCR α of the generation of dna sequence dna shown in Fig. 5 b and the 5c and β chain born of the same parents respectively;
The dna sequence dna of the sophisticated people IL-10 of Fig. 7 a-;
The aminoacid sequence of the sophisticated people IL-10 of Fig. 7 b-;
Fig. 8 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-10 through the Pro-Gly joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Pro-Gly joint is shown in underscore;
Fig. 8 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-10 through the Pro-Gly joint.The halfcystine of introducing is with shadow representation.The Pro-Gly joint is shown in underscore;
Fig. 9 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-10 through the Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Fig. 9 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-10 through the Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 10 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-10 through Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 10 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-10 through Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
The dna sequence dna of the sophisticated people IL-4 of Figure 11 a-;
The aminoacid sequence of the sophisticated people IL-4 of Figure 11 b-;
Figure 12 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine forms the novel interchain key that sophisticated people IL-4 links to each other through the Pro-Gly joint with participation.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Pro-Gly joint is shown in underscore;
Figure 12 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-4 through the Pro-Gly joint.The halfcystine of introducing is with shadow representation.The Pro-Gly joint is shown in underscore;
Figure 13 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-4 through the Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 13 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-4 through the Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 14 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-4 through Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 14 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-4 through Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
The dna sequence dna of the sophisticated people IL-13 of Figure 15 a-;
The aminoacid sequence of the sophisticated people IL-13 of Figure 15 b-;
Figure 16 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-13 through the Pro-Gly joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Pro-Gly joint is shown in underscore;
Figure 16 b-contains the aminoacid sequence of the AH1.23 TCR β chain of non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-13 through the Pro-Gly joint.The halfcystine of introducing is with shadow representation.The Pro-Gly joint is shown in underscore;
Figure 17 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-13 through the Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 17 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-13 through the Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 18 a-contains the dna sequence dna of the AH1.23 TCR β chain of a non-natural halfcystine, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-13 through Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.The dna sequence dna of coding Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 18 b-contains the aminoacid sequence of the AH1.23 TCR β chain of a non-natural halfcystine codon, and this halfcystine participates in forming the novel interchain key that links to each other with sophisticated people IL-13 through Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint.The halfcystine of introducing is with shadow representation.Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro joint is shown in underscore;
Figure 19 has described the dna sequence dna of pEX821 plasmid in detail;
Figure 20 provides the plasmid figure of pEX821 carrier, and its dna sequence dna is seen Figure 19;
Figure 21 has described the dna sequence dna of pEX954 plasmid in detail;
Figure 22 provides the plasmid figure of pEX954 carrier, and its dna sequence dna is seen Figure 21;
Figure 23 a has described the dna sequence dna of coding high-affinity c61 NY-ESO MTCR β chain in detail, and Figure 23 b has described the aminoacid sequence of dna sequence encoding shown in Figure 23 a in detail;
Figure 24 a has described the dna sequence dna of the high-affinity c61 NY-ESOMTCR β chain that the coding C-terminal links to each other with IL-18 through peptide linker in detail.Figure 24 b described in detail this fusion rotein aminoacid sequence, peptide linker is shown in underscore;
Figure 25 a has described the former proteic dna sequence dna of IL-18 that the coding C-terminal links to each other with high-affinity c61 NY-ESO MTCR β chain through peptide linker in detail.This former-IL-18 DNA is through change and the cleavage site of the factor X that encoded.Figure 25 b described in detail this fusion rotein aminoacid sequence, peptide linker is shown in underscore;
Figure 26 a has described the dna sequence dna of the high-affinity c61 NY-ESOMTCR β chain that the coding C-terminal links to each other with IL-10 through peptide linker in detail.Figure 26 b has described the aminoacid sequence of this fusion rotein in detail, and peptide linker is shown in underscore;
Figure 27 a has described the dna sequence dna of the high-affinity c61 NY-ESOMTCR β chain that the coding C-terminal links to each other with IL-13 through peptide linker in detail.Figure 27 b has described the aminoacid sequence of this fusion rotein in detail, and peptide linker is shown in underscore;
Figure 28 a has described the dna sequence dna of the high-affinity c61 NY-ESO MTCR β chain that the coding C-terminal links to each other through peptide linker ectotoxic with PE38 " KDEL " variant in detail.Figure 28 b has described the aminoacid sequence of this fusion rotein in detail, and peptide linker is shown in underscore;
Figure 29 a has described the dna sequence dna of coding high-affinity c58 NY-ESO MTCR α chain in detail, and Figure 29 b has described the aminoacid sequence of dna sequence encoding shown in Figure 29 a in detail.
The mutagenesis of embodiment 1-design of primers and A6 TAX TCR α and β chain
For the A6 Tax Threonine 48 with the TRAC*01 exons 1 sports halfcystine, designed following primer (sudden change shows with small letter):
5’-C ACA GAC AAA tgT GTG CTA GAC AT(SEQ ID NO：3)
5’-AT GTC TAG CAC Aca TTT GTC TGT G(SEQ ID NO：4)
For the A6 Tax Serine 57 with TRBC1*01 and TRBC2*01 exons 1 sports halfcystine, designed following primer (sudden change shows with lowercase):
5’-C AGT GGG GTC tGC ACA GAC CC(SEQ ID NO：5)
5’-GG GTC TGT GCa GAC CCC ACT G(SEQ ID NO：6)
PCR mutagenesis:
As follows, utilize the sudden change of above-mentioned α-strand primer and beta chain primer to contain the expression plasmid of A6 Tax TCR α or β chain gene respectively.100ng plasmid and 5 μ l 10mM dNTP, 25 μ l, 10 * Pfu-damping fluids (Stratagene), 10 Pfu of unit polysaccharases (Stratagene) are mixed, use H 2O is adjusted to 240 μ l with final volume.Adding primer in 48 these mixtures of μ L, to be diluted to final concentration be 0.2 μ M, and the end reaction volume is 50 μ L.Through 95 ℃, behind 30 seconds the initial denaturing step, reaction mixture carries out sex change (95 ℃, 30 seconds), annealing (55 ℃, 60 seconds) and extends (73 ℃, 8 minutes) with Hybaid PCR fast PCR instrument, and 15 take turns.37 ℃ with 10 DpnI of unit Restriction Enzymes (New England Biolabs) digestion product 5 hours then.The reaction (product) of 10 μ l digestion is transformed in the competence XL1-Blue bacterium, and bacterium was 37 ℃ of growths 18 hours.Choose a bacterium colony, at 5ml TYP+ penbritin (16g/l Bacto-Tryptones, 16g/l yeast extract, 5g/l NaCl, 2.5g/l K 2HPO 4, the 100mg/l penbritin) in grow overnight.According to manufacturer's working instructions with the little preparative column plasmid DNA purification of Qiagen, automatic sequencing checking sequence.Fig. 1 a and 2a and Fig. 1 b and 2b have shown the nucleotide sequence and the aminoacid sequence of α chain and the sudden change of β chain respectively.
Embodiment 2-soluble T CR expression, refolding and purifying
Be transformed among intestinal bacteria (E.coli) the bacterial strain BL21pLysS containing the α-chain of sudden change and the expression plasmid of beta chain respectively, 37 ℃ with TYP (Ampicillin Trihydrate 100 μ g/ml) the culture medium culturing Ampicillin Trihydrate single bacterium colony of resistance to OD 600Be 0.4, express with 0.5mM IPTG induced protein then.Induce back 3 hours with the BeckmanJ-6B whizzer with centrifugal 30 minutes collecting cells of 4000rpm.Cell precipitation is resuspended in and contains 50mMTris-HCl, 25% (w/v) sucrose, 1mM NaEDTA, 0.1% (w/v) sodium azide, 10mM DTT, in the damping fluid of pH 8.0.After freeze thawing was spent the night, the 12mm diameter probe with standard in Milsonix XL2020 ultrasonoscope carried out 1 minute explosive ultrasonication to re-suspended cell, about 10 minutes altogether.Reclaimed the inclusion body throw out with Beckman J2-21 whizzer in centrifugal 30 minutes with 13000rpm.Remove cell debris and membrane component 3 times with detergent washing then.At every turn with inclusion body at Triton damping fluid (50mM Tris-HCl, 0.5%Triton-X100,200mM NaCl, 10mM NaEDTA, 0.1% (w/v) sodium azide, 2mM DTT, pH 8.0) middle homogenate, use Beckman J2-21 whizzer with 13000rpm centrifugation 15 minutes then.Carry out similar washing to remove washing composition and salt with following damping fluid then: 50mM Tris-HCl, 1mM NaEDTA, 0.1% (w/v) sodium azide, 2mM DTT, pH 8.0.At last, inclusion body is divided into the 30mg equal portions ,-70 ℃ freezing.With the dissolving of 6M guanidine-HCl and with the Bradford dyestuff in conjunction with the protein yield of testing (PerBio) quantitative assay inclusion body.
The sex change of soluble T CR: from freezing reserve, melt 30mg dissolved TCR β chain inclusion body and 60mg dissolved TCR α chain inclusion body.With 6M guanidine solution inclusion body being diluted to final concentration is 5mg/ml, add DTT (2 M storing solution) to final concentration be 10mM.Mixture was cultivated 30 minutes at 37 ℃.
The refolding of soluble T CR: 1L refolding damping fluid is 5 ℃ ± 3 ℃ vigorous stirring.Add redox couple reagent (2-mercaptoethylamine and cystamine) to final concentration and be respectively 6.6mM and 3.7mM, add the TCR chain of sex change after about 5 minutes.5 ℃ ± 3 ℃ stir abouts 5 hours ± 15 minutes are with this protein of refolding.
The soluble T CR of dialysis refolding: the TCR of refolding is with Spectrapor 1 film (Spectrum; Production number .132670) dialysed 18-20 hour in 5 ℃ ± 3 ℃ with 10L 10mM Tris, pH 8.1.Be fresh 10mM Tris pH 8.1 (10L) then with the dialysis buffer fluid exchange, continue 5 ℃ ± 3 ℃ dialysis 20-22 hour.
The Biacore surface plasmon resonance characteristics of embodiment 3-and specificity pMHC bonded sTCR is identified
(BIAcore 3000 with surperficial plasmon resonance biological sensor TM) analyze the situation that combines of sTCR and its peptide-MHC part.Single pMHC mixture (being described in hereinafter) that preparation is fixed in the semi-directional mode on the mating surface of Streptavidin bag quilt helps this analysis, thus tested effectively soluble T-cell receptor and 4 kinds of different pMHC (being fixed on the different flow cell) nearly in conjunction with situation.Manually inject the accurate level that the HLA mixture can easily be controlled fixed I quasi-molecule.
This fixed mixture can be injected into the two the solution phase simultaneously in conjunction with T-cell receptors and accessory receptor CD8 α α.Even also can obtain the specificity combination of TCR at lower concentration (at least 40 μ g/ml), TCR is more stable for hint.If adopt solution mutually or the sTCR of stationary phase, the pMHC binding characteristic of observing sTCR in quality with quantitatively similar.This part activity to control solubility (TCR) kind is very important, and the biologic activity of also pointing out biotinylation pMHC mixture is identical with abiotic elementization mixture.
Biotinylation I class HLA-A2-peptide complex in the inclusion body that contains composing type protein subunit and synthetic peptide of external refolding bacterial expression, carry out behind the purifying vitro enzyme (catalysis) biotinylation (O ' Callaghan etc., (1999), Anal.Biochem.266:9-15).The HLA-heavy chain of expressing contains and has replaced this proteinic terminal biotinylation label of C-of striding film and cytoplasmic structure territory in the suitable construction.The inclusion body expression level is about 75mg/ and rises bacterial cultures.HLA light chain or B2M also rise inoculum with about 500mg/ in intestinal bacteria horizontal expression is the inclusion body of suitable construction.
The cracking intestinal bacteria, with inclusion body purification to about 80% pure.The protein of inclusion body 6M guanidine-HCl, 50mM Tris pH 8.1,100mM NaCl, 10mM DTT, 10mM EDTA sex change, by denatured protein being added in the refolding damping fluid with pulsatile once＜5 ℃ the time, with 30mg/ rise concentration that heavy chain, 30mg/ rise β 2m 0.4M L-arginine-HCl, 100mM Tris pH 8.1,3.7mM cystamine, mM cysteamine, 4mg/ml peptide (for example, tax11-19) in refolding.Refolding is carried out 1 hour side at least at 4 ℃ and is finished.
10mM Tris pH 8.1 dialysis exchange buffering liquid with 10 times of volumes.For fully reducing the ionic strength of this solution, damping fluid need be changed twice.Then protein soln is passed through 1.5 μ m rhodia membrane filtrations, application of sample is (bed volume 8ml) on POROS 50HQ anion-exchange column.With 0-500mM NaCl linear gradient liquid elute protein.The HLA-A2-peptide complex about 250mM NaCl place wash-out, is collected all peaks component greatly, adds protease inhibitor cocktail (Calbiochem), and each component refrigerates on ice.
Is 10mM Tris pH 8.1,5mM NaCl in order to the Pharmacia quick desalination post of same buffer pre-equilibration with the buffer exchange of the HLA mixture of biotinylation mark.To contain proteinic component behind the wash-out immediately and refrigerate, add protease inhibitor cocktail (Calbiochem) on ice.Add biotinylation reagent then: 1mM vitamin H, 5mM ATP (being buffered to pH 8), 7.5mM MgCl 2With 5 μ g/ml BirA enzymes (according to O ' Callaghan etc., (1999), Anal.Biochem.266:9-15 purifying).Then mixture is cultivated in room temperature and spent the night.
Adopt the HLA mixture of gel permeation chromatography purifying biological elementization.With filtering PBS pre-equilibration Pharmacia Superdex 75 HR 10/30 post, add 1ml biotinylation reaction mixture, use PBS with 0.5ml/ minute wash-out.Biotinylated HLA mixture is as the about 15ml of unimodal wash-out.Merge and contain proteinic component,, add protease inhibitor cocktail in refrigeration on ice.Adopt coomassie to measure protein concn in conjunction with test (PerBio), biotinylated HLA mixture is divided into sample aliquot and is kept at-20 ℃.The amine coupling method of employing standard is Streptavidin fixedly.
At BIAcore 3000 TMAnalysis contains the A6 Tax sTCR of novel interchain key and the interaction between its part/MHC mixture or the incoherent HLA-peptide combination (above-mentioned product) on surface plasmon resonance (SPR) biosensor.SPR can detect near the variation of the refractive index the sensor surface in the small flow chamber, and this changes with reacton (RU) expression, and this principle can be used for detecting receptor-ligand interaction and analyzes their avidity and kinetic parameter.Each HLA-peptide complex is fixed on prepares the probe flow cell in the different flow cell by crosslinked vitamin H and the keying action between the Streptavidin (the two is crosslinked in the activating surface of flow cell with chemical process) on β 2m.Make sTCR flow through the surface of different flow cell then, measure SPR when doing like this again and react and carry out this test with constant flow rate.At first make sTCR flow through two different surfaces and verify this interactional specificity with the constant flow rate of 5 μ l per minutes; Surface specific peptide-HLA mixture bag quilt of about 5000RU, second the non-specific peptide with about 5000RU-HLA mixture bag quilt.Available constant flow rate injects sTCR with different concentration and compares to determine background resonance with peptide-HLA mixture.The detected value that deducts these contrasts from the numerical value that obtains with specific peptide-HLA mixture calculates binding affinity (Price and the Dwek that represents with dissociation constant Kd, " biochemist's physical and chemical principle and problem " (Principles andProblems in Physical Chemistry for Biochemists), (second edition), 1979, Clarendon Press, the Oxford).
The value that the Kd value (1.8 μ M) that obtains is reported near interacting between A6 Tax sTCR that does not contain novel disulfide linkage and the pMHC (0.91 μ M-Ding etc., 1999, Immunity, 11:45-56).
Embodiment 4-preparation contains the solubility JM22 TCR of novel disulfide linkage
The β chain of the solubility A6 TCR of embodiment 1 preparation contains the native sequences BglII restriction site (AAGCTT) that is suitable as connection site.
The following detailed description in detail carried out 5 ' the locating of α chain novel cysteine codon that solubility A6TCR is introduced BamH1 restriction site (GGATCC) in PCR mutagenesis.Embodiment 1 described sequence is as the template of this mutagenesis.Use following primer:
BamHI
5’-ATATCCAGAACCCgGAtCCTGCCGTGTA-3’(SEQ ID NO：7)
5’-TACACGGCAGGAaTCcGGGTTCTGGATAT-3’(SEQ ID NO：8)
100ng plasmid and 5 μ l 10mM dNTP, 25 μ l, 10 * Pfu-damping fluids (Stratagene), 10 Pfu of unit polysaccharases (Stratagene) are mixed, use H 2O is adjusted to 240 μ l with final volume.Adding primer in 48 these mixtures of μ L, to be diluted to final concentration be 0.2 μ M, and the end reaction volume is 50 μ L.Through 95 ℃, behind 30 seconds the initial denaturing step, the sex change that reaction mixture carries out with Hybaid PCR fast PCR instrument (95 ℃, 30 seconds), annealing (55 ℃, 60 seconds) and extension (73 ℃, 8 minutes) 15 are taken turns totally.Then at 37 ℃ with 10 DpnI of unit Restriction Enzymes (New England Biolabs) digestion product 5 hours.10 μ l digestion reactions (product) are transformed in the competence XL1-Blue bacterium, and bacterium was 37 ℃ of growths 18 hours.Choose a bacterium colony, at 5ml TYP+ penbritin (16g/l Bacto-Tryptones, 16g/l yeast extract, 5g/l NaCl, 2.5g/lK 2HPO 4, the 100mg/l penbritin) in grow overnight.With the little preparative column plasmid DNA purification of Qiagen, automatic sequencing is verified this sequence according to manufacturer's working instructions.The sudden change of introducing the α chain is " silence " sudden change, thus the aminoacid sequence of this chain and Fig. 2 a describe in detail compare no change.Fig. 3 a has shown the dna sequence dna of the α chain of this sudden change.
Mixed the solubility JM22 TCR of novel disulfide linkage for generation, the A6 TCR plasmid that will contain α chain BamH1 and β chain BglII restriction site is as template.Used following primer:
|NdeI|
5’-GGAGATATACATATGCAACTACTAGAACAA-3’(SEQ ID NO：9)
5’-TACACGGCAGGATCCGGGTTCTGGATATT-3’(SEQ ID NO：10)
|BamHI|
|Nde1|
5’-GGAGATATACATATGGTGGATGGTGGAATC-3’(SEQ ID NO：11)
5’-CCCAAGCTTAGTCTGCTCTACCCCAGGCCTCGGC-3’(SEQ ID NO：12)
|BglII|
As follows, obtain JM22 TCR α and beta chain construction by the PCR clone.Utilize above-mentioned primer and the template that contains JM22 TCR chain to carry out the PCR reaction.With relevant this PCR product of Restriction Enzyme restrictive diges-tion, it is cloned into pGMT7 obtains expression plasmid.The sequence of this plasmid inset of automated DNA sequence verification.Fig. 3 b and 3c have shown the sudden change α of JM22 TCR and the dna sequence dna of β chain respectively, and Fig. 4 a and 4b have shown the aminoacid sequence that obtains.
As expression as described in embodiment 1 and 2, folded together and each TCR chain of purifying.
As described in embodiment 3, carry out JM22 TCR and pMHC bonded Biacore analysis.The TCR that this disulfide linkage of HLA-flu mixture connects its Kd after measured is 7.9 ± 0.51 μ M.
Embodiment 5-preparation contains the solubility AH-1.23 TCR of novel interchain disulfide bond
The T cellular segregation that provides from Hill Gaston (Medical School, Addenbrooke ' s Hospital, Cambridge) according to known technology obtain the encoding cDNA of AH-1.23 TCR.Handle this HiRNA obtain encoding cDNA of NY-ESO TCR with reversed transcriptive enzyme.
As described in embodiment 4, mixed the solubility AH-1.23 TCR of novel disulfide linkage for generation, the TCR plasmid that will contain α chain BamHI and β chain BglII restriction site is as framework.Used following primer:
|NdeI|
5’-GGGAAGCTTACATATGAAGGAGGTGGAGCAGAATTCTGG-3’(SEQ ID
NO：13)
5’-TACACGGCAGGATCCGGGTTCTGGATATT-3’(SEQ ID NO：14)
|BamHI|
|NdeI|
5’-TTGGAATTCACATATGGGCGTCATGCAGAACCCAAGACAC-3(SEQ ID
NO：15)
5’-CCCAAGCTTAGTCTGCTCTACCCCAGGCCTCGGC-3’(SEQ ID NO：16)
|BglII|
As follows, carry out the PCR clone and obtain AH-1.23 TCR α and beta chain construction.Utilize above-mentioned primer and the template that contains AH-1.23 TCR chain to carry out the PCR reaction.With relevant Restriction Enzyme restrictive diges-tion PCR product, it is cloned into pGMT7 obtains expression plasmid.Sequence by this plasmid inset of automated DNA sequence verification.Fig. 5 a and 5b have shown the sudden change α of AH1.23 TCR and the dna sequence dna of β chain respectively, and Fig. 6 a and 6b have shown the aminoacid sequence that obtains.
As expression as described in the embodiment 2, folded together and each TCR chain of purifying.
Embodiment 6-prepares solubility AH-1.23 TCR-IL-10 fusion rotein
Can prepare then comprise the one-tenth acquaintance IL-10 dna sequence dna that Fig. 7 a described in detail and be positioned at IL-10 dna sequence dna 5 ' end many DNA extension it-synthetic gene.This 5 ' end DNA extension is the joint sequence that is used to connect IL-10DNA and coding AH1.23 TCR β chain.
Joint sequence:
Cccggg-its coding contains the Pro-Gly joint of Xma1 restriction enzyme site
Its coding of ggatccggcggtccg-(SEQ ID NO:17) contains Gly-Ser-Gly-Gly-Pro (the SEQ ID NO:18) joint of BamHI restriction enzyme site.
Its coding of ggatccggtgggggcggaagtggaggcagcggtggatccggcggtccg-(SEQ ID NO:19) contains Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro (the SEQ ID NO:20) joint of two BamH1 restriction enzyme sites.
Then one of above-mentioned synthetic gene subclone is gone in the pGMT7 plasmid that contains AH1.23 TCR β chain as preparation as described in the embodiment 5, thereby form the dna sequence dna of coding TCR β chain-joint-IL-10 fusion rotein.
Fig. 8 a and 8b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Pro-Gly-IL-10 syzygy respectively in detail.
Fig. 9 a and 9b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Gly-Ser-Gly-Gly-Pro (SEQ ID NO:18)-IL-10 syzygy respectively in detail.
Figure 10 a and 10b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro (SEQ ID NO:20)-IL-10 syzygy respectively in detail.
Thereby the method that adopts embodiment 2 to describe in detail then makes these AH1.23 TCR β chain-joint-IL-10 fusion roteins and the refolding of AH1.23 TCR α chain produce complete solubility AH1.23 TCR-IL-10 fusion rotein.
More than the method for Xiang Shuing has been described the C-end for preparing TCR β chain and has been connected with the monomeric solubility α β of IL-10 TCR.Common IL-10 is the homodimer form.Therefore, make the IL-10 polypeptide dimerization possibility that links to each other with solubility AH1.23 TCR useful.This can realize in many ways.For example, the one-tenth acquaintance IL-10 homodimer and the TCR β of single stranded form can be merged, then with the refolding of TCR α chain.Perhaps, can be in solution the people IL-10 of mature form be added TCR β chain-IL-10 fusion rotein according to above-mentioned formation,, perhaps add the α β TCR-IL-10 fusion rotein of refolding then with the refolding of soluble T CR α chain.Perhaps, can adopt the described method of present embodiment that other IL-10 molecule is added TCR α chain formation fusion rotein and produce TCR β chain-IL-10 fusion rotein.Can adopt embodiment 2 described methods to make two kinds of TCR chains-IL-10 fusion rotein refolding together then.At last, can contain two TCR mixture of (respectively containing an IL-10 polypeptide that links to each other with TCR β chain) by the homologous dimerization formation of IL-10 polypeptide.This will cause the mixture of following type:
α β TCR-IL-10 homodimer-α β TCR.
Embodiment 7-prepares solubility AH-1.23 TCR-IL-4 and AH-1.23 TCR-IL-13 fusion rotein
Also can adopt embodiment 6 described method preparations to contain the fusion rotein of the solubility AH-1.23 TCR that links to each other with other polypeptide.
Can make up the synthetic gene that comprises one of 5 ' cited end DNA extension sequence of one-tenth acquaintance IL-4 dna sequence dna that Figure 11 a described in detail and embodiment 6, its subclone is gone in the pGMT7 plasmid that contains AH1.23TCR β chain as preparation as described in the embodiment 5, thereby form the dna sequence dna of coding TCR β chain-joint-IL-4 fusion rotein.
Figure 12 a and 12b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Pro-Gly-IL-4 syzygy respectively in detail.
Figure 13 a and 13b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Gly-Ser-Gly-Gly-Pro-(SEQ ID NO:18)-IL-4 syzygy respectively in detail.
Figure 14 a and 14b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro (SEQ ID NO:20)-IL-4 syzygy respectively in detail.
Can make up the synthetic gene that comprises one of 5 ' cited end DNA extension sequence of one-tenth acquaintance IL-13 dna sequence dna that Figure 15 a described in detail and embodiment 6, its subclone is gone in the pGMT7 plasmid that contains AH1.23TCR β chain as preparation as described in the embodiment 5, thereby form the dna sequence dna of coding TCR β chain-joint-IL-4 fusion rotein.
Figure 16 a and 16b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Pro-Gly-IL-13 syzygy respectively in detail.
Figure 17 a and 17b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Gly-Ser-Gly-Gly-Pro-(SEQ ID NO:18)-IL-13 syzygy respectively in detail.
Figure 18 a and 18b have described the dna sequence dna and the aminoacid sequence of AH1.23 TCR β chain-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Pro (SEQ ID NO:20)-IL-13 syzygy respectively in detail.
Thereby the method that adopts embodiment 2 to describe in detail then makes these AH1.23 TCR β chain-joint-interferon fusion proteins and the refolding of AH1.23 TCR α chain produce complete solubility AH1.23 TCR-interferon fusion protein.
Embodiment 10-assessment AH1.23 TCR-IL-10 fusion rotein causes the thymidine of mastocyte multiplication capacity to mix test
In the presence of IL-4 will to people IL-10 react can breed 5 * 10 6The cell cultures that individual D36 mouse hypertrophy cell is is in RPMI 1640 substratum.As a series of AH1.23TCR-IL-10 fusion roteins of preparation (0,0.01,0.1,0.5 and 1 μ M) as described in the embodiment 9, IL-4 is added above-mentioned substratum.(Schlaak etc., (1994), J Immunological Methods, 168 49-54)
Then, at 1 * 10 of 96 orifice plates 5The H that adds 1.85MBq/ml in the individual above-mentioned culturing cell 3Thymidine.37 ℃, 5%CO 2Cultivated these cultures again 8 hours down.Utilize cell harvesting machine (cell-harvester) collecting cell, utilize Top Count β counter to detect the thymidine level of mixing cell.
Finding is compared when not having fusion rotein, and AH1.23 TCR-IL-10 fusion rotein mixes the D36 cell when existing thymidine reduces, and shows that the IL-10 of this fusion rotein partly has activity, can cause D36 mastocyte propagation.
Embodiment 11-prepares high affinity ny-eso MTCR-therapeutic substance fusion rotein
The synthetic gene that synthesizes the dna sequence dna of the coding solubility high-affinity c61 NY-ESO TCR β chain that contains Figure 23 a detailed description, the dna sequence dna of its encoded peptide linker links to each other with the DNA of the many immunomodulators of coding.
There are many companies that suitable DNA service, for example Geneart (Germany) are provided.
Figure 24 a has described the dna sequence dna of the high-affinity c61 NY-ESOMTCR β chain that the coding C-terminal links to each other with IL-18 through peptide linker in detail.Figure 24 b described in detail this fusion rotein aminoacid sequence, peptide linker is shown in underscore.
Figure 25 a has described the former proteic dna sequence dna of IL-18 that the coding C-terminal links to each other with high-affinity c61 NY-ESO MTCR β chain through peptide linker in detail.Former-IL-18 dna sequence dna helps to remove some amino acid whose factor X cleavage sites in this former sequence after the translation through changing with coding.Figure 25 b described in detail this fusion rotein aminoacid sequence, peptide linker is shown in underscore.
Figure 26 a has described the dna sequence dna of the high-affinity c61 NY-ESOMTCR β chain that the coding C-terminal links to each other with IL-10 through peptide linker in detail.Figure 26 b has described the aminoacid sequence of this fusion rotein in detail, and peptide linker is shown in underscore.
Figure 27 a has described the dna sequence dna of the high-affinity c61 NY-ESOMTCR β chain that the coding C-terminal links to each other with IL-13 through peptide linker in detail.Figure 27 b has described the aminoacid sequence of this fusion rotein in detail, and peptide linker is shown in underscore.
Figure 28 a has described the dna sequence dna of the high-affinity c61 NY-ESO MTCR β chain that the coding C-terminal links to each other through peptide linker ectotoxic with PE38 " KDEL " variant in detail.Figure 28 b has described the aminoacid sequence of this fusion rotein in detail, and peptide linker is shown in underscore.
The dna sequence dna of above c61 NY-ESO TCR β chain can be connected into the pEX821 carrier.(dna sequence dna of this carrier and plasmid figure see Figure 19 and 20 respectively)
Basically prepare the α β TCR-therapeutic substance that disulfide linkage connects according to embodiment 2 described methods then.In brief, the dna sequence dna of the coding high-affinity c61 NY-ESO MTCR α chain that composite diagram 29a describes in detail connects the carrier into pEX954.(dna sequence dna of this carrier and plasmid figure see Figure 21 and 22 respectively) be the above-mentioned TCR β of refolding chain fusion protein in the presence of c61 NY-ESO TCR α chain then.
Figure 29 a has described the dna sequence dna of coding high-affinity c58 NY-ESO MTCR α chain in detail, and Figure 29 b has described the aminoacid sequence of dna sequence encoding shown in Figure 29 a in detail.
The cell toxicity test of embodiment 12-MTCR-PE-38 fusion rotein
With 1 * 10 -6Individual required target cell (for example SK-MEL tumour cell or J82 cells) is suspended in 10mlRPMI substratum+10% foetal calf serum (FCS).If desired, 37 ℃ with 10 these target cells of μ M related peptides pulse 2 hours.Use the RPMI+10%FCS washing sample then three times, centrifugal 5 minutes of each time washing room 1200rpm.Again the cell of counting washing and be resuspended in the RPMI+10%FCS substratum of suitable volumes and obtain 2 * 10 then 5The final cell density of individual cell/ml.
To be diluted to 2 * 10 as the MTCR-PE38 fusion rotein of preparation as described in the embodiment 11 with RPMI substratum+10%FCS -6The M final concentration obtains working standard.Utilize this working standard to prepare a series of serial dilutions then.
Preparation experiment and control sample in the microtitre plate hole:
In the laboratory sample hole, inject the mTCR-PE38 of 50 μ l substratum preparation and the cell of 50 μ l substratum preparation.For in the flat White-opalescent orifice plates in 96 holes (Nunc 136101), obtaining the cumulative volume of 100 μ l.Utilize the mTCR-PE38 serial dilution of above preparation that a series of mTCR-PE38 concentration are provided in these holes.
Utilize 100 μ l cells (having only the contrast of cell) or 100 μ l mTCR-PE38 and substratum (having only effector cell's contrast) preparation control sample hole.
Then at 37 ℃, 5%CO 2Cultivate experiment and control sample 48 or 96 hours.Working instructions according to the manufacturer adopt CellTiter-Glo then Luminous test (Promega catalog number (Cat.No.): G7572) assess the viable cell number that retains in each hole.
The result
Figure 30 a and 30b show that 1G4 MTCR-PE38 fusion rotein can kill and wound NY-ESO +SK-MEL 37 and Mel 624 tumor cell lines.
The data computation that provides from Figure 30 a goes out 1G4 MTCR-PE38 fusion rotein respectively with 5.9 * 10 -9With 1 * 10 -8M cultivates after 48 hours the EC50 value that SK-MEL 37 and Mel 624 tumor cell lines are renderd a service.
The data computation that provides from Figure 30 b goes out 1G4 MTCR-PE38 fusion rotein respectively with 5.7 * 10 -9With 2.1 * 10 -8M cultivates after 96 hours the EC50 value that SK-MEL 37 and Mel 624 tumor cell lines are renderd a service.
Figure 30 a all proves with the result that 30b provides and uses observed the comparing of J82 target cell that is not subjected to pulse, causes NY-ESO TCR-PE38 construction more effectively to kill and wound these cells with relevant SLLMWITQC NY-ESO peptide pulse J82 target cell.

Claims (37)

  1. One kind with therapeutic substance bonded dimer TCR (dTCR) or strand TCR (scTCR), wherein said material is selected from: IL-1, IL-1 α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-β, IFN-γ, lymphotoxin, TNF α, anti--CD2 antibody, anti-CD 3 antibodies, anti-CD 4 antibodies, anti--CD8 antibody, anti--CD44 antibody, anti--CD45RA antibody, anti--CD45RB antibody, anti--CD45RO antibody, anti--Thy 1.2 antibody, antilymphocyte globulin (ALG), anti--α β TCR antibody, anti--gamma delta T CR antibody, anti--CD49a antibody, anti--CD49b antibody, anti--CD49c antibody, anti--CD49d antibody, anti--CD49e antibody, anti--CD49f antibody, anti--TCR V β 8 antibody, anti--CD16 antibody, anti--CD28 antibody, CTLA-4-Ig, anti--B7.2 antibody, anti-CD 40 L antibody, anti--ICAM-1 antibody, ICAM-I, anti--Mac antibody, anti--LFA-1 antibody, anti--the IFN-gamma antibodies, IFN-γ, IFN-γ R/IgG1 syzygy, anti--IL-2R antibody, IL-2R antibody, IL-2 diphtheria-toxin protein, anti--IL-12 antibody, IL-12 antagonist (p40), anti--IL-1 antibody, the IL-1 antagonist, L-Glutamic decarboxylase (GAD), anti--GAD antibody, viral protein and peptide, bacterioprotein or peptide, A-galactosyl-ceramide, thyrocalcitonin, niacinamide, anti--oxygenant (vitamin-E, the probucol analogue, probucol+deflazacort or aminoguanidine), anti--inflammatory drugs (pentoxifylline or rolipram), immunomodulator (linomide, Ling-zhi-8, the D-dextran, multifunctional protein 14, Ciamexon, cholera toxin B, vanadate or vitamin D 3 analogs, small molecules CD80 inhibitor, male sex hormone, IGF-1, thing (natural antibody) is controlled in immunity, the lupus idiotype, lipopolysaccharides), sulfatide, bee venom, the Kampo preparation, silica, Sphingolipids,sialo, anti-asialo GM-1 antibody, Unidasa, concanavalin A, anti--I class MHC antibody, or anti--II class MHC antibody, S-Neoral, FK-506, azathioprine, rapamycin or Gusperimus, the PE38 Pseudomonas exotoxin, wherein said TCR comprises:
    By first section that the aminoacid sequence corresponding to TCR α variable region sequences constitutes, this aminoacid sequence merges with aminoacid sequence N-terminal corresponding to the outer sequence of TCR α chain constant region born of the same parents;
    By second section that the aminoacid sequence corresponding to TCR β variable region sequences constitutes, this aminoacid sequence merges with aminoacid sequence N-terminal corresponding to the outer sequence of TCR β chain constant region born of the same parents;
    A disulfide linkage is arranged between this first and second chain, and described disulfide linkage does not have Equivalent in natural α β TXi Baoshouti;
    And, also contain at described scTCR under the situation of the joint sequence that connects this first section C-terminal and this second section N-terminal, or under the situation that vice versa, the length of this joint sequence and the position of this disulfide linkage make variable region sequences directed each other basically as natural α β TXi Baoshouti of described first and second sections.
  2. 2. as claimed in claim 1 and therapeutic substance bonded dTCR or scTCR, it is characterized in that described therapeutic substance is selected from: IL-1, IL-1 α, IL-3, IL-5, IL-6, IL-7, IL-11, IL-12, TGF-β, lymphotoxin, TNF α, anti--CD2 antibody, anti-CD 4 antibodies, anti--CD8 antibody, anti--CD44 antibody, anti--CD45RA antibody, anti--CD45RB antibody, anti--CD45RO antibody, anti--Thy 1.2 antibody, antilymphocyte globulin (ALG), anti--α β TCR antibody, anti--gamma delta T CR antibody, anti--CD49a antibody, anti--CD49b antibody, anti--CD49c antibody, anti--CD49d antibody, anti--CD49e antibody, anti--CD49f antibody, anti--TCR V β 8 antibody, anti--CD16 antibody, anti--CD28 antibody, CTLA-4-Ig, anti--B7.2 antibody, anti-CD 40 L antibody, anti--ICAM-1 antibody, ICAM-1, anti--Mac antibody, anti--LFA-1 antibody, anti--the IFN-gamma antibodies, IFN-γ, IFN-γ R/IgG1 syzygy, anti--IL-2R antibody, IL-2R antibody, IL-2 diphtheria-toxin protein, anti--IL-12 antibody, IL-12 antagonist (p40), anti--IL-1 antibody, the IL-1 antagonist, L-Glutamic decarboxylase (GAD), anti--GAD antibody, viral protein and peptide, bacterioprotein or peptide, A-galactosyl-ceramide, thyrocalcitonin, niacinamide, anti--oxygenant (vitamin-E, the probucol analogue, probucol+deflazacort or aminoguanidine), anti--inflammatory drugs (pentoxifylline or rolipram), immunomodulator (Roquinimex, Ling-zhi-8, the D-dextran, multifunctional protein 14, Ciamexon, cholera toxin B, vanadate or vitamin D 3 analogs, small molecules CD80 inhibitor, male sex hormone, IGF-1, thing (natural antibody) is controlled in immunity, the lupus idiotype, lipopolysaccharides), sulfatide, bee venom, the Kampo preparation, silica, Sphingolipids,sialo, anti-asialo GM-1 antibody, Unidasa, concanavalin A, anti--I class MHC antibody, or anti--II class MHC antibody, S-Neoral, FK-506, azathioprine, rapamycin or Gusperimus.
  3. 3. as claimed in claim 1 and therapeutic substance bonded dTCR or scTCR is characterized in that described therapeutic substance is one of IL-10, IL-4 or IL-13.
  4. 4. described and therapeutic substance bonded dTCR or scTCR as above each claim is characterized in that described dTCR or scTCR are tissue-specific.
  5. 5. as claimed in claim 4 and therapeutic substance bonded dTCR or scTCR, it is characterized in that described dTCR or scTCR have tissue specificity to the target position tissue of autoreactive T cell in autoimmune disease, organ rejection or the graft versus host disease (GVH disease) (GVHD).
  6. 6. as claim 4 or 5 described and therapeutic substance bonded dTCR or scTCR, it is characterized in that described dTCR or scTCR are that islet cells is specific.
  7. 7. as claimed in claim 1 and therapeutic substance bonded dTCR or scTCR is characterized in that described therapeutic substance is selected from one of IL-15, IL-21, IL-23, PE38 Pseudomonas exotoxin, IFN-γ or anti-CD 3 antibodies.
  8. As above each claim described with therapeutic substance bonded dTCR.
  9. As above each claim described with therapeutic substance bonded scTCR.
  10. 10. as claimed in claim 9 and therapeutic substance bonded scTCR is characterized in that described joint sequence connects the C-terminal of described first section and the N-terminal of described second section.
  11. 11. as claimed in claim 9 and therapeutic substance bonded scTCR is characterized in that, described joint sequence is suc as formula-PGGG-(SGGGG) nShown in-the P-, wherein n is 5 or 6, and P is a proline(Pro), and G is a glycine, and S is a Serine.
  12. 12. as claimed in claim 8 and therapeutic substance bonded dTCR is characterized in that described dTCR contains:
    First polypeptide is wherein corresponding to the sequence of TCR α chain variable region sequence and corresponding to the TCR α chain constant region born of the same parents sequence N-terminal fusion of sequence outward; With
    Second polypeptide is wherein corresponding to the sequence of TCR β chain variable region sequence and corresponding to the TCR β chain constant region born of the same parents sequence N-terminal fusion of sequence outward;
    This first and second polypeptide connects by a disulfide linkage that does not have Equivalent in natural α β TXi Baoshouti.
  13. 13. as claimed in claim 8 and therapeutic substance bonded TCR is characterized in that described TCR is dTCR, it contains:
    First polypeptide, wherein corresponding to the sequence of TCR α chain variable region sequence and corresponding to the TCR α chain constant region born of the same parents sequence N-terminal fusion of sequence outward,
    Second polypeptide, wherein corresponding to the sequence of TCR β chain variable region sequence and corresponding to the TCR β chain constant region born of the same parents sequence N-terminal fusion of sequence outward,
    This first and second polypeptide by replacing the TRAC*01 exons 1 Thr 48 and a cysteine residues or the disulfide linkage between its inhuman Equivalent of the Ser 57 that replaces TRBC1*01 or TRBC2*01 exons 1 link to each other.
  14. 14. as above each claim is described and therapeutic substance bonded TCR, it is characterized in that, the aminoacid sequence of described dTCR or scTCR is corresponding to outer constant region of the born of the same parents of people α β TCR and variable region sequences.
  15. 15. as claimed in claim 14 and therapeutic substance bonded TCR is characterized in that, a disulfide linkage that does not have Equivalent in natural TCR connects the amino-acid residue of described constant region sequence.
  16. 16. as claimed in claim 15 and therapeutic substance bonded TCR is characterized in that, described disulfide linkage in corresponding to natural TCR contained β carbon atom between cysteine residues less than the amino-acid residue of 0.6nm.
  17. 17. as claimed in claim 15 and therapeutic substance bonded TCR, it is characterized in that described disulfide linkage is between the cysteine residues or its inhuman Equivalent of the Ser 57 of the Thr 48 that replaces the TRAC*01 exons 1 and replacement TRBC1*01 or TRBC2*01 exons 1.
  18. 18. as above each claim described with therapeutic substance bonded TCR, it is characterized in that, described dTCR or scTCR with natural TCR in contain a disulfide linkage between the corresponding residue of residue that disulfide linkage is connected.
  19. 19. as above each claim is described and therapeutic substance bonded TCR, it is characterized in that described dTCR or scTCR do not contain the sequence of striding film or tenuigenin sequence corresponding to natural TCR.
  20. 20. as above each claim is described and therapeutic substance bonded TCR, it is characterized in that described therapeutic substance is the PE38 extracellular toxin.
  21. 21. as claimed in claim 20 and PE38 extracellular toxin bonded TCR is characterized in that it contains aminoacid sequence shown in (SEQ ID NO:73) and (SEQ ID NO:71).
  22. 22. as above each claim is described and therapeutic substance bonded TCR, it is characterized in that described TCR combines with at least one polyalkylene glycol chain.
  23. 23. as claimed in claim 22 and therapeutic substance bonded TCR is characterized in that described one or more polyalkylene glycol chain links to each other with the TCR covalency.
  24. 24., it is characterized in that described one or more polyalkylene glycol chain contains at least two polyoxyethylene glycol repeating units as claim 22 or 23 described and therapeutic substance bonded TCR.
  25. 25. a multivalence TCR mixture, its contain at least two as above each claim described with therapeutic substance bonded TCR.
  26. 26. a multivalence TCR mixture, it contains at least two as each described and therapeutic substance bonded TCR among the claim 1-24, and described at least two TCR link to each other by nonpeptidic polymer chain or peptide linker sequence.
  27. 27. multivalence TCR mixture as claimed in claim 26, it is characterized in that, described polymer chain or peptide linker sequence and the amino-acid residue of therapeutic substance or its functional variant or each TCR of fragment bonded between extend, but be not arranged in the variable region sequences of TCR.
  28. 28. as claim 26 or 27 described multivalence TCR mixtures, it is characterized in that, describedly link to each other by the peptide linker of polyalkylene glycol chain or derived from human multimerization structural domain with therapeutic substance bonded TCR.
  29. 29. multivalence TCR mixture as claimed in claim 28 is characterized in that, has been connected between the tie point of TCR of therapeutic substance to have the divalent alkyl spacer groups in polyalkylene glycol chain and itself and mixture.
  30. 30., it is characterized in that described polyalkylene glycol chain contains at least two polyoxyethylene glycol repeating units as claim 28 or the described multivalence TCR mixture of claim 29.
  31. 31. a pharmaceutical composition, it contains just like each described and therapeutic substance bonded TCR among the claim 1-24 or as each described its multivalence mixture among the claim 25-30, and pharmaceutically acceptable carrier.
  32. 32. treatment method for cancer, it comprise the object significant quantity of suffering from described cancer as each is described with therapeutic substance bonded TCR or as each described its multivalence mixture among the claim 25-30 among the claim 1-24, wherein said therapeutic substance be selected from claim 2 defined those.
  33. 33. among the claim 1-24 each described and therapeutic substance bonded TCR or as the purposes of each described its multivalence mixture among the claim 25-30 in preparing cancer treatment compositions, wherein said therapeutic substance be selected from claim 2 defined those.
  34. 34. a treatment method for cancer, it comprise the object significant quantity of suffering from described cancer as claim 20 or 21 described fusion roteins.
  35. 35. claim 20 or the 21 described fusion roteins purposes in the preparation cancer treatment compositions.
  36. 36. method for the treatment of autoimmune disease, organ rejection or GVHD, it comprise among the object of suffering from autoimmune disease, organ rejection or GVHD such as the claim 1-24 each described and therapeutic substance bonded TCR or as among the claim 25-30 each described more than its valency mixtures, wherein said therapeutic substance be selected from claim 3 defined those.
  37. 37. each described and therapeutic substance bonded TCR are used for the treatment of purposes in the composition of autoimmune disease, organ rejection or GVHD in preparation among the claim 1-24, wherein said therapeutic substance be selected from claim 3 defined those.
CNA2005800393813A 2004-10-01 2005-09-29 T-cell receptors containing a non-native disulfide interchain bond linked to therapeutic agents Pending CN101061138A (en)

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GB0421836.8 2004-10-01
GBGB0421836.8A GB0421836D0 (en) 2004-10-01 2004-10-01 T cell receptors containing a non-native disulfide interchain bond linked to immunomodulatory agents
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GB0427584.8 2004-12-16

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101250575B (en) * 2008-03-07 2011-09-14 秋生堂生物科技（北京）有限公司 Method for preparing vanadium-containing peptides and products produced thereby
CN104159923A (en) * 2012-01-13 2014-11-19 乌利班-马克西姆利安大学 Dual antigen-induced bipartite functional complementation
WO2017201635A1 (en) * 2016-05-23 2017-11-30 蔡胜和 Cellular expression of hyaluronidase and use thereof in solid tumour cell therapy
CN108003237A (en) * 2016-11-01 2018-05-08 中国科学院广州生物医药与健康研究院 The Humanized anti-human CD3 single-chain antibodies and its preparation method of high stability and application

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101250575B (en) * 2008-03-07 2011-09-14 秋生堂生物科技（北京）有限公司 Method for preparing vanadium-containing peptides and products produced thereby
CN104159923A (en) * 2012-01-13 2014-11-19 乌利班-马克西姆利安大学 Dual antigen-induced bipartite functional complementation
CN104159923B (en) * 2012-01-13 2018-01-23 乌利班-马克西姆利安大学 The difunctional complementation of double antigen inductions
WO2017201635A1 (en) * 2016-05-23 2017-11-30 蔡胜和 Cellular expression of hyaluronidase and use thereof in solid tumour cell therapy
CN108003237A (en) * 2016-11-01 2018-05-08 中国科学院广州生物医药与健康研究院 The Humanized anti-human CD3 single-chain antibodies and its preparation method of high stability and application
CN108003237B (en) * 2016-11-01 2023-03-10 中国科学院广州生物医药与健康研究院 High-stability humanized anti-human CD3 single-chain antibody and preparation method and application thereof

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