CN101084014A - Single domain antibodies against tnfr1 and methods of use therefor - Google Patents

Single domain antibodies against tnfr1 and methods of use therefor Download PDF

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Publication number
CN101084014A
CN101084014A CN200580042042.0A CN200580042042A CN101084014A CN 101084014 A CN101084014 A CN 101084014A CN 200580042042 A CN200580042042 A CN 200580042042A CN 101084014 A CN101084014 A CN 101084014A
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tar2h
seq
dab
tnfr1
monomer
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N·D·布鲁易斯
B·P·伍尔文
S·霍尔姆斯
I·M·汤林森
J·李
C·埃尼弗
A·巴斯兰
K·琼斯
R·德维尔德特
S·布莱恩
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Domantis Ltd
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Domantis Ltd
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Priority claimed from PCT/GB2004/004253 external-priority patent/WO2005035572A2/en
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Abstract

The invention provides methods for treating inflammatory diseases (e.g., chronic inflammatory diseases) comprising administering an antagonist of Tumor Necrosis Factor Receptor 1. The invention also provides ligands that contain an immunoglobulin single variable domain (domain antibody, dAb) monomer that binds Tumor Necrosis Factor Receptor 1, and methods of using the ligands. Also provided are nucleic acids encoding the ligands, recombinant host cells and methods for preparing the ligands.

Description

The single domain antibody of anti-tumor necrosis factor receptor 1 and using method thereof
Related application
The application is that it is in the part continuation application of the Application No. 10/985,847 of application on November 10th, 2004:
1) in the part continuation application of the international patent application no PCT/GB2004/004253 (designated state is the U.S.) of on October 8th, 2004 application; With
2) in the part continuation application of the international patent application no PCT/GB2003/005646 (designated state is the U.S.) of December in 2003 application on the 24th, and require in the Britain application number GB 0230202.4 of December in 2002 application on the 27th with in the priority of the Britain application number GB 0327706.8 of application on November 28th, 2003, it is
In the part continuation application of the international patent application no PCT/GB2003/002804 (designated state is the U.S.) of on June 30th, 2003 application, and require in the priority of the Britain application GB 0230202.4 of December in 2002 application on the 27th, it is
Part continuation application in the international patent application no PCT/GB02/03014 (designated state is the U.S.) of on June 28th, 2002 application.
Whole disclosures of above-mentioned application all are attached to herein by reference.
Background of invention
The antigen binding domain of antibody comprises two independent districts: variable region of heavy chain (V H) and variable region of light chain (V L: can be V κ or V λ).Antigen binding site itself is made of six polypeptide rings: 3 from V HDistrict (H1, H2 and H3), 3 from V LDistrict (L1, L2 and L3).Coding V HDistrict and V LThe changeable elementary storehouse (diverse primary repertoire) of the V gene in district is reset and is produced by the constant gene segment C combination.V HGene is by V H, D and J HThree constant gene segment C reorganization produce.The mankind, 51 functional V are arranged approximately HSection (Cook and Tomlinson (1995) Immunol Today, 16:237), 25 functional D sections (Corbett etc. (1997) J.Mol.Biol., 268:69) and 6 functional J H(Ravetch etc. (1981) Cell, 27:583), this depends on haplotype (haplotype) to section.V HSection coded polypeptide sequence constitutes V HThe first and second antigen coupling collars (H1 and H2) in district, and V H, D and J HSection is combined to form V HThe antigen iii coupling collar (H3) in district.V LGene is only by V LAnd J LTwo constant gene segment C reorganization produce.The mankind, have approximately 40 functional V κ sections (Sch  ble and Zachau (1993) Biol.Chem.Hoppe-Seyler, 374:1001), 31 functional V λ sections (Williams etc. (1996) J.Mol.Biol., 264:220; Kawasaki etc. (1997) Genome Res., 7:250), 5 functional J κ section (Hieter etc. (1982) J.Biol.Chem., 257:1516) (Vasicek and Leder (1990) J.Exp.Med., 172:609), this depends on haplotype with 4 functional J λ sections.V LSection coded polypeptide sequence constitutes V LThe first and second antigen coupling collars (L1 and L2) in district, and V LAnd J LSection is combined to form V LThe antigen iii coupling collar (L3) in district.It is believed that the antibody of selecting from this elementary storehouse has enough multiformity, making can be with medium at least affinity in conjunction with nearly all antigen.High-affinity antibody is produced by " affinity maturation " of resetting gene, wherein produces point mutation and selects according to improved combination by immune system.
The analysis of antagonist structure and sequence shows have 5 (H1, H2, L1, L2, L3) to have the limited main chain conformation of number or canonical structure (Chothia and Lesk (1987) J.Mol.Biol., 196:901 in 6 antigen coupling collars; Chothia etc. (1989) Nature, 342:877).The main chain conformation come to be determined by following 2: (i) length of antigen coupling collar and the (ii) specific residue or the residue kind of certain key position in antigen coupling collar and the antibody framework.Ring length and Key residues are analyzed, and let us can be predicted main chain conformation (Chothia etc. (1992) J.Mol.Biol., the 227:799 by the coded H1 of most of human sequence antibodies, H2, L1, L2 and L3; Tomlinson etc. (1995) EMBO J., 14:4628; Williams etc. (1996) J.Mol.Biol., 264:220).Although H3 district multiformity on sequence, length and structure much higher (because having used the D section), but it also constitutes the main chain conformation of the limited becate length of number, this depends on the length of specific residue on the ring and the key position of antibody framework and whether exists, or residue kind (Martin etc. (1996) J.Mol.Biol., 263:800; Shirai etc. (1996) FEBS Letters, 399:1).
Comprise V HDistrict and V LThe bi-specific antibody of district's complementary pair is known in the art.These bi-specific antibodys must comprise two couples of V HAnd V L, every couple of V H/ V LIn conjunction with antigen or epi-position.This method relates to hybrid hybridoma (Milstein and Cuello AC, Nature 305:537-40), miniantibody (minibody) (Hu etc. (1996) Cancer Res 56:3055-3061), double-stranded antibody (diabody) (Holliger etc. (1993) Proc.Natl.Acad.Sci.U.S.A., 90,6444-6448; WO 94/13804), chelating recombinant antibodies (CRAb; (Neri etc. (1995) J.Mol.Biol.246,367-373), biscFv ((1996) Mol.Immunol.33 such as Atwell for example, 1301-1312), antibody that " pestle mortar structure (knobs in holes) " is stable (Carter etc. (1997) Protein Sci.6,781-788).Under multiple situation, every kind of antibody all comprises two antigen binding sites, each free V HDistrict and V LDistrict's complementary pair forms.Therefore, each antibody capable is simultaneously in conjunction with two not synantigen or epi-positions, and is by V with combining of EACH antigen or epi-position HAnd complementary V LDistrict's mediation.The each have their own shortcoming of these technology; For example with regard to the hybrid hybridoma, non-activity V H/ V LTo can greatly reducing bispecific IgG part.In addition, most of bispecific methods depend on different V H/ V LRight association or V HChain and V LThe association of chain is to obtain two different V H/ V LBinding site.Therefore, ratio that can not control binding site and each antigen or epi-position in the assembling molecule is so many assembling molecular energies are in conjunction with antigen or epi-position, but not in conjunction with other antigen or epi-position.In some cases, can transform the subunit interface (Carter etc., 1997) of heavy chain or light chain, two antigens or epi-position are had the molecular amounts of binding site, but this but can not make all molecules can both be in conjunction with two antigens or epi-position so that improve.
Some evidence shows that two different antibody binding specificities can be incorporated into same binding site, but two or more specificitys of these general proxies, and described specificity is corresponding to the antigen or the epi-position of structurally associated or have the antibody of extensive cross reaction.For example cross reacting antibody is existing describes, usually, relevant on sequence and structure (for example hen egg-white lysozyme and turkey lysozyme (McCafferty etc. of two antigen wherein, WO 92/01047)), perhaps corresponding to free hapten and hapten (the Griffiths AD etc. that are conjugated on the carrier, EMBO J.1994,13:14 3245-60).In another example, WO 02/02773 (AbbottLaboratories) has described the antibody molecule with " bispecific ".Described antibody molecule is meant the antibody that produces or select at multiple antigen, makes their specificity cross over more than a kind of antigen.The complementary V of in the antibody of WO 02/02773 each H/ V LRight, have unijunction at two or more structurally associated antigens and close specificity; V in such complementary pair HDistrict and V LThe district does not have independent specificity separately.Therefore, antibody has at two antigenic monospecifics widely of structurally associated.In addition, the NAA of multiple reactionness is existing to be described (Casali and Notkins, Ann.Rev.Immunol., 7,515-531), can react with the not synantigen or the epi-position of at least two (more usually) structure-irrelevants.Also know, adopt display technique of bacteriophage that monoclonal antibody is carried out the selection of random peptide library, will identify the peptide sequence of a series of suitable antigen binding sites.Some sequence is a height correlation, is fit to consensus sequence, and other sequence is then very different, be called mimic epitope (mimotope) (Lane and Stephen, Current Opinion in Immunology, 1993,5,268-271).Therefore, obviously natural four chain antibodies comprise connecting and complementary V HDistrict and V LThe district has and the bonded potentiality of many not synantigens interior, and these antigens are from various known antigens.Still not clear in same antibody, how the generation at two given antigenic binding sites, especially when they might not be structurally associated.
It is relevant with these aspects that protein engineering method has shown.For example, also known and can produce catalytic antibody, it has by a variable region and the bonded activity of metal ion, and have by contacting with complementary variable region and the bonded activity of hapten (substrate) (Barbas etc. with metal ion, 1993, Proc.Natl.Acad.Sci.U.S.A., 90,6385-6389).Yet in this case, think and the combining and catalysis of substrate (first antigen), need with the combining of metal ion (second antigen).Therefore, with V H/ V LRight combination relates to one pack system antigen rather than multi-component antigen.
Existing description of method from camel heavy chain of antibody single domain produces bi-specific antibody produces the first antigenic combination contact a variable region in this method, produces second antigenic in conjunction with contact in second variable region.Yet the variable region is not complementary.Therefore, at first selection of antigen, first variable region of heavy chain, at second selection of antigen, second variable region of heavy chain, again these two districts are connected on the same chain together, obtain bispecific antibody fragment (Conrath etc., J.Biol.Chem.270,27589-27594).Yet the camel single domain heavy chain is thundering, because they derive from the natural camel antibody that does not have light chain, and single domain heavy chain can't combine the complementary V of formation with the camel light chain HAnd V LRight.
Also described single variable region of heavy chain, derived from natural antibody, be connected with light chain (from monoclonal antibody or from the domain storehouse usually; Referring to EP-A-0368684).Know that these variable region of heavy chaines and one or more related antigen have specificity and interact, but can not be in conjunction with other heavy chain or variable region of light chain, with produce to two or more not synantigen have specific part.In addition, know that the half-life is very short in the body of these single domains.Therefore, the therapeutic value of these domains is limited.
Know, link together, can prepare bispecific antibody fragment (as mentioned above) by having not homospecific variable region of heavy chain.The shortcoming of this method is exactly: the isolated antibody variable region may have hydrophobic interfaces, and this interface is usually with the light chain interaction and be exposed to solvent, and is " viscosity ", makes single domain to combine with hydrophobic surface.In addition, when the gametophyte light chain does not exist, the combination of two or more different heavy chains variable regions and combination (may pass through its hydrophobic interfaces) thereof can stop rather than two in their binding partners, and they can be in conjunction with two parts when separating.In addition, in this case, variable region of heavy chain will not combine with complementary variable region of light chain, thus stability decreases and separate easily folding (Worn and Pluckthun, 1998, Biochemistry 37,13120-7).
Summary of the invention
The present invention relates to the using method of tumor necrosis factor 1 (TNFR1, p55, CD120a, P60, TNF receptor superfamily member 1A, TNFRSF1A) antagonist and described antagonist.Preferred antagonist treatment, suppress or the prevention chronic inflammatory disease in effectively, and antagonism tumor necrosis factor 2 (TNFR2, P75, P80, CD120b, TNF receptor superfamily member 1B, TNFRSF1B) not basically.In certain embodiments, described antagonist is monovalent.
In other embodiments, described antagonist is antibody or its Fab, for example TNFR1 is had the monovalent antigen binding fragment (for example scFv, Fab, Fab ', dAb) of binding specificity.
Other preferred antagonist be as herein described can be in conjunction with the part of TNFR1.Part comprise to TNFR1 have the immunoglobulin list variable region of binding specificity or domain antibodies (singlevariable domain or domain antibody, dAb), or the complementary determining region of the suitable form of described dAb.In certain embodiments, described part is the dAb monomer, is made up of the immunoglobulin list variable region or the dAb that TNFR1 are had binding specificity, perhaps is made up of it basically.In other embodiments, described part is the polypeptide of the dAb (or CDR of dAb) that comprises suitable form (for example antibody formation).
In certain embodiments, described part is the bispecific part, and it comprises can be in conjunction with the dAb and the 2nd dAb with binding specificity different with a dAb of TNFR1.In an example, comprise can be in conjunction with a dAb of last first epi-position of TNFR1 and can be in conjunction with the 2nd dAb of the epi-position on the different targets for the bispecific part.In another example, the 2nd dAb can be in conjunction with the epi-position on the serum albumin.
In other embodiments, described part is the polyspecific part, it comprise first epi-position that TNFR1 is had a binding specificity in conjunction with territory and at least one other epi-position in conjunction with the territory, the latter has and is different from the binding specificity of first epi-position in conjunction with the territory.For example, first epi-position can be dAb in conjunction with TNFR1 in conjunction with the territory, perhaps can be to comprise (for example to be transplanted to the CDR on suitable protein support or the skeleton in conjunction with the domain of the CDR of the dAb of TNFR1, for example affine body (affibody), SpA support, ldl receptor category-A district or EGF district), perhaps can be the domain in conjunction with TNFR1, wherein said domain be selected from affine body, SpA district, ldl receptor category-A district or EGF district.
In certain embodiments, described part or dAb monomer have one or more following features: the 1) dissociation constant (K that disintegrates down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -12) suppress tumor necrosis factor (TNF α) and the bonded IC of TNFR1 50Be 500nM～50pM; 3) in standard L929 raji cell assay Raji, in and the ND of people TNFR1 50Be 500nM～50pM; 4) in standard cell lines is measured, the active ND of antagonism TNFR1 50≤ 100nM, in described mensuration, when concentration≤10 μ M, the activity of exciting TNFR1≤5%; 5) Y-suppressed lethal rate in the septic shock model that mice LPS/D-galactosamine brings out; 6) anti-aggregation; 7) when expressing in escherichia coli (E.coli) or Pichia sp. (for example pichia pastoris phaff (P.pastoris)), its secretory volume is at least about 0.5mg/L; 8) can reversibly separate folding; 9) effective in being selected from following chronic inflammatory disease model: the inflammatory bowel model that collagen-induced arthritis model, mouse arthritis Δ ARE model, mice inflammatory bowel Δ ARE model, the mice dextran sulfate sodium of mice brought out, mice caused by smoking chronic obstructive pulmonary disease model and suitable primates model (for example collagen-induced arthritis model of primates); And/or 10) effective in treatment, inhibition or prevention chronic inflammatory disease.
In specific embodiment, the dissociation constant (K that described part or dAb monomer disintegrate down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1Suppress tumor necrosis factor (TNF α) and the bonded IC of TNFR1 50Be 500nM～50pM; In standard L929 raji cell assay Raji, in and the ND of people TNFR1 50Be 500nM～50pM.In other specific embodiment, the dissociation constant (K that described part or dAb monomer disintegrate down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1Suppress tumor necrosis factor (TNF α) and the bonded IC of TNFR1 50Be 500nM～50pM; Effective in being selected from following chronic inflammatory disease model: the inflammatory bowel model that collagen-induced arthritis model, mouse arthritis Δ ARE model, mice inflammatory bowel Δ ARE model, the mice dextran sulfate sodium of mice brought out, mice caused by smoking chronic obstructive pulmonary disease model and suitable primates model (for example collagen-induced arthritis model of primates).In other specific embodiment, the dissociation constant (K that described part or dAb monomer disintegrate down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1In standard L929 raji cell assay Raji, in and the ND of people TNFR1 50Be 500nM～50pM; In standard cell lines is measured, the active ND of antagonism TNFR1 50≤ 100nM, in described mensuration, when concentration≤10 μ M, the activity of exciting TNFR1≤5%.
In a more particular embodiment, the monomeric aminoacid sequence of described part or dAb has at least about 90% homology: TAR2h-12 (SEQID NO:32) with the aminoacid sequence that is selected from following dAb, TAR2h-13 (SEQ ID NO:33), TAR2h-14 (SEQ ID NO:34), TAR2h-16 (SEQ ID NO:35), TAR2h-17 (SEQ ID NO:36), TAR2h-18 (SEQIDNO:37), TAR2h-19 (SEQ ID NO:38), TAR2h-20 (SEQ ID NO:39), TAR2h-21 (SEQ ID NO:40), TAR2h-22 (SEQ ID NO:41), TAR2h-23 (SEQ ID NO:42), TAR2h-24 (SEQ ID NO:43), TAR2h-25 (SEQ ID NO:44), TAR2h-26 (SEQ ID NO:45), TAR2h-27 (SEQ ID NO:46), TAR2h-29 (SEQ ID NO:47), TAR2h-30 (SEQ ID NO:48), TAR2h-32 (SEQ ID NO:49), TAR2h-33 (SEQ ID NO:50), TAR2h-10-1 (SEQ ID NO:51), TAR2h-10-2 (SEQ ID NO:52), TAR2h-10-3 (SEQ IDNO:53), TAR2h-10-4 (SEQ ID NO:54), TAR2h-10-5 (SEQ ID NO:55), TAR2h-10-6 (SEQ ID NO:56), TAR2h-10-7 (SEQ ID NO:57), TAR2h-10-8 (SEQ ID NO:58), TAR2h-10-9 (SEQ ID NO:59), TAR2h-10-10 (SEQ ID NO:60), TAR2h-10-11 (SEQ ID NO:61), TAR2h-10-12 (SEQ ID NO:62), TAR2h-10-13 (SEQ ID NO:63), TAR2h-10-14 (SEQ ID NO:64), TAR2h-10-15 (SEQ ID NO:65), TAR2h-10-16 (SEQ ID NO:66), TAR2h-10-17 (SEQ ID NO:67), TAR2h-10-18 (SEQ ID NO:68), TAR2h-10-19 (SEQ ID NO:69), TAR2h-10-20 (SEQ ID NO:70), TAR2h-10-21 (SEQ ID NO:71), TAR2h-10-22 (SEQ ID NO:72), TAR2h-10-27 (SEQ ID NO:73), TAR2h-10-29 (SEQ ID NO:74), TAR2h-10-31 (SEQ ID NO:75), TAR2h-10-35 (SEQ ID NO:76), TAR2h-10-36 (SEQ ID NO:77), TAR2h-10-37 (SEQ ID NO:78), TAR2h-10-38 (SEQ ID NO:79), TAR2h-10-45 (SEQ ID NO:80), TAR2h-10-47 (SEQ ID NO:81), TAR2h-10-48 (SEQ ID NO:82), TAR2h-10-57 (SEQ ID NO:83), TAR2h-10-56 (SEQ ID NO:84), TAR2h-10-58 (SEQ ID NO:85), TAR2h-10-66 (SEQ ID NO:86), TAR2h-10-64 (SEQ ID NO:87), TAR2h-10-65 (SEQ ID NO:88), TAR2h-10-68 (SEQ ID NO:89), TAR2h-10-69 (SEQ ID NO:90), TAR2h-10-67 (SEQ ID NO:91), TAR2h-10-61 (SEQ ID NO:92), TAR2h-10-62 (SEQ ID NO:93), TAR2h-10-63 (SEQ ID NO:94), TAR2h-10-60 (SEQ ID NO:95), TAR2h-10-55 (SEQ ID NO:96), TAR2h-10-59 (SEQ ID NO:97), TAR2h-10-70 (SEQ ID NO:98), TAR2h-34 (SEQ ID NO:373), TAR2h-35 (SEQ ID NO:374), TAR2h-36 (SEQ ID NO:375), TAR2h-37 (SEQ ID NO:376), TAR2h-38 (SEQ ID NO:377), TAR2h-39 (SEQID NO:378), TAR2h-40 (SEQ ID NO:379), TAR2h-41 (SEQ ID NO:380), TAR2h-42 (SEQ ID NO:381), TAR2h-43 (SEQ ID NO:382), TAR2h-44 (SEQ ID NO:383), TAR2h-45 (SEQ ID NO:384), TAR2h-47 (SEQ ID NO:385), TAR2h-48 (SEQ ID NO:386), TAR2h-50 (SEQID NO:387), TAR2h-51 (SEQ ID NO:388), TAR2h-66 (SEQ ID NO:389), TAR2h-67 (SEQ ID NO:390), TAR2h-68 (SEQ ID NO:391), TAR2h-70 (SEQ ID NO:392), TAR2h-71 (SEQ ID NO:393), TAR2h-72 (SEQ ID NO:394), TAR2h-73 (SEQ ID NO:395), TAR2h-74 (SEQID NO:396), TAR2h-75 (SEQ ID NO:397), TAR2h-76 (SEQ ID NO:398), TAR2h-77 (SEQ ID NO:399), TAR2h-78 (SEQ ID NO:400), TAR2h-79 (SEQ ID NO:401) and TAR2h-15 (SEQ ID NO:431).
In other embodiments, the monomeric aminoacid sequence of described part or dAb has at least about 90% homology: TAR2h-131-8 (SEQID NO:433) with the aminoacid sequence that is selected from following dAb, TAR2h-131-24 (SEQ ID NO:434), TAR2h-15-8 (SEQ IDNO:435), TAR2h-15-8-1 SEQ ID NO:436), TAR2h-15-8-2 (SEQ ID NO:437), TAR2h-185-23 (SEQ ID NO:438), TAR2h-154-10-5 (SEQ ID NO:439), TAR2h-14-2 (SEQ ID NO:440), TAR2h-151-8 (SEQ ID NO:441), TAR2h-152-7 (SEQ ID NO:442), TAR2h-35-4 (SEQ ID NO:443), TAR2h-154-7 (SEQ ID NO:444), TAR2h-80 (SEQ ID NO:445), TAR2h-81 (SEQ ID NO:446), TAR2h-82 (SEQ ID NO:447), TAR2h-83 (SEQ ID NO:448), TAR2h-84 (SEQ ID NO:449), TAR2h-85 (SEQID NO:450), TAR2h-86 (SEQ ID NO:451), TAR2h-87 (SEQ ID NO:452), TAR2h-88 (SEQ ID NO:453), TAR2h-89 (SEQ ID NO:454), TAR2h-90 (SEQ ID NO:455), TAR2h-91 (SEQ ID NO:456), TAR2h-92 (SEQ ID NO:457), TAR2h-93 (SEQ ID NO:458), TAR2h-94 (SEQID NO:459), TAR2h-95 (SEQ ID NO:460), TAR2h-96 (SEQ ID NO:461), TAR2h-97 (SEQ ID NO:462), TAR2h-99 (SEQ ID NO:463), TAR2h-100 (SEQ ID NO:464), TAR2h-101 (SEQ ID NO:465), TAR2h-102 (SEQ ID NO:466), TAR2h-103 (SEQ ID NO:467), TAR2h-104 (SEQ ID NO:468), TAR2h-105 (SEQ ID NO:469), TAR2h-106 (SEQ ID NO:470), TAR2h-107 (SEQ ID NO:471), TAR2h-108 (SEQ ID NO:472), TAR2h-109 (SEQ ID NO:473), TAR2h-110 (SEQ ID NO:474), TAR2h-111 (SEQ ID NO:475), TAR2h-112 (SEQ ID NO:476), TAR2h-113 (SEQ ID NO:477), TAR2h-114 (SEQ ID NO:478), TAR2h-115 (SEQ ID NO:479), TAR2h-116 (SEQ ID NO:480), TAR2h-117 (SEQ ID NO:481), TAR2h-118 (SEQ ID NO:482), TAR2h-119 (SEQ ID NO:483), TAR2h-120 (SEQ ID NO:484), TAR2h-121 (SEQ ID NO:485), TAR2h-122 (SEQ ID NO:486), TAR2h-123 (SEQ ID NO:487), TAR2h-124 (SEQ ID NO:488), TAR2h-125 (SEQ ID NO:489), TAR2h-126 (SEQ ID NO:490), TAR2h-127 (SEQ ID NO:490), TAR2h-128 (SEQ ID NO:492), TAR2h-129 (SEQ ID NO:493), TAR2h-130 (SEQ ID NO:494), TAR2h-131 (SEQ ID NO:495), TAR2h-132 (SEQ ID NO:496), TAR2h-133 (SEQ ID NO:497), TAR2h-151 (SEQ ID NO:498), TAR2h-152 (SEQ ID NO:499), TAR2h-153 (SEQ ID NO:500), TAR2h-154 (SEQ ID NO:501), TAR2h-159 (SEQ ID NO:502), TAR2h-165 (SEQ ID NO:503), TAR2h-166 (SEQ ID NO:504), TAR2h-168 (SEQ ID NO:505), TAR2h-171 (SEQ ID NO:506), TAR2h-172 (SEQ ID NO:507), TAR2h-173 (SEQ ID NO:508), TAR2h-174 (SEQ ID NO:509), TAR2h-176 (SEQ ID NO:510), TAR2h-178 (SEQ ID NO:511), TAR2h-201 (SEQ ID NO:512), TAR2h-202 (SEQ ID NO:513), TAR2h-203 (SEQ ID NO:514), TAR2h-204 (SEQ ID NO:515), TAR2h-185-25 (SEQ ID NO:516), TAR2h-154-10 (SEQ ID NO:517) and TAR2h-205 (SEQ ID NO:627).
The present invention relates to tumor necrosis factor 1 (TNFR1) antagonist, described antagonist can and suppress signal transduction by TNFR1 in conjunction with tumor necrosis factor 1 (TNFR1), and wherein said antagonist does not suppress combining of TNF α and TNFR1.In certain embodiments, described antagonist comprises first domain antibodies (dAb) monomer and the 2nd dAb monomer, a wherein said dAb monomer can be in conjunction with the TNFR1 territory that is selected from territory 1, territory 2, territory 3 and territory 4, described the 2nd dAb monomer can be in conjunction with the TNFR1 territory that is selected from territory 1, territory 2, territory 3 and territory 4, wherein in standard L929 cytotoxic assay or standard HeLa IL-8 mensuration, when concentration is about 1 μ M, the not exciting TNFR1 of described antagonist.
In certain embodiments, the present invention is domain antibodies (dAb) monomer or the part that comprises dAb, and it can and suppress signal transduction by TNFR1 in conjunction with tumor necrosis factor 1 (TNFR1), and wherein said dAb monomer does not suppress combining of TNF α and TNFR1.
In other embodiments, the present invention is domain antibodies (dAb) monomer or the part that comprises dAb, it can be in conjunction with tumor necrosis factor 1 (TNFR1), and wherein said dAb can combine mice TNFR1 or combine people TNFR1 with TAR2h-205 competitiveness in conjunction with the territory 1 of TNFR1 and with TAR2m-21-23 competitiveness.
In other embodiments, the present invention is domain antibodies (dAb) monomer or the part that comprises dAb, it can be in conjunction with tumor necrosis factor 1 (TNFR1), and wherein said dAb can combine people TNFR1 in conjunction with the territory 3 of TNFR1 and with TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 or TAR2h-185-25 competitiveness.
The present invention also relates to antibody or its Fab, they have binding specificity and effective in treatment, inhibition or prevention chronic inflammatory disease to TNFR1.In certain embodiments, described antibody or Fab are the monovalent antigen binding fragments.
The present invention also provides the dAb monomer and comprises the monomeric part of dAb, the signal conduction that it has binding specificity to TNFR1 and can suppress TNFR-1 mediation, but do not suppress combining of TNF α and TNFR1 basically.In certain embodiments, the dAb monomer suppresses the crosslinked or cluster by TNFR1 on the inductive cell surface of TNF α.
The present invention also provides separation and/or the nucleic acid molecules of reorganization and the carrier that comprises described recombinant nucleic acid molecules of code book invention part.Host cell that comprises recombinant nucleic acid molecules of the present invention or carrier and the method that is used to prepare part also are provided.
The present invention also relates to pharmaceutical composition, described compositions comprises on antagonist of the present invention or part and the pharmacology, on the physiology or pharmaceutically acceptable carrier.
The present invention also relates to the method for treatment, inhibition or prevent disease or obstacle (for example chronic inflammatory disease, autoimmune disease, inflammatory diseases, arthritis, multiple sclerosis, inflammatory bowel, chronic obstructive pulmonary disease, pneumonia, septic shock), this method comprises the antagonist of the present invention or the part of the mammal treatment effective dose that needs are arranged.
The present invention also relates to the antagonist of the present invention or the part that are used for the treatment of or diagnose, and antagonist of the present invention or part are used for the treatment of, suppress or prevent purposes in the medicine of aforesaid disease or obstacle (for example chronic inflammatory disease, autoimmune disease, inflammatory diseases, arthritis, multiple sclerosis, inflammatory bowel, chronic obstructive pulmonary disease, pneumonia or septic shock) in preparation.In other embodiments, described disease can be cystic fibrosis or serious steroidal opposing type asthma.
The invention still further relates to the pharmaceutical composition that is used for the treatment of, suppresses or prevent disease as herein described or obstacle (for example chronic inflammatory disease, autoimmune disease, inflammatory diseases, arthritis, multiple sclerosis, inflammatory bowel, chronic obstructive pulmonary disease, pneumonia or septic shock), described compositions comprises antagonist of the present invention or part as active component.In other embodiments, described disease can be cystic fibrosis or serious steroidal opposing type asthma.
Part to TNFR1 and part have single variable region of binding specificity or domain antibodies (dAb) and comprise these single variable regions or dAb has some advantages.For example, single variable region or the dAb energy antagonism TNFR1 that TNFR1 is had binding specificity as herein described.Therefore, the curative (for example be used for the treatment of, diagnose or prevent purpose) that can comprise the anti-TNFR1 immunoglobulin of the present invention list variable region or dAb, the side effect of described curative (for example immunosuppressant) dangerous because in conjunction with and/or antagonism TNFR2 and basic the reduction.The curative of targeting TNF α is ENBREL  (entarecept for example; Immunex Corporation) antagonism TNFR1 and TNFR2 give these medicines and can produce immunosuppressant and related side effects (for example severe infections).These side effects limit the use of this class medicine, especially for the chronic disease that needs long term administration.(Kollias G. and Kontoyiannis D., Cytokine GrowthFactor Rev., 73 (4-5): 315-321 (2002)).By contrast, because ligand specificity's antagonism TNFR1 of the present invention, so for chronic disease, they can give for a long time, the danger of its side effect reduces, and can provide the advantage for the treatment of inflammatory disease and chronic inflammatory disease (comprising the prolonged sickness that is characterized as resting stage and activeness inflammatory phase, for example inflammatory bowel and arthritis).
The accompanying drawing summary
Fig. 1 shows V H/ HAS is in the variation (DVT or NNK encode respectively) of position H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, and they are at V HIn the antigen binding site of HAS.(SEQ ID NO:1, nucleotide sequence; SEQ IDNO:2, aminoacid sequence).V κ sequence has multiformity at position L50, L53.
Fig. 2 sketch map shows the structure of the plasmid pIT1/pIT2 be used to prepare strand Fv (scFv) library, and shows to cross over and express control and the plasmid nucleotide sequence (SEQ ID NO:3) in clone district and coded aminoacid sequence (SEQ ID NO:4).This plasmid is used for preparation
Library 1: kind is V κ/DVT V H,
Library 2: kind is V κ/NNK V H,
Library 3: kind is V H/ DVT V κ and
Library 4: kind is V H/ NNK V κ is with phage display/ScFv form.
According to conjugated protein A and the general part of albumen L, prescreen is carried out in these libraries, make that most of clone and selected library are functional.At HSA (first round) and β-gal (second takes turns) or HSA β-gal selects or at β-gal (first round) and HSA (second takes turns) β-gal HSA selection, the library is selected.These are from PCR clone's solubility scFv in the extension increasing sequence.The clone who selects a coding bi-specific antibody K8 carries out further work.
Fig. 3 shows the comparison of following sequence: V HChain (V HDummy (SEQ ID NO:5), K8 (SEQ ID NO:6), VH2 (SEQ ID NO:7), VH4 (SEQ ID NO:8), VHC11 (SEQ ID NO:9), VHA10sd (SEQ ID NO:10), VHAlsd (SEQ ID NO:11), VHA5sd (SEQ ID NO:12), VHC5sd (SEQ ID NO:13), VHCllsd (SEQ ID NO:14), VHCllsd (SEQ ID NO:15)) and V κ chain (Vk dummy (SEQ ID NO:16), K8 (SEQ ID NO:17), E5sc (SEQ ID NO:18), C3 (SEQ ID NO:19)).
Fig. 4 shows the sign of the binding characteristic of K8 antibody, and the binding characteristic of K8 antibody characterizes through monoclonal phage E LISA, and discovery bispecific K8 antibody capable is in conjunction with HSA and β-gal and be illustrated in phage surface, and its absorption signal is greater than 1.0.Do not detect and other proteinic cross reactivity.
Fig. 5 shows the result of solubility scFv ELISA, adopts the K8 antibody fragment of concentration known.96 orifice plates concentration 100 μ g HSA, 10 μ g/ml BSA and 100 μ g/ml β-gal and 1 μ g/ml protein A bag quilt.Adopt the K8 scFv of 50 μ g serial dilutions, detect the binding antibody fragment with albumen L-HRP.ELISA result has confirmed the bispecific of K8 antibody.
Fig. 6 shows clone K8V κ/dummy V HIn conjunction with feature, analyze to adopt solubility scFvELISA.Solubility scFv fragment is induced generation by IPTG, referring to Harrison etc., Methods Enzymol.1996; 267:83-109 directly measures the supernatant that contains scFv then.Described according to embodiment 1, carry out solubility scFv ELISA, measure in conjunction with scFv with albumen L-HRP.ELISA result shows that this clone still can then be eliminated in conjunction with BSA in conjunction with β-gal.
Fig. 7 shows the sequence (SEQ ID NO:2 and SEQ ID NO:3) of variable region carrier 1 and 2.
Fig. 8 is used to make up V H1/V HThe C of 2 polyspecific parts HThe carrier collection of illustrative plates.
Fig. 9 is the C κ carrier collection of illustrative plates that is used to make up V κ 1/V κ 2 polyspecific parts.
Figure 10 shows the TNF receptor determination, compares with TAR1-5 dimer 4, TAR1-5-19 dimer 4 and TAR1-5-19 monomer.
Figure 11 shows the TNF receptor determination of comparison TAR1-5 dimer 1-6.All dimers are all through the FPLC purification, the result of display optimization dimer kind.
Figure 12 shows the TNF receptor determination of multi-form TAR1-519 homodimer: have the dAb-joint-dAb form of 3U, 5U or 7U joint, Fab form and cysteine knuckle joint form.
Figure 13 shows the Dummy V in library 1 HSequence.(aminoacid sequence ((SEQ ID NO:5; Nucleotide sequence: coding strand (SEQ ID NO:20), noncoding strand (SEQ ID NO:21).The VH frame sequence is sequence D P47-JH4b based on kind.NNK randomization (N=A or T or C or G nucleotide; K=G or T nucleotide) position that is attached in the library 1 adds that with runic underscore represents.
Figure 14 shows the Dummy V in library 2 HSequence.(aminoacid sequence ((SEQ ID NO:22; Nucleotide sequence: coding strand (SEQ ID NO:23), noncoding strand (SEQ ID NO:24).The VH frame sequence is sequence D P47-JH4b based on kind.NNK randomization (N=A or T or C or G nucleotide; K=G or T nucleotide) position that is attached in the library 2 adds that with runic underscore represents.
Figure 15 shows the Dummy V κ sequence in library 3.(aminoacid sequence ((SEQ ID NO:16; Nucleotide sequence: coding strand (SEQ ID NO:25), noncoding strand (SEQ ID NO:26).V κ frame sequence is sequence D P κ 9-J κ 1 based on kind.NNK randomization (N=A or T or C or G nucleotide; K=G or T nucleotide) position that is attached in the library 3 adds that with runic underscore represents.
Figure 16 shows nucleotide sequence and aminoacid sequence (nucleotide sequence (SEQ ID NO:27), aminoacid sequence (SEQ ID NO:28) and the MSA 26 (nucleotide sequence (SEQ ID NO:29), aminoacid sequence (SEQ ID NO:30)) of anti-MSA dAb MSA 16.
Figure 17 shows the inhibition biacore of MSA16 and 26.By suppressing biacore purification dAb MSA16 and MSA26 are analyzed, draw K dIn brief, measure dAb, on biacore CM5 chip, reach the required dAb concentration of 200RU response with high density MSA bag quilt to obtain.In case measure required dAb concentration, will expect K dThe MSA antigen of left and right sides concentration range and dAb premix merge overnight incubation.Then with 30 μ l/ minutes high flow rate, measure the combining of biacore chip of dAb and MSA bag quilt in each pre-composition.
Figure 18 shows the serum levels of injection back MSA16.Measure the dAb MSA16 serum half-life of mice.Give the about 1.5mg/kg MSA16 of CD1 mice single intravenous injection.With 2 compartment model modelings, show that the t  α of MSA16 is 0.98 hour, t  β is 36.5 hours, AUC is 913 hours .mg/ml.Compare with HEL4 (a kind of anti-hen egg-white lysozyme dAb), the half-life of MSA16 is quite long, and t  α is 0.06 hour, and t  β is 0.34 hour.
Figure 19 a-19c shows that (Figure 19 a) and TNF receptor determination (Figure 19 b, 19c) shows that TNF is suppressed with segmental combination of Fab sample that comprises MSA26Ck and TAR1-5-19CH to ELISA.Adding has the segmental MSA of Fab sample and can reduce the inhibition level.The elisa plate of usefulness 1 μ g/mlTNF α bag quilt bispecific V κ C HSurvey with V κ C κ Fab print section, also detect in conjunction with dAb with contrast TNF α simultaneously, its concentration is for providing the concentration of similarity signal as calculated in ELISA.In 2mg/ml MSA existence or not, adopt bispecific dAb and contrast dAb to survey elisa plate.The signal in bispecific hole descends and surpasses 50%, but the signal in dAb hole does not descend (referring to Figure 19 a) fully.Be with or without under the situation of MSA, identical dual specificity protein is also being carried out receptor determination, and also showing and MSA competition (referring to Figure 19 c).This proof MSA competitiveness is in conjunction with dual specificity protein and TNF α.
Figure 20 shows the TNF receptor determination, shows that TNF is suppressed with the TAR1-5-19dAb and the combining of MSA16 dAb heterodimer of disulfide bonding.Add and have dimeric MSA, reduce the inhibition level in the dose dependent mode.In the presence of the MSA of the heterodimer (18nM) of constant density and serial dilution and HAS, carry out TNF receptor determination (Figure 19 (b)).The existence of the HAS of finite concentration scope (2mg/ml at most) does not cause that dimer suppresses the ability drop of TNF α.Yet add MSA, dose dependent ground has reduced the ability of dimer inhibition TNF α, and (Figure 19 a).This proof MSA and TNF α competitiveness are in conjunction with TAR1-5-19, the MSA16 dimer of cys bonding.In mensuration, MSA and HAS singly use TNF in conjunction with not influence of level.
Figure 21 A-21M shows that some have human normal immunoglobulin's amino acid sequences (SEQ ID NO:31-98 and SEQ ID NO:373-401 and 431) of binding specificity to people TNFR1.Shown aminoacid sequence is successive, does not have the room; Inserted in the sequence symbol～, the expression complementary determining region (CDR) the position.The both sides of CDR1 are～, the both sides of CDR2 are～～, the both sides of CDR3 are～～～.
Figure 22 A-22T shows the nucleotide sequence (SEQ ID NO:99-166 and SEQ ID NO:402-430 and 432) of the nucleic acid of the human normal immunoglobulin variable region shown in some code pattern 21A-21M.Shown nucleotide sequence is successive, does not have the room; Inserted in the sequence symbol～, the position of the sequence of presentation code CDR.The both sides of sequence of coding CDR1 are～, the both sides of the sequence of coding CDR2 are～～, the both sides of the sequence of coding CDR3 are～～～.
Figure 23 A-23B shows that some have human normal immunoglobulin's amino acid sequences (SEQ ID NO:167-179) of binding specificity to mice TNFR1.Shown aminoacid sequence is successive, does not have the room; In some series, inserted symbol～, the expression complementary determining region (CDR) the position.The both sides of CDR1 are～, the both sides of CDR2 are～～, the both sides of CDR3 are～～～.
Figure 24 A-24C shows the nucleotide sequence (SEQ ID NO:180-192 and 626) of the nucleic acid of the human normal immunoglobulin variable region shown in some code pattern 23A-23B.All the encode aminoacid sequence of SEQ ID NO:173 of SEQ ID NO:186 and SEQ ID NO:626.The both sides of SEQ ID NO:626 sequence of coding CDR1 are～, the both sides of the sequence of coding CDR2 are～～, the both sides of the sequence of coding CDR3 are～～～.
The nucleotide sequence of some immune globulin variable regions of Figure 25 A-25L code displaying and coded human normal immunoglobulin's amino acid sequences (SEQ ID NO:193-198 and 200-295).
Figure 26 curve chart is presented at the not exciting basically TNFR1 of anti-TNFR1 dAb form in the L929 mensuration.The L929 cell is cultivated in containing the culture medium of following composition: the anti-TNFR1 dAb monomer (TAR2m-21-23) of variable concentrations, crosslinked anti-SA dAb of the anti-TNFR1 dAb/ of TAR2m-21-23 monomer, bispecific (TAR2m-21-23 3U TAR7m-16) or the anti-TNFR1 dAb of the PEGization monomer (TAR2m-21-23 40KPEG) of commercial anti myc antibody (9E10).With regard to the antibody linked TAR2m-21-23 monomer of anti-myc, before cultivating, with dAb and antibody by 2: 1 mixed and preincubate 1 hour at room temperature, with immune cross-linked effect in the analogue body.(the TAR2m-21-23 monomer comprises the myc epi-position).With concentration is 3, and the TAR2m-21-23 monomer of 000nM is hatched with the L929 cell.TAR2m-21-23 monomer and anti-Myc antibody are hatched together, and dAb concentration is 3,000nM.With concentration is 25nM, 83.3nM, 250nM, 833nM and 2, and the TAR2m-21-23 3U TAR7m-16 of 500nM is hatched with cell.With concentration is 158.25nM, 527.5nM, 1582.5nM, 5,275nM and 15, and the TAR2m-21-23 40K PEG of 825nM is hatched with cell.After the overnight incubation, estimate cell survival rate.The result shows, can be crosslinked and commercial anti TNFR1 IgG antibody (the catalog number (Cat.No.) AF-425-PB of exciting TNFR1 with L929 cell and 10nM, 1nM or 0.1nM; R﹠amp; D system, Minneapolis MN) is hatched together, and causing increases the debility cell in dosage dependence mode, has therefore proved the sensitivity of these cells to the TNFR1 agonist.By contrast, hatch with the anti-TNFR1 form of varying number, antagonism TNFR1 does not cause the increase of debility cell quantity in the culture yet, even when adopting above 1000 times of commercial anti TNFR1 IgG antibody concentration.
Figure 27 A-27I shows that some have human normal immunoglobulin's amino acid sequences (SEQ ID NO:433-517 and 627) of binding specificity to people TNFR1.Shown aminoacid sequence is successive, does not have the room; Inserted in the sequence symbol～, the expression complementary determining region (CDR) the position.The both sides of CDR1 are～, the both sides of CDR2 are～～, the both sides of CDR3 are～～～.
Figure 28 A-28O shows the nucleotide sequence (SEQ ID NO:518-602 and 628) of the nucleic acid of the human normal immunoglobulin variable region shown in some code pattern 27A-27H.Shown nucleotide sequence is successive, does not have the room; Inserted in the sequence symbol～, the position of the sequence of presentation code CDR.The both sides of sequence of coding CDR1 are～, the both sides of the sequence of coding CDR2 are～～, the both sides of the sequence of coding CDR3 are～～～.
Detailed Description Of The Invention
In specification of the present invention, describe many embodiments, write mode so that specification of the present invention can be not only clear but also simple and clear, but should be appreciated that and do not departing from the situation of the present invention, can carry out various combinations or separately to these embodiments.
Definition
" complementation ", when two immunoglobulin domains belong to can consist of related to or during the structure family of associated group, when perhaps they derived from such family and have kept this feature, then they were " complementations ". For example, the V of antibodyHDistrict and VLThe district is complementary; Two VHQu Ze is not complementary, two VLDistinguish also not complementary. In other member of immunoglobulin superfamily, also can find complementary district, for example the V of φt cell receptorαAnd Vβ(or γ and δ) district. In the situation of the present invention's second general layout, the not collaborative binding target molecule in incomplementarity district, but the different target epi-positions of independent action on identical or different molecule. The artificial structure territory for example based on the domain (they are not in conjunction with epi-position, unless make them in conjunction with epi-position through transformation) of protein scaffolds, is non-complementary. Equally, two domains based on (for example) immunoglobulin domains and fibronectin domain are not complementary.
" immunoglobulin (Ig) " refers to such peptide family: the immunoglobulin folding feature that it keeps antibody molecule, contain two β-pleated sheets, and generally also contain a conservative disulfide bond. The immunoglobulin superfamily member participates in cell and the interactional many aspects of acellular in vivo, be included in the immune system (such as antibody, φt cell receptor molecule etc.) and have wide application, participate in cell adherence (for example ICAM molecule) and intracellular signal transduction (for example acceptor molecule, for example pdgf receptor). The present invention is applicable to that all have the immunoglobulin superfamily molecule in conjunction with the territory. Preferably the present invention relates to antibody.
" combination ", each variable region of the present invention combine and consist of one group of domain; For example, complementary district can combine, for example VLDistrict and VHThe district combines. The incomplementarity district also can combine. Domain can combine in a large amount of modes, comprises that each domain connects by mode covalently or non-covalently.
" domain (territory, domain, district) " is that it has kept tertiary protein structure, but is independent of the remainder of protein through folding protein structure. Usually, each domain is responsible for separation functional of protein, and can add, removes or be transferred in many cases other oroteins, and does not lose the function of the remainder of this protein and/or this domain. Monoclonal antibody body variable region (single antibody variable domain) refers to comprise the sequence that is characterized as antibody variable region through folding peptide zone. Therefore, it comprises the complete antibody variable region and modifies variable region (for example wherein one or more rings sequence of not had an antibody variable region feature replaces), perhaps by brachymemma or comprise N-end or antibody variable region that the C-end extends, and at least part of fold segments in conjunction with active and specific variable region that keeps the total length domain.
" storehouse (repertoire) ", the set of the diversified variant (for example polypeptide variants) that primary sequence is different. The used library of the present invention can comprise the peptide library that contains at least 1000 members.
" library (library) ", the term library refers to the mixture of heterologous polypeptide or nucleic acid. The library is by member composition, and each member has a polypeptide or nucleotide sequence. With regard to this one side, library (library) and storehouse (repertoire) synonym. Sequence difference between the member of library causes the diversity in library. The library can be the form of the simple mixtures of polypeptide or nucleic acid, perhaps can be with the organism of nucleic acid library conversion or the form of cell, such as bacterium, virus, animal and plant cells etc. Preferred each organism or cell only contain the library member of a library member or Limited Number. Preferably nucleic acid is incorporated in the expression vector, to express the coded polypeptide of this nucleic acid. Therefore, one preferred aspect, the library can be the form of host organisms colony, each organism contains the expression vector of one or more copies, described carrier contains a member in the library that is the nucleic acid form, and described nucleic acid can be expressed and be produced its corresponding polypeptide member. Therefore, host organisms colony has the potentiality in the very big polypeptide variants storehouse with genetic diversity of coding.
" closed conformation polyspecific part (closed conformation multi-specific ligand) " is used for describing polyspecific part defined herein, comprises at least two epi-positions defined herein in conjunction with territory (epitope binding domain). The epi-position that term " closed conformation " (polyspecific part) refers to part is to arrange like this in conjunction with the territory: so that by the epi-position combination of an epi-position in conjunction with the territory, and be combined the epi-position in territory by another epi-position in conjunction with competing. That is to say that related epi-position can be passed through each epi-position in conjunction with the independent combination in territory, rather than simultaneously combination. Adopt method as herein described, can obtain the closed conformation of part.
" antibody ", antibody (for example IgG, IgM, IgA, IgD or IgE) or its fragment (for example Fab, F (ab ')2, Fv, disulfide bond Fv, the scFv, closed conformation multi-specificity antibody, the disulfide bond that the connect scFv, the double-chain antibody that connect), no matter be the antibody from the natural generation of any species, or the antibody that is produced by recombinant DNA technology; Which kind of no matter from following sample, separate: serum, B cell, hybridoma, transfectoma, yeast or bacterium.
" bispecific part ", refer to comprise the part of the first immunoglobulin (Ig) list variable region and the second immunoglobulin (Ig) list variable region defined herein, wherein the variable region can be in conjunction with two two epi-positions on synantigen or the same antigen not, and they are not combined with the monospecific immunoglobulin (Ig) usually. For example, two epi-positions can be on same haptens, rather than same epi-position or enough near so that in conjunction with the monospecific part. Bispecific part of the present invention forms by having not homospecific variable region, but does not comprise the variable region pair with mutually homospecific mutual complementation.
" antigen ", a kind of can with the molecule of ligand binding of the present invention. Usually, antigen is combined with antibody ligand and can be produced in vivo antibody response. Antigen can be polypeptide, protein, nucleic acid or other molecule. Generally speaking, for the target-specific of specific antigen and select bispecific part of the present invention. With regard to conventional antibody and fragment thereof, can conjugated antigen by the antibody combining site that variable loop (L1, L2, L3 and H1, H2, H3) limits.
" epi-position " is with immunoglobulin (Ig) VH/V LConstruction unit to conventional combination. Epi-position limits the minimum binding site for antibody, has therefore represented the specific target of antibody. With regard to single domain antibody, epi-position has represented the construction unit of being combined with the variable region of separating.
" general part (generic ligand) " can be in conjunction with the part of all members in a certain storehouse. Generally speaking, the not combination by above-mentioned antigen binding site. Limiting examples comprises albumin A, albumen L and Protein G.
" selection " comes self-sizing or from Darwin's selection course, wherein between domain and antigen or the epi-position or between antibody and antigen or epi-position binding interactions taking place. Therefore, in the existence of complementary variable region or not, can select first variable region of conjugated antigen or epi-position.
" general framework (universal framework) ", monoclonal antibody body frame sequence, corresponding to the conservative antibody district in the sequence, define according to Kabat (" Sequences of Proteins of ImmunologicalInterest ", US Department of Health and Human Services (U.S. HHS)); Be immunoglobulin (Ig) storehouse or structure corresponding to ethnic group perhaps, according to Chothia and Lesk, (1987) J.Mol.Biol.196:910-917 defines. The invention provides the purposes of single framework or one group of such framework, have been found that they allow the in fact source of any binding specificity, although variation is only in hypervariable region.
" half-life ", refer to that the serum-concentration of part was down to for 50% needed time in vivo, for example because by the ligand degradation due to the natural mechanism and/or removing or chelating. Part of the present invention is stable in vivo, and its half-life prolongs because of the molecule in conjunction with anti-degraded and/or removing or chelating. Usually, this quasi-molecule is naturally occurring protein, and they itself have the long half-life in vivo. If it is longer that the molecule of the functional activity of part duration comparison prolong half-life does not in vivo have specific similar part, then the part Increased Plasma Half-life. Therefore, will have specific part to HSA and target molecule, and and HAS not had specificity and be not combined HAS and comparing in conjunction with the identical ligands of other molecule. For example, second epi-position on its energy binding target molecule. Usually, Increased Plasma Half-life 10%, 20%, 30%, 40%, more than 50%. The Increased Plasma Half-life scope at 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, be possible more than the 50x. Perhaps, in addition, the Increased Plasma Half-life scope also is possible up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x.
" homogeneous immunoassays (homogeneous immunoassay) ", need not to separate combination and not the step of binding reagents just can detect the immunoassays of analyte.
" basic identical (or " basic homology ") ", first amino acid or nucleotide sequence contain the identical or suitable of sufficient amount with second amino acid or nucleotide sequence and (for example have similar side chain, for example conserved amino acid replaces) amino acid residue or nucleotides, so that first and second amino acid or nucleotide sequence have similar activity. With regard to antibody, SA has identical binding specificity and has at least 50% of first antibody affinity with first antibody.
Term used herein " low stringency ", " medium stringency ", " high stringency " or " high stringency " have been described nucleic acid hybridization and wash conditions. The guide that carries out hybridization reaction can be referring to Current Protocols in Molecular Biology, John Wiley and Sons, and N.Y. (1989), 6.3.1-6.3.6, described document all is attached to herein by reference. Described aqueous methods and anhydrous process in this list of references, can adopt. Concrete hybridization conditions used herein is as follows: (1) low stringency hybridization condition is in 6X sodium chloride/sodium citrate (SSC), at about 45 ℃, again in 0.2X SSC, 0.1%SDS, at least 50 ℃ of (for low stringency, wash temperature can be increased to 55 ℃) washed twice; (2) medium stringency hybridization condition is in 6X SSC, at about 45 ℃, again in 0.2X SSC, 0.1%SDS, at 60 ℃ of washing one or many; (3) high stringency hybridization condition at about 45 ℃, is washed one or many at 65 ℃ again in 0.2X SSC, 0.1%SDS in 6X SSC; With preferred (4) high stringency hybridization condition be among 0.5M sodium phosphate, the 7%SDS, at 65 ℃, again in 0.2X SSC, 1%SDS, at 65 ℃ of washing one or many. High stringency (4) is preferred condition, except as otherwise noted, otherwise should adopt this condition.
Term used herein " closed conformation " (polyspecific part) refers to that the epi-position of part is connected to each other or combination in conjunction with the territory, optional mode by protein scaffolds, so that by the epi-position combination in conjunction with the territory of epi-position, and be combined the epi-position in territory by another epi-position in conjunction with competing. That is to say that the epi-position that related epi-position can be by separately is in conjunction with the independent combination in territory, rather than simultaneously combination. Adopt method as herein described, can obtain the closed conformation of part.
" open conformation (open conformation) ", the epi-position that is meant part is connected to each other or combination in conjunction with the territory, optional mode by protein scaffolds makes by the epi-position combination of an epi-position in conjunction with the territory, does not compete with the epi-position combination that combines the territory by another epi-position.
Term used herein " competition " be meant when the related epi-position of second epi-position with it in conjunction with the territory in conjunction with the time, the related epi-position with it of first epi-position is suppressed in conjunction with the combination in territory.For example, in conjunction with being to be suppressed on the space, for example, its affinity to epi-position (affinity) or affinity (avidity) are reduced by in conjunction with the physics blocking-up in territory or by changing structure or environment in conjunction with the territory.
Term " immunoglobulin list variable region " is meant can be independent of other V district (V region, V domain) and the antibody variable region (V of specificity conjugated antigen or epi-position H, V HH, V L); Yet, term immunoglobulin list used herein variable region can with other variable region (variableregion, variable domain) presents for example homopolymer or special-shaped polymeric form, wherein (region is not that the antigen of immunoglobulin list variable region is in conjunction with necessary (promptly wherein immunoglobulin list variable region is independent of other variable region with antigenic the combination) domain) in other district." immunoglobulin list variable region " not only comprises the single variable region of isolated antibody polypeptide, and comprises the one or more monomeric big polypeptide that contains antibody list variable region peptide sequence.Term used herein " domain antibodies " or " dAb " and term used herein " immunoglobulin list variable region " polypeptide synonym.Immunoglobulin list used herein variable region polypeptide is meant mammal (preferred people, but also comprising rodent) immunoglobulin list variable region polypeptide (for example is disclosed in WO 00/29004, the content of described document all is attached to herein by reference) or camellid (camelid comprises camel, vigone and relevant kind of system) V HHDAb.Camellid dAb is an immunoglobulin list variable region polypeptide, it is from camel (camel), yamma (llama), alpaca (alpaca), dromedary camel (dromedary) and chestnut color vigone species such as (guanaco), and comprises the heavy chain antibody of natural shortage light chain: V HHV HHMolecule is littler about 10 times than IgG molecule, and as single polypeptide, they are highly stable, can tolerate extreme pH and temperature conditions.
Term used herein " Tumor Necrosis Factor Receptors 1 (Tumor Necrosis FactorReceptor 1, TNFR1) antagonist " is meant and a kind ofly can and can suppresses the medicine (for example molecule, chemical compound) of one or more functions of TNFR1 in conjunction with TNFR1.For example, the TNFR1 antagonist can suppress combining of TNF α and TNFR1 and/or suppress by the TNFR1 Mediated Signal Transduction.Therefore, the process and the cell response (for example inductive cell death of TNF α in standard L929 cytotoxic assay) of TNFR1 mediation can be suppressed by the TNFR1 antagonist.The TNFR1 antagonist can be for example organic molecule, natural product, protein, peptide or peptide mimics (peptidomimetic).Can or adopt other appropriate method by screening library as herein described or elements collection (for example Chemical Repository of the National Cancer Institute), identify the TNFR1 antagonist.Preferred TNFR1 antagonist is antibody as herein described, antigen-binding fragments of antibodies, part and dAb monomer.
With the sequence of sequence similarity disclosed herein or homology (for example at least about 70% sequence homogeneity) also be ingredient of the present invention.In certain embodiments, the sequence homogeneity on the amino acid levels can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more than 99%.On nucleic acid level, sequence homogeneity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more than 99%.Perhaps, when nucleic acid segment when selected hybridization conditions (for example high stringency hybridization condition) is hybridized with complementary strand down, then have basic homogeneity.Nucleic acid may reside in the intact cell, is present in the cell lysate, perhaps is the form of partial purification or basic purification.
" homology " or " sequence homogeneity " or being calculated as follows of " similarity " (these terms being used interchangeably at this paper) are carried out between two sequences.In order to optimize the comparison purpose, with sequence alignment (for example in order to compare purpose, for the best comparison, can introduce the room in first and second aminoacid or nucleotide sequence, non-homogeneous sequence can be ignored).In a preferred embodiment, in order to compare purpose, the reference sequences length of being compared accounts at least 30%, preferably at least 40%, more preferably at least 50% even more preferably at least 60% even more preferably at least 70%, 80%, 90%, 100% of reference sequences total length.Then, amino acid residue or the nucleotide to corresponding amino acid position or nucleotide position compares.When certain position in first sequence was occupied with the same amino acid residue of the second sequence relevant position or nucleotide, molecule was identical (aminoacid used herein or nucleic acid " homology " are equal to aminoacid or nucleic acid " homogeneity ") on this position so.% homogeneity between two sequences is the function of the same position number of considering that two sequences after room number and each room length are shared, needs to introduce these rooms to carry out the best comparison of two sequences.
Preferably adopt BLAST algorithm (2.0 editions) to carry out sequence alignment, parameter setting is to default value.The BLAST algorithm goes up in the WWW (" www ") of the state-run biotechnology of National Institutes of Health (" the nih ") information centre (National Center for Biotechnology Information, " .ncbi ") of U.S. government (" .gov "), under "/Blast/ " catalogue, in " blast_help.html " file, detailed introduction is arranged.Search argument is defined as follows, and preferably is set in the default parameters of having reserved.
BLAST (Basic Local Alignment Search Tool) is the heuristic searching algorithm that blastp, blastn, blastx, tblastn and tblastx program are adopted; These programs all mainly give the credit to and adopt Karlin and Altschul, and 1990, the discovery of the statistical method of Proc.Natl.Acad.Sci.USA87 (6): 2264-8 (referring to aforesaid " blast_help.html " file) has only reinforcement seldom.Blast program is specifically designed to the sequence similarity retrieval, for example identifies the congener of search sequence.These programs generally are not used in the retrieval of motif style.The discussion of the basic problem of relevant sequence library similarity retrieval is referring to (1994) such as Altschul.
Obtainable 5 blast programs in website are finished following task in the U.S. state-run biotechnology information centre:
" blastp " comparing amino acid search sequence and protein sequence database;
" blastn " be nucleotide query sequence and nucleotide sequence database relatively;
" blastx " be six the frame conceptual translation products and the protein sequence database of nucleotide query sequence (two chain) relatively;
" tblastn " comparison protein search sequence and all six frames (two chain) are the nucleotide sequence database of translation dynamically.
Six frames of " tblastx " comparison nucleotide query sequence translate and six frames of nucleotide sequence database are translated.
BLAST adopts following search argument:
HISTOGRAM shows the rectangular histogram of each retrieval score; Be defaulted as yes (being).(referring to the Parameter H in the BLAST handbook).
The DESCRIPTIONS restriction is to the quantity of the matching sequence Short Description of specified quantity report; Default limit is 100 descriptions.(referring to the V parameter of handbook).Other sees EXPECT and CUTOFF.
ALIGNMENTS is limited to the report skyer with database sequence and divides section to (high-scoring segment pairs, specified quantity HSP); Default limit is 50.If database sequence is more than this, for the report threshold value (referring to following EXPECT and CUTOFF) that satisfies statistical significance, only report belongs to the coupling of maximum statistical significance.(referring to the B parameter of BLAST handbook).
EXPECT is at the threshold value of the statistical significance of the coupling of database sequence report; Default value is 10, makes expection only find 10 couplings accidentally, according to the stochastic model of Karlin and Altschul (1990).If the statistical significance that belongs to coupling greater than the EXPECT threshold value, then will not reported coupling.The EXPECT threshold value is low more, and stringency is high more, and the MM that causes reporting can be few more.Fractional value is acceptable.(referring to the parameter E of BLAST handbook).
CUTOFF be used to report skyer divide section right by (Cutoff) score value.Default value is obtained from EXPECT value (referring to above).HSP reports for database sequence, only when the statistical significance that belongs to them the same with isolated HSP at least when high with the score value that equals the CUTOFF value.The CUTOFF value is high more, and stringency is high more, and the MM that causes reporting can be more little.(referring to the parameter S of BLAST handbook).Usually, the significance threshold value can be controlled more intuitively with EXPECT.
MATRIX specifies and substitutes the marking matrix, is used for BLASTP, BLASTX, TBLASTN and TBLASTX.Default matrix be BLOSUM62 (Henikoff and Henikoff, 1992, Proc.Natl.Aacad.Sci.USA 89 (22): 10915-9).Effectively alternative comprises: PAM40, PAM120, PAM250 and IDENTITY.Substitute the marking matrix and be not suitable for BLASTN; Specify the direction of MATRIX in the BLASTN request to return errored response.
STRAND just in time is limited in the TBLASTN retrieval top chain or the end chain of database sequence; Or BLASTN, BLASTX or TBLASTX retrieval just in time is limited in the top chain of search sequence or the frame of end chain.
FILTER shelters the search sequence section (the SEG program by Wootton and Federhen (1993) Computers and Chemistry 17:149-163 is determined) with low complicated component, or the section of being made up of short period property internal repeat is (by Claverie and States, 1993, the XNU program of Computers and Chemistry 17:191-201 is determined, perhaps, determine (referring to the internet sites of NCBI)) by the DUST program of Tatusov and Lipman for BLASTN.Filtration can be eliminated the report (for example at common be rich in hitting of acidity, alkalescence or proline district) that has significance,statistical but do not have biological significance from blast output, stay the search sequence district that has more biological significance, be applicable to specific coupling at database sequence.
The low-complexity sequence of finding in the filter substitutes (for example " N " repeats 13 times) with letter " N " in nucleotide sequence, substitute (for example " X " repeats 9 times) with letter " X " in protein sequence.
Filtration is only applicable to search sequence (or its translation product), and is not suitable for database sequence.For BLASTN, it is DUST that acquiescence is filtered, and is SEG for other program.
When being used for the sequence of SWISS-PROT, what SEG, XNU or the two do not shelter fully, and this is not to be uncommon, always do not have the result so should not wish to filter.In addition, in some cases, sequence is masked fully, and this significance,statistical that shows any coupling of reporting at not filtering search sequence should fall under suspicion.
NCBI-gi Causes NCBI gi identifier shows in output, except searching number and/or location name.
The simple BLAST searching algorithm that is provided under "/BLAST " catalogue of above-mentioned NCBI network address most preferably is provided, carries out sequence relatively.
Except as otherwise noted, otherwise all scientific and technical terminologies used herein all have this area (for example cell culture, molecular genetics, nucleic acid chemistry, hybridization technique and biochemistry) known identical meanings of those of ordinary skill.Standard technique is used for molecule, heredity and biochemical method (generally referring to Sambrook etc., Molecular Cloning:A Laboratory Manual, the 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. with Ausubel etc., Short Protocols in Molecular Biology (1999) the 4th edition, JohnWiley﹠amp; Sons, Inc., described document is attached to herein by reference) and chemical method.
TNFR1 is a transmembrane receptor, and it contains extracellular region and intracellular region, and wherein extracellular region can binding partner, and intracellular region lacks inherent signal transduction activity but can the binding signal transduction molecule.The TNFR1 complex that has in conjunction with TNF contains 3 TNFR1 chains and 3 TNF chains.(Banner etc., Cell, 73 (3) 431-445 (1993)).Tnf ligand exists with trimeric form, it and 3 TNFR1 chain combinations (ibid).These 3 TNFR1 chains in the receptor-ligand complex tight bunch gather together, this bunch of collection is the prerequisite of TNFR1 Mediated Signal Transduction.In fact, when TNF does not exist, can induce TNFR1 cluster and signal transduction in conjunction with the multivalence material (for example anti-TNFR1 antibody) of TNFR1, and usually as the TNFR1 agonist.(referring to for example Belka etc., EMBO, 14 (6): 1156-1165 (1995); Mandik-Nayak etc., J.Immunol., 167:1920-1928 (2001)).Therefore, with the bonded multivalence material of TNFR1 be not effective TNFR1 antagonist usually, even when the combining of their blocking-up TNF α and TNFR1.
The extracellular region of TNFR1 comprises 13 amino acid whose aminoterminal section (amino acid/11s-13 of SEQ ID NO:603 (people); The amino acid/11-13 of SEQ ID NO:604 (mice)), territory 1 (the amino acid/11 4-53 of SEQID NO:603 (people); The amino acid/11 4-53 of SEQ ID NO:604 (mice)), territory 2 (the aminoacid 54-97 of SEQ ID NO:603 (people); The aminoacid 54-97 of SEQ ID NO:604 (mice)), territory 3 (the aminoacid 98-138 of SEQ ID NO:603 (people); The aminoacid 98-138 of SEQED NO:604 (mice)) and territory 4 (the amino acid/11 39-167 of SEQ ID NO:603 (people); The amino acid/11 39-167 of SEQ ID NO:604 (mice)), then be film proximal end region (the amino acid/11 68-182 of SEQ ID NO:603 (people) thereafter; The amino acid/11 68-183 of SEQ ID NO:604 (mice)).(referring to Banner etc., Cell 73 (3) 431-445 (1993) and Loetscher etc., Cell 61 (2) 351-359 (1990)).Territory 2 contacts binding partner (TNF β, TNF α) with territory 3.(Banner etc., Cell, 73 (3) 431-445 (1993)).The TNFR1 extracellular region also contains district's (amino acid/11-53 of SEQ ID NO:603 (people) that is called preceding part in conjunction with assembly section or PLAD district; The amino acid/11-53 of SEQ ID NO:604 (mice)) (U.S. government, WO 01/58953; Deng etc., Nature Medicine, doi:10.1038/nm1304 (2005)).
TNFR1 comes off from cell surface in vivo by processes such as Proteolytic enzyme, produces the TNFR1 of soluble form, and described process comprises that TNFR1 is in the territory 4 or at film proximal end region (the amino acid/11 68-182 of SEQ IDNO:603; The amino acid/11 68-183 of SEQ ID NO:604) Proteolytic enzyme.Soluble TNF R1 has kept the ability in conjunction with TNF α, therefore plays the effect of the endogenous inhibitor of TNF alpha active.
The present invention relates to antibody or its Fab (for example dAb) or part, it is in conjunction with TNFR1, but does not combine TNFR1 with TNF competitiveness.For example, antibody or its Fab (for example dAb) or part can be in conjunction with the territory 1 of TNFR1 or the territories 4 of TNFR1.Therefore such antibody or its Fab (for example dAb) or part have the advantage as diagnostic reagent, can be used for the TNFR1 in combination and detection, quantification or the working sample, but not with sample in TNF competitiveness combine TNFR1.Therefore, accurately whether have in TNFR1 and the sample how many TNFR1 are arranged in the working sample.In certain embodiments, in conjunction with TNFR1 but do not combine antibody or its Fab (for example dAb) or the part of TNFR1, be exactly TNFR1 antagonist as herein described with TNF competitiveness.
The present invention also relates to a kind of diagnostic kit and operation instructions (for example existence of TNFR1 and/or quantity in the working sample), be used for determining whether sample exists in TNFR1 and the sample how many TNFR1 are arranged, described test kit comprises in conjunction with TNFR1 but does not combine antibody or its Fab (for example dAb) or the part of TNFR1 with TNF competitiveness.In certain embodiments, test kit also comprises one or more auxiliary reagents, for example suitable reducing or suitable detectable (for example detectable label antibody or its Fab, its binding energy in conjunction with TNFR1 but do not combine antibody or its Fab (for example dAb) or the part of TNFR1) with TNF competitiveness.
The present invention also relates to a kind of device, described device comprises the surface of solids, wherein, make that immobilized antibody or its Fab (for example dAb) or part can be in conjunction with TNTR1 can be in conjunction with TNFR1 but do not combine the antibody of TNFR1 or its Fab (for example dAb) or part and be fixed on the described surface of solids with TNF competitiveness.Can use any suitable surface of solids that antibody or its Fab (for example dAb) or part can be fixed thereon, for example glass, plastics, carbohydrate (for example sepharose 4B).If necessary, holder can contain required functional group or pass through and modify to contain required functional group, so that sessile antibody or its Fab (for example dAb) or part.Device and/or holder can have any suitable shape, for example sheet, little bar, band, plate, slide, globule, piller, dish, gel, test tube, ball, chip, plate or ware etc.In certain embodiments, device is to soak rod (dipstick).
The present invention relates to Tumor Necrosis Factor Receptors 1 (TNFR1; P55; CD120a) has the TNFR1 antagonist (part for example as herein described) of binding specificity.Preferred antagonist of the present invention does not have binding specificity to tumor necrosis factor 2 (TNFR2), perhaps antagonism TNFR2 not basically.In standard cell lines is measured, when antagonist (1nM, 10nM, 100nM, 1 μ M, 10 μ M or 100 μ M) cause being no more than about 5% at by the active inhibition of the inductive TNFR2 mediation of TNF α (100pg/ml) time, the TNFR1 antagonist is antagonism TNFR2 not basically.Particularly preferred TNFR1 antagonist is the effective medicine (be effectively, have curative effect) that is used for the treatment of chronic inflammatory disease.For example, in certain embodiments, effective in the chronic inflammatory disease model of TNFR1 antagonist below for example: the inflammatory bowel model that collagen-induced arthritis model, mouse arthritis Δ ARE model, the mice dextran sulfate sodium of mice brought out, mice inflammatory bowel Δ ARE model, mice caused by smoking chronic obstructive pulmonary disease model or suitable primates model (for example collagen-induced arthritis of primates).
The TNFR1 antagonist can be a unit price or polyvalent.In certain embodiments, described antagonist is monovalent, contains one and the interactional binding site of TNFR1.The unit price antagonist can be in conjunction with a TNFR1, and the crosslinked or cluster of inducing cell surface TNFR1 not, and so crosslinked or cluster can cause receptor activation and signal transduction.In specific embodiment, TNFR1 unit price antagonist is in conjunction with the territory 1 of TNFR1.In a more particular embodiment, TNFR1 unit price antagonist is in conjunction with the territory 1 of TNFR1, and combines mice TNFR1 with TAR2m-21-23 competitiveness or combine people TNFR1 with TAR2h-205 competitiveness.
In other embodiments, the TNFR1 antagonist is polyvalent.TNFR1 multivalence antagonist can contain two or more copies at the particular combination site of TNFR1 or contain two or more can be in conjunction with the different binding sites of TNFR1.For example, TNFR1 antagonist as herein described can be dimer, trimer or polymer, and what it comprised two or more copies can be in conjunction with the different dAb of TNFR1 in conjunction with the specific dAb of TNFR1 or two or more.In standard cell lines is measured, the preferred not exciting basically TNFR1 of TNFR1 multivalence antagonist (playing the effect of TNFR1 agonist) is (promptly in mensuration, when concentration is 1nM, 10nM, 100nM, 1 μ M, 10 μ M, 100 μ M, 1000 μ M or 5, during 000 μ M, cause being no more than about 5% activity) by the inductive TNPR1 mediation of TNF α (100pg/ml).
In certain embodiments, TNFR1 multivalence antagonist contains two or more at the required epi-position of TNFR1 or the binding site of domain.For example, TNFR1 multivalence antagonist can comprise two or more can be in conjunction with the binding site of same epi-position in the territory 1 of TNFR1.
In other embodiments, TNFR1 multivalence antagonist contain two or more can be in conjunction with the different epi-positions of TNFR1 or the binding site of domain.In an example, TNFR1 multivalence antagonist comprises first binding site and second binding site, and wherein first binding site can be in conjunction with first epi-position in the territory 1 of TNFR1, and second binding site can be in conjunction with second different in the territory 1 epi-positions.In other example, TNFR1 multivalence antagonist can comprise can be in conjunction with two or more the required epi-positions of TNFR1 or the binding site of domain.For example, TNFR1 multivalence antagonist can comprise the binding site at the domain of following TNFR1: territory 1 and territory 2, territory 1 and territory 3, territory 1 and territory 4, territory 2 and territory 3, territory 2 and territory 4 or territory 3 and territory 4.For example, TNFR1 multivalence antagonist can comprise the binding site in territory 1, territory 2 and territory 3 at TNFR1, at the binding site in territory 1, territory 2 and the territory 4 of TNFR1, or at the binding site in territory 1, territory 3 and the territory 4 of TNFR1.In certain embodiments, the TNFR1 antagonist is the bispecific part, and it comprises can be in conjunction with the dAb in the territory 1 of TNFR1 and can be in conjunction with the dAb in the territory 3 of TNFR1.In standard L929 cytotoxic assay as herein described or standard HeLa IL-8 mensuration, when preferred described multivalence antagonist is about 1nM or about 10nM or about 100nM or about 1 μ M or about 10 μ M when concentration, not exciting TNFR1.
Some TNFR1 antagonisies can and suppress combining of TNF α and TNFR1 in conjunction with TNFR1.In certain embodiments, such TNFR1 antagonist can be in conjunction with territory 2 and/or the territory 3 of TNFR1.In specific embodiment, described antagonist combines TNFR1 with TAR2h-10-27, TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 or TAR2h-185-25 competitiveness.
Other part (wherein embodiment preferred is the TNFR1 antagonist) does not suppress combining of TNF α and TNFR1.This class part (and antagonist) has the advantage as diagnostic reagent because they can be used for the TNFR1 in combination and detection, quantification or the working sample, but not with sample in TNF competitiveness combine TNFR1.Therefore, accurately whether there is TNFR1 and how many TNFR1 are arranged in the working sample.
Some TNFR1 antagonisies do not suppress combining of TNF α and TNFR1, but suppress by the TNFR1 Mediated Signal Transduction.For example, the TNFR1 antagonist can suppress the inductive TNFR1 cluster of TNF α, and this is by the step before the signal transduction of TNFR1.This class antagonist has some advantages.For example, in the presence of such antagonist, TNF α can be combined in TNFR1 cell surface expression and that leave cellular environment, but can not activate the TNFR1 Mediated Signal Transduction.Therefore, the generation of the extra TNF of TNFR1 signal induction and other inflammatory mediator will be suppressed.Equally, can and suppress by the TNFR1 Mediated Signal Transduction but not suppress TNF α and the bonded TNFR1 antagonist of TNFR1 in conjunction with TNFR1, with do not suppress endogenous generation soluble TNF R1 TNF α combination and suppress activity.Therefore, such antagonist is had the mammal that needs, can replenish endogenous adjusting approach, described approach suppresses the activity of TNF α and the activity of TNFR1 in vivo.The present invention also relates to part, described part (i) can be in conjunction with TNFR1 (for example in the territory 1), (ii) the not activation of antagonism TNFR1 Mediated Signal Transduction and (iii) do not suppress combining of TNF α and TNFR1.This class part can be in conjunction with soluble TNF R1, but do not stop soluble recepter in conjunction with TNF α, therefore such antagonist is had the mammal that needs, can replenish endogenous adjusting approach, described approach suppresses the activity of TNF α in vivo by the half-life that prolongs soluble recepter in the serum.These advantages are especially relevant with such part: described part is refined into to having big hydrodynamics size (hydrodynamic size), for example by connecting PEG group, serum albumin, transferrins, TfR or its transferrins bound fraction, antibody Fc district at least, perhaps by puting together with the antibody district.For example, can have refining the becoming of the material with following characteristic (for example polypeptide) bigger antigen-binding fragments of antibodies or become antibody (for example refining Fab, Fab ', the F (ab) of becoming 2, F (ab ') 2, IgG, scFv): (i) can be in conjunction with TNFR1 (promptly in the territory 1), the (ii) not activation of antagonism TNFR1 Mediated Signal Transduction and (iii) do not suppress combining of TNF α and TNFR1 (for example dAb monomer).Also can by with the TNFR1 bonding agent with can in conjunction with as herein described can extension body in the antigen of half-life or the puting together or be connected of epi-position in conjunction with territory (for example antibody or antibody fragment), thereby the hydrodynamics size and the serum half-life (referring to appendix 1) thereof of increase part.For example, TNFR1 bonding agent (for example polypeptide) can be puted together or be connected with antiserum albumin or anti-new born animal Fc receptor antibody or antibody fragment (for example anti-SA or anti-new born animal Fc receptor dAb, Fab, Fab ' or scFv) or with affine body of anti-SA or the affine body of anti-new born animal Fc receptor.
The example of suitable albumin, albumin fragment or albumin variant that is used for TNFR1 binding partner of the present invention is referring to WO 2005/077042A2, and described document all is attached to herein by reference.Specifically, following albumin, albumin fragment or albumin variant can be used for the present invention:
● SEQ ID NO:1 (be disclosed in WO 2005/077042A2, this sequence is attached in this description by reference clearly);
● albumin fragment or variant, it comprises the amino acid/11-387 of SEQID NO:1 among the WO 2005/077042A2 or is made up of it;
● albumin or its fragment or variant, it comprises and is selected from following aminoacid sequence: (a) the aminoacid 54-61 of SEQ ID NO:1 among the WO 2005/077042A2; (b) the aminoacid 76-89 of SEQ ID NO:1 among the WO 2005/077042A2; (c) the aminoacid 92-100 of SEQ ID NO:1 among the WO 2005/077042A2; (d) the amino acid/11 70-176 of SEQ ID NO:1 among the WO 2005/077042A2; (e) the aminoacid 247-252 of SEQ ID NO:1 among the WO 2005/077042A2; (f) the aminoacid 266-277 of SEQ ID NO:1 among the WO 2005/077042A2; (g) the aminoacid 280-288 of SEQ ID NO:1 among the WO 2005/077042A2; (h) the aminoacid 362-368 of SEQ ID NO:1 among the WO 2005/077042A2; (i) the aminoacid 439-447 of SEQ ID NO:1 among the WO 2005/077042A2; (j) the aminoacid 462-475 of SEQ ID NO:1 among the WO 2005/077042A2; (k) the aminoacid 478-486 of SEQ ID NO:1 among the WO 2005/077042A2; (l) the aminoacid 560-566 of SEQ ID NO:1 among the WO 2005/077042A2.
Other example of suitable albumin, its fragment and variant that is used for TNFR1 binding partner of the present invention is referring to WO 03/076567A2, and described document all is attached to herein by reference.Specifically, following albumin, fragment or variant can be used for the present invention:
● the human serum albumin, referring to WO 03/076567A2, for example in Fig. 3 (this sequence information is attached in this description by reference clearly);
● human serum albumin (HA), it is made up of 585 amino acid whose single non-glycosylated polypeptide chains, and molecular weight is 66,500 (referring to Meloun etc., FEBS Letters58:136 (1975); Behrens etc., Fed.Proc.34:591 (1975); Lawn etc., Nucleic Acids Research 9:6102-6114 (1981); Minghetti etc., J.Biol.Chem.261:6747 (1986));
● albumin polymorphic variant or its analog or fragment, referring to Weitkamp etc., Ann.Hum.Genet.37:219 (1973);
● albumin fragment or variant, referring to EP 322094, the fragment between HA (1-373), HA (1-388), HA (1-389), HA (1-369) and HA (1-419) and 1-369 and 1-419 for example;
● albumin fragment or variant, referring to EP 399666, the fragment between HA (1-177) and HA (1-200) and the HA (1-X) for example, wherein X is the Any Digit between 178-199.
When one or more half-life prolongations (for example albumin, transferrins and fragment thereof and analog) are used for TNFR1 binding partner of the present invention, can it be puted together with any appropriate method, for example by directly merging with TNFR1 bound fraction (for example anti-TNFR1 dAb or antibody fragment), for example by using the single constructs of encoding fusion protein, wherein the fusion rotein coding becomes single polypeptide chain and half-life prolongation (being positioned at the N end or the C end of TNFR1 bound fraction).Perhaps, can (for example (whole disclosures of these joints be attached in this description by reference for WO 03/076567 A2 or WO 2004/003019 described peptide linker by using peptide linker between each several part, to be provided for example of the present invention)), finish conjugation reaction.
In a more particular embodiment, can and suppress by the TNFR1 Mediated Signal Transduction but do not suppress TNF α and the bonded TNFR1 antagonist of TNFR1 in conjunction with TNFR1, can be in conjunction with the territory 1 of TNFR1 or the territory 4 of TNFR1.In certain embodiments, this TNFR1 antagonist is can be in conjunction with the dAb monomer or the part in the territory 4 of the territory 1 of TNFR1 or TNFR1.
In a specific embodiments, TNFR1 antagonist (for example dAb monomer or part) can be in conjunction with the territory 1 of TNFR1, and in case in conjunction with suppressing to pass through the TNFR1 Mediated Signal Transduction behind the TNF α.Such antagonist can suppress the signal transduction by TNFR1, but does not suppress TNF α and combining of TNFR1 and/or coming off of TNFR1, to produce soluble TNF R1.Therefore, such antagonist is had the mammal that needs, can replenish endogenous adjusting approach, described approach suppresses the activity of TNF α and the activity of TNFR1 in vivo.
Other TNFR1 antagonist can be in conjunction with TNFR1, but not in conjunction with territory 4.This class antagonist can suppress the function of TNFR1, but does not suppress coming off of soluble TNF R1.Therefore, such antagonist is had the mammal that needs, can replenish endogenous adjusting approach, described approach suppresses the activity of TNF α and the activity of TNFR1 in vivo.
In certain embodiments, described antagonist (for example chemical compound, new chemical entity, dAb monomer, part) can combine mice TNFR1 or combine people TNFR1 with TAR2h-205 competitiveness in conjunction with the territory 1 of TNFR1 and with TAR2m-21-23 competitiveness.In other embodiments, described antagonist (for example chemical compound, new chemical entity, dAb monomer, part) can be in conjunction with territory 2 or the territory 4 of TNFR1.In other embodiments, described antagonist (for example chemical compound, new chemical entity, dAb monomer, part) can combine TNFR1 (for example people and/or mice TNFR1) in conjunction with the territory 3 of TNFR1 and with TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10, TAR2h-185-25 or TAR2h-27-10 competitiveness.
Some parts (being the TNFR1 antagonist in preferred embodiments) can be in conjunction with people TNFR1 and mice TNFR1.This class part (for example antagonist, dAb monomer) provides such advantage: available identical ligands is carried out clinical preceding and clinical research, and does not need to carry out preclinical study with suitable alternative part.
In other embodiments, described antagonist or part are to TNFR1 or its Fab (for example Fab fragment, Fab ' fragment, F (ab ') 2Fragment or Fv fragment (for example scFV)) have the antibody of binding specificity.In other embodiments, described antagonist or part are monovalent, for example the monovalent antigen binding fragment of dAb or antibody, for example Fab fragment, Fab ' fragment or Fv fragment.
In described in this manual other embodiment of the present invention, in antagonist of the present invention or part, do not use " dAb ", be to consider that the technical staff can use to comprise and (for example to be transplanted to the CDR on suitable protein support or the skeleton in conjunction with the domain of the CDR of the dAb of TNFR1, for example affine body, SpA support, ldl receptor category-A district or EGF district) or can be the protein domain that comprises at the binding site of TNFR1, for example wherein said domain is selected from affine body, SpA district, ldl receptor category-A district or EGF district.Therefore, generally speaking, disclosure of the present invention should be considered as providing antagonist, part and use described domain to substitute the method for dAb.
Preferred TNFR1 antagonist is a part as herein described.Part comprises the complementary determining region that TNFR1 is had the suitable form of the immunoglobulin list variable region of binding specificity or domain antibodies (dAb) or described dAb.The polypeptide that part can be formed or be made up of such dAb basically by such dAb.Part also can be to comprise suitable form (antibody formation (for example IgG sample form, scFv, Fab, Fab ', F (ab ') for example 2)) the polypeptide of dAb (or CDR of dAb), comprising can be in conjunction with the dAb of TNFR1 and can be in conjunction with the bispecific part of the 2nd dAb of another target protein, antigen or epi-position (for example serum albumin), polyspecific part perhaps as herein described.
The TNFR1 antagonist, the dAb monomer that comprises the part of any aspect of the present invention and be used to make up described part all can be advantageously disintegrates down from their related target, according to the mensuration of surface plasma resonance, K dBe 300nM～5pM (promptly 3 * 10 -7M～5 * 10 -12M), preferred 50nM～20pM or 5nM～200pM or 1nM～100pM, 1 * 10 -7M is following, 1 * 10 -8M is following, 1 * 10 -9M is following, 1 * 10 -10M is following, 1 * 10 -11Below the M; And/or K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1, preferred 1 * 10 -2s -1～1 * 10 -6s -1, or 5 * 10 -3s -1～1 * 10 -5s -1, or 5 * 10 -1s -1Below or 1 * 10 -2s -1Below or 1 * 10 -3s -1Below or 1 * 10 4s -1Below or 1 * 10 -5s -1Below or 1 * 10 -6s -1Below.K dSpeed constant is defined as K Dissociate/ K Associate
In other embodiments, described antagonist is in conjunction with TNFR1 and suppress one or more functions (for example conduction of receptor clustering, receptor signal or TNF α and TNFR1's combines) of TNFR1, and in conjunction with another member of TNF receptor superfamily.Preferred such antagonist also suppresses other member's of TNF receptor superfamily function (for example combination of member's bunch collection, signal transduction or this member and its related part).The TNF receptor superfamily is a histone matter well known in the art, they comprise TNFR1 (p55, CD120a, p60, TNF receptor superfamily member 1A, TNFRSF1A), TNFR2 (p75, p80, CD120b, TNF receptor superfamily member 1B, TNFRSF1B),
CD18(TNFRSF3，LTBR，TNFR2-RP，TNFR-RP，TNFCR，TNF-R-III)，OX40(TNFRSF4，ACT35，TXGP1L)，CD40(TNFRSF5，p50，Bp50)，Fas(CD95，TNFRSF6，APO-1，APTI)，DcR3(TNFRSF6B)，CD27(TN-FRSF7，Tp55，S152)，CD30(TNFRSF8，Ki-1，D1S166E)，CD137(TNFRSF9，4-1BB，ILA)，TRAILR-1(TNFRSF10A，DR4，Apo2)，TRAIL-R2(TNFRSF10B，DR5，KILLER，TRICK2A，TRICKB)，TRAILR3(TNFRSF10C，DcR1，LIT，TRID)，TRAILR4(TNFRSF10D，DcR2，TRUNDD)，RANK(TNFRSF11A)，OPG(TNFRSF11B，OCIF，TR1)，DR3(TNFRSF12，TRAMP，WSL-1，LARD，WSL-LR，DDR3，TR3，APO-3)，DR3L(TNFRSF12L)，TAC1(TNFRSF13B)，BAFFR(TNFRSF13C)，HVEM(TNFRSF14，ATAR，TR2，LIGHTR，HVEA)，NGFR(TNFRSF16)，BCMA(TNFRSF17，BCM)，AITR(TNFRSF18，GITR)，TNFRSF19，FLJ14993(TNFRSF19L，RELT)，DR6(TNFRSF21)，SOBa(TNFRSF22，Tnfrh2，2810028K06Rik)，mSOB(THFRSF23，Tnfrh1)
In certain embodiments, described antagonist comprise can in conjunction with TNFR1 and suppress the TNFR1 function a dAb and can be in conjunction with another member's of TNF receptor superfamily the 2nd dAb, TNFR2 (CD120b), OX40, CD40, Fas (CD95), TRAILR-1, TRAILR-2, TAC1, BCMA etc. that described another member enumerates more than for example.In another embodiment, described antagonist comprises and can and suppress the TNFR1 function and also can be in conjunction with another member's of TNF receptor superfamily dAb monomer (described function is combining of receptor clustering, receptor signal conduction or TNF α and TNFR1 for example) in conjunction with TNFR1, TNFR2 (CD120b), OX40, CD40, Fas (CD95), TRAILR-1, TRAILR-2, TAC1, BCMA etc. that described another member enumerates more than for example.
With bonded part of TNFR1 and dAb monomer
The invention provides and comprise the monomeric part of anti-TNFR1 dAb (the bispecific part that for example comprises this dAb), according to the mensuration of surface plasma resonance, described monomer and the bonded K of TNF receptor I dBe 300nM～5pM (promptly 3 * 10 -7M～5 * 10 -12M), preferred 50nM～20pM, more preferably 5nM～200pM, 1nM～100pM most preferably, for example 1 * 10 -7M is following, preferred 1 * 10 -8M is following, more preferably 1 * 10 -9M is following, advantageously 1 * 10 -10M is following, most preferably 1 * 10 -11Below the M; And/or K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1, preferred 1 * 10 -2s -1～1 * 10 -6s -1, more preferably 5 * 10 -3s -1～1 * 10 -5s -1For example 5 * 10 -1s -1Below, preferred 1 * 10 -2s -1Below, advantageously 1 * 10 -3s -1Below, more preferably 1 * 10 -4s -1Below, also more preferably 1 * 10 -5s -1Below, most preferably 1 * 10 -6s -1Below.
Preferred part or dAb monomer inhibition TNF α combine its inhibition concentration 50 (IC with TNF α receptor 1 (p55 receptor) 50) be 500nM～50pM, preferred 100nM～50pM, more preferably 10nM～100pM, 1nM～100pM advantageously; For example following, the preferred 5nM of 50nM following, more preferably 500pM following, advantageously 200pM following, most preferably below the 100pM.The target of preferred TNF receptor 1 is a human TNF alpha.
Preferred part or dAb are in conjunction with people TNFR1 and suppress combining of human TNF alpha and people TNFR1, perhaps suppress to conduct in conjunction with the signal that passes through TNFR1 because of response TNF α.For example, in certain embodiments, described part or dAb monomer can and suppress the signal conduction of TNFR1 mediation in conjunction with TNFR1, but do not suppress combining of TNF α and TNFR1 basically.In certain embodiments, described part or dAb monomer suppress the crosslinked or cluster by TNFR1 on the inductive cell surface of TNF α.This class part or dAb (TAR2m-21-23 for example as herein described) they are favourable, but because the TNFR1 of their antagonism cell surfaces, but do not reduce the inhibition activity of endogenous soluble TNF R1 basically.For example, in receptors bind was measured, described part or dAb can be in conjunction with TNFR1, but suppress TNF α and combining of TNFR1 be no more than about 10%, be no more than about 5%, be no more than about 4%, be no more than about 3%, be no more than about 2% or be no more than about 1%.And, in these embodiments, in standard cell lines was measured, described part or dAb suppressed the crosslinked and/or TNFR1 Mediated Signal Transduction of the inductive TNFR1 of TNF α at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99%.
As described herein, part can be monovalent (a for example dAb monomer) or polyvalent (for example bispecific, polyspecific).In specific embodiment, described part is can be in conjunction with the dAb monomer in the territory 1 of TNFR1.With respect to other combining form (for example monoclonal antibody), can have little footprint (smallfootprint) in conjunction with the domain antibodies monomer in the territory 1 of TNFR1.Therefore, such dAb monomer is alternative blocks territory 1, but does not disturb the function of other domain of TNFR1.For example, but can be in conjunction with the dAb monomer antagonism TNFR1 in the territory 1 of TNFR1, but do not suppress TNF α and combining of TNPR1 or coming off of TNFR1.
In a more particular embodiment, described part is the dAb monomer that can combine mice TNFR1 in conjunction with the territory 1 of TNFR1 and with TAR2m-21-23 competitiveness or combine people TNFR1 with TAR2h-205 competitiveness.
In other embodiments, described part is polyvalent, and comprising two or more can be in conjunction with the dAb monomer of TNFR1.What multivalent ligand can contain two or more copies can be in conjunction with the specific dAb of TNFR1, and perhaps containing two or more can be in conjunction with the dAb of TNFR1.For example, part as herein described can be dimer, trimer or polymer, and what it comprised two or more copies can be in conjunction with the specific dAb of TNFR1, and perhaps two or more can be in conjunction with the different dAb of TNFR1.In some example kinds, part is homodimer or homotrimer, and what they comprised two or three copies respectively can be in conjunction with the specific dAb of TNFR1.In standard cell lines is measured, the preferred not exciting basically TNFR1 of multivalent ligand (as the TNFR1 agonist) is (promptly in mensuration, when concentration is 1nM, 10nM, 100nM, 1 μ M, 10 μ M, 100 μ M, 1000 μ M or 5, during 000 μ M, cause being no more than about 5% activity) by the inductive TNFR1 mediation of TNF α (100pg/ml).
In certain embodiments, multivalent ligand contain two or more can be in conjunction with the dAb of required epi-position of TNFR1 or domain.What for example, multivalent ligand can comprise two or more copies can be in conjunction with the dAb of the territory 1 required epi-position of TNFR1.
In other embodiments, multivalent ligand contain two or more can be in conjunction with the dAb of different epi-positions of TNFR1 or domain.In an example, multivalent ligand comprises a dAb and the 2nd dAb, and wherein a dAb can be in conjunction with first epi-position in the territory 1 of TNFR1, the 2nd dAb can territory 1 in conjunction with TNFR1 in the second different epi-positions.In other example, multivalent ligand comprises can be in conjunction with two or more the required epi-positions of TNFR1 or the dAb of domain.For example, multivalent ligand can comprise the dAb in conjunction with the domain of following TNFR1: territory 1 and territory 2, territory 1 and territory 3, territory 1 and territory 4, territory 2 and territory 3, territory 2 and territory 4 or territory 3 and territory 4.
In certain embodiments, multivalent ligand is the bispecific part, and described part comprises can be in conjunction with the dAb in the territory 1 of TNFR1, and can be in conjunction with the dAb in the territory 3 of TNFR1.Such part can high-affinity in conjunction with TNFR1, and compared with other part form (for example dAb monomer), at its surperficial high density overexpression TNFR1 or express cell higher of TNFR1 in conjunction with selectivity.
In other specific embodiment, multivalent ligand comprises two or more can be in conjunction with the dAb in the territory 1 of TNFR1 or the specific dAb of two or more copies.Such multivalent ligand can low-affinity in conjunction with the TNFR1 monomer, but with high affinity bind receptor polymer (for example viewed trimer in the receptors ligand complex).Therefore, the part of this form can be given the efficient targeting receptor, described receptor is cluster or association and/or part (for example TNF α) bunch collection or the association required with the TNFR1 Mediated Signal Transduction each other.
Some parts or dAb monomer are in conjunction with TNFR1 and suppress combining of TNF α and TNFR1.In certain embodiments, such part or dAb monomer can be in conjunction with territory 2 and/or the territories 3 of TNFR1.In specific embodiment, described part or dAb monomer can be in conjunction with the territories 3 of TNFR1.In a more particular embodiment, described part or dAb monomer can combine TNFR1 in conjunction with the territory 3 of TNFR1 and with TAR2h-10-27, TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 or TAR2h-185-25 competitiveness.
Other part or dAb monomer do not suppress combining of TNF α and TNFR1.This class antagonist has the advantage as diagnostic reagent because they can be used for the TNFR1 in combination and detection, quantification or the working sample, but not with sample in TNF competitiveness combine TNFR1.Therefore, accurately whether there is TNFR1 and how many TNFR1 are arranged in the working sample.
Some parts and dAb monomer do not suppress combining of TNF α and TNFR1, but suppress by the TNFR1 Mediated Signal Transduction.For example, part or dAb monomer can suppress the inductive TNFR1 cluster of TNF α, and this is the step before passing through the signal transduction of TNFR1.This class part or dAb monomer have some advantages, as relevant antagonist with these characteristics discussed in this article.In specific embodiment, such part or dAb monomer can be in conjunction with the territory 1 of TNFR1 or the territories 4 of TNFR1.In certain embodiments, described part is can be in conjunction with the dAb monomer in the territory 4 of the territory 1 of TNFR1 or TNFR1.
In a specific embodiment, described part or dAb monomer can be in conjunction with the territories 1 of TNFR1, and in case in conjunction with just suppressing to pass through the TNFR1 Mediated Signal Transduction behind the TNF α.This class part or dAb monomer can suppress the signal transduction by TNFR1, but do not suppress TNF α and TNFR1 combine and/or coming off of TNFR1 produces soluble TNF R1.Therefore, such part or dAb monomer are had the mammal that needs, can replenish endogenous adjusting approach, described approach suppresses the activity of TNF α and the activity of TNFR1 in vivo.
Other part or dAb monomer can be in conjunction with TNFR1, but not in conjunction with territory 4.This class part or dAb monomer can suppress the function of TNFR1, but do not suppress coming off of soluble TNF R1.Therefore, such antagonist is had the mammal that needs, can replenish endogenous adjusting approach, described approach suppresses the activity of TNF α and the activity of TNFR1 in vivo.
In standard test (standard L929 for example as herein described or standard HeLa IL-8 measure), in preferred part or the dAb monomer and TNF α or TNFR1 (suppressing its activity), its nertralizer amount 50 (ND 50) be 500nM～50pM, preferred 100nM～50pM, more preferably 10nM～100pM, 1nM～100pM advantageously; For example following, the preferred 5nM of 50nM following, more preferably 500pM following, advantageously 200pM following, most preferably below the 100pM.
In certain embodiments, described part or dAb monomer specificity are in conjunction with human tumor necrosis factor receptor 1 (TNFR1; P55), according to the mensuration of surface plasma resonance, the dissociation constant (K that described part or dAb monomer disintegrate down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1
In other embodiments, described part or dAb monomer specificity are in conjunction with TNFR1 (its K dAs described herein) and in the septic shock model that standard mice LPS/D-galactosamine brings out Y-suppressed lethal rate (promptly compare with appropriate control, its prevention causes death or reduces fatality rate at least about 10%).In the septic shock model that standard mice LPS/D-galactosamine brings out, compare with appropriate control, as about 5mg/kg or more preferably from about during the dAb monomer of 1mg/kg, preferred dAb monomer Y-suppressed lethal rate is at least about 25% or at least about 50%.
In other embodiments, in standard cell lines was measured, described part or dAb monomer were in conjunction with TNFR1 and the active ND of antagonism TNFR1 50≤ 100nM, when concentration≤10 μ M, activity≤5% of the exciting TNFR1 of dAb.
In specific embodiment, in standard cell lines is measured, the not exciting basically TNFR1 of described part or dAb monomer (playing the effect of TNFR1 agonist) is (promptly in mensuration, when concentration is 1nM, 10nM, 100nM, 1 μ M, 10 μ M, 100 μ M, 1000 μ M or 5, during 000 μ M, cause being no more than about 5% activity) by the inductive TNPR1 mediation of TNF α (100pg/ml).
In certain embodiments, described part or dAb monomer can be resisted gathering basically.For example, in certain embodiments, when with 1-5mg/ml, 5-10mg/ml, 10-20mg/ml, 20-50mg/ml, 50-100mg/ml, 100-200mg/ml or 200-500mg/ml part or dAb solution are dissolved in medicine preparation common solvent (saline for example, buffer saline, citric acid buffer brine, water, emulsifying agent and any of these contain the solvent of acceptable excipient, the excipient that described excipient is for example ratified by FDA) time, will be less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% part or dAb monomer aggregation thing, at about 22 ℃, 22-25 ℃, 25-30 ℃, 30-37 ℃, 37-40 ℃, 40-50 ℃, 50-60 ℃, 60-70 ℃, 70-80 ℃, 15-20 ℃, 10-15 ℃, 5-10 ℃, 2-5 ℃, 0-2 ℃,-10 ℃ to 0 ℃,-20 ℃ to-10 ℃,-40 ℃ to-20 ℃,-60 ℃ to-40 ℃ or-80 ℃ are to-60 ℃ temperature, keep a period of time, for example 10 minutes, 1 hour, 8 hours, 24 hours, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, January, February, March, April, June, 1 year or 2 years.
Can adopt microscope inspection, detect any appropriate method such as deciding solution turbidity, gathering is estimated by range estimation or spectroscope.Preferably estimate gathering by dynamic light scattering.Anti-accumulative part or dAb monomer have some advantages.For example, express by adopting suitable biological production system (for example escherichia coli), form with soluble protein produces this class part or dAb monomer and yield height easily, can the concentration higher than conventional polypeptide prepare and/or store then, gathering and active loss are lower simultaneously.
In addition, produce anti-accumulative part or dAb monomer, can be more more economical in conjunction with polypeptide (for example conventional antibody) than producing other antigen or epi-position.For example, be used for the antigen used in the body or epi-position and generally include the process (example gel filtration) of assembling polypeptide of removing in conjunction with the preparation of polypeptide.If can not remove such aggregation, preparation is not suitable in the body uses, for example, if, can play the effect of agonist by the crosslinked or cluster of inducing target antigen because wish to play the accumulative words of antigen-binding polypeptides of antagonist action.Gathering albumen also can reduce the effect of therapeutical peptide by induce immune response in accepting their patient's body.
By contrast, can prepare anti-gathering part of the present invention or dAb monomer, be used for using in the body, and need not to comprise the process of removing aggregation, and can be used for using in the body, and do not assemble the caused above-mentioned shortcoming of polypeptide.
In certain embodiments, described part or dAb monomer can reversibly be separated folding when being heated to temperature (Ts) and being cooled to temperature (Tc), and wherein Ts is higher than the melting temperature (Tm) of dAb, and Tc is lower than the melting temperature of dAb.For example, the dAb monomer is when being heated to 80 ℃ and can reversibly separate when being cooled to about room temperature folding.Reversibly separate folding polypeptide in that separate when folding can loss of function, in case and can restore funcitons after the refolding.This class polypeptide is different from separating and assembles when folding or the polypeptide (polypeptide of misfolding) of inappropriate refolding (can not restore funcitons).
Can adopt any appropriate method, estimate polypeptide by direct or indirect mensuration polypeptide structure and separate folding and refolding.For example, can pass through circular dichroism (CD) (UV CD for example far away, nearly UV CD), fluorescence (for example fluorescence of tryptophan side chain), sensitivity, nuclear magnetic resonance, NMR (NMR) to proteolysis, perhaps by detect or measure depend on suitable folding polypeptide function (for example with the combining of target ligands, with the combining of general part), measure polypeptide structure.In an example, adopt functional examination, estimate polypeptide and separate foldingly, the wherein forfeiture of combined function (for example in conjunction with general part and/or target ligands, bound substrates) shows that this polypeptide separates folding.
Can adopt and separate folding or degeneration curve, measure the monomeric degree of separating folding and refolding of part or dAb.Can be that temperature and abscissa are folding polypeptide relative concentration mapping by vertical coordinate, folding curve be separated in drafting.Any suitable method (for example CD, fluorescence, combination are measured) be can adopt, folding part or the monomeric relative concentration of dAb directly or indirectly measured.For example, can prepare part or dAb monomer solution, measure the ellipticity of solution then by CD.Folding part of gained ellipticity value representation or the monomeric relative concentration of dAb are 100%.Again by the solution temperature that raises gradually, it is folding that part in the solution or dAb monomer are separated, and measures ellipticity with suitable increment (for example the every rising of temperature once back) then.By reducing solution temperature gradually, make part or the refolding of dAb monomer in the solution then, measure ellipticity with suitable increment again.Can map to data, draw and separate folding curve and refolding curve.Separate folding curve and refolding curve and have distinctive S shape, it comprises part or dAb monomer molecule folded part, part or dAb monomer molecule are separated the folding/refolding conversion portion of separating when folding in various degree, and part or dAb monomer molecule are separated folded part.The Y-axis intercept of refolding curve is refolding accessories or the monomeric relative populations of recovering of dAb.At least about 50% or at least about 60% or at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% recovery, show that part or dAb monomer reversibly separate folding.
In a preferred embodiment, add the folding and refolding curve of pyrolysis by preparing part or dAb monomer solution and drawing, mensuration part or dAb monomer are separated folding reversibility.Can prepare part or dAb monomer solution in any suitable solvent, described solvent is water-containing buffering liquid for example, and its pH is suitable for allowing part or dAb monomer dissolve (for example pH is than high or low approximately 3 units of isoelectric point, IP (pI)).Part or dAb monomer solution are enough dense, so as can to detect separate folding/folding.For example, part or dAb monomer solution can be about 0.1 μ M to about 100 μ M or preferred about 1 μ M to about 10 μ M.
If the monomeric melting temperature of part or dAb (Tm) is known, then solution can be heated to low about 10 degree (Tm-10) than Tm, then by ellipticity or fluorescence (for example far-UV CD can be from 200nm to 250nm, fixed wave length CD is 235nm or 225nm; The tryptophan fluorescence emission spectrum is 300-450nm, excites to be 298nm) estimate folding, so that 100% folding relatively part or dAb monomer to be provided.By predetermined increment (for example about 0.1 degree is to the increase of about 1 degree), solution is heated to than Tm height 10 degree (Tm+10) at least again, when each increment, measures ellipticity or fluorescence.Then,,, make part or the refolding of dAb monomer, when each increment, measure ellipticity or fluorescence by being cooled to Tm-10 at least by predetermined increment.If do not know the monomeric melting temperature of part or dAb, can pass through from about 25 ℃ extremely about 100 ℃ increment heating gradually, it is folding that solution is separated, and increment is cooled to make its refolding at least about 25 ℃ gradually again, measures ellipticity or fluorescence when each heating and cooling increment.Can the mapping of gained data be drawn and be separated folding curve and refolding curve, wherein the Y-axis intercept of refolding curve is the proteinic relative populations of recovering of refolding.
In certain embodiments, part of the present invention or dAb monomer are when giving with effective dose, and be effective in the chronic inflammatory disease model.Usually effective dose is that about 1mg/kg is to about 10mg/kg (for example about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg or about 10mg/kg).Chronic inflammatory disease model as herein described is well known to a person skilled in the art, is used for prediction in the intravital curative effect of people.Whether effectively prior art is not given in the enlightenment of using TNFR1 antagonist (unit price antagonist for example as herein described, part) in these models, do not provide their enlightenments yet.
In specific embodiment, described part or dAb monomer are effective in the collagen-induced arthritis model of standard mice (embodiment 15A).For example compare with appropriate control, in the collagen-induced arthritis model of standard mice, the part that gives effective dose can reduce the average arthritis score summation for example about 1 of extremity to about 16, about 3 to about 16, about 6 to about 16, about 9 to about 16 or about 12 to about 16.In another example, compare with appropriate control, in the collagen-induced arthritis model of standard mice, the part that gives effective dose can postpone the arthritic symptom outbreak for example reach about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days or about 28 days.In another example, in the collagen-induced arthritis model of standard mice, it is 0 to about 3, about 3 to about 5, about 5 to about 7, about 7 to about 15, about 9 to about 15, about 10 to about 15, about 12 to about 15 or about 14 to about 15 that the part that gives effective dose can cause the average arthritis score summation of extremity.
In other embodiments, described part or dAb monomer are effectively (embodiment 15B) in mouse arthritis Δ ARE model.For example, compare with appropriate control, in mouse arthritis Δ ARE model, the part that gives effective dose can reduce average arthritis score and for example reach about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about 2.5 or about 2 to about 2.5.In another example, compare with appropriate control, in mouse arthritis Δ ARE model, the part that gives effective dose can postpone the arthritic symptom outbreak for example reach about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days or about 28 days.In another example, in mouse arthritis Δ ARE model, it is 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2 or about 2 to about 2.5 that the part that gives effective dose can cause average arthritis score.
In other embodiments, described part or dAb monomer are effective in mice inflammatory bowel (IBD) Δ ARE model (embodiment 15B).For example, compare with appropriate control, in mice IBD Δ ARE model, the part that gives effective dose can reduce average acute and/or the chronic inflammatory disease scoring for example reaches about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about 2.5 or about 2 to about 2.5.In another example, compare with appropriate control, in mice IBD Δ ARE model, the part that gives effective dose can postpone the IBD paresthesia epilepsy for example reach about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days or about 28 days.In another example, in mice IBD Δ ARE model, the part that gives effective dose can cause average acute and/or the chronic inflammatory disease scoring is 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2 or about 2 to about 2.5.
In other embodiments, described part or dAb monomer are effectively (embodiment 15C) in the IBD model that mice dextran sulfate sodium (DSS) is brought out.For example, compare with appropriate control, in mice DSS IBD model, the part that gives effective dose can reduce average seriousness scoring and for example reach about 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about 2.5 or about 2 to about 2.5.In another example, compare with appropriate control, in mice DSS IBD model, the part that gives effective dose can postpone the IBD paresthesia epilepsy for example reach about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days or about 28 days.In another example, in mice DSS IBD model, it is 0 to about 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2 or about 2 to about 2.5 that the part that gives effective dose can cause average seriousness scoring.
In specific embodiments, described part or dAb monomer are effective (embodiment 15D) in mice caused by smoking chronic obstructive pulmonary disease (COPD) model.For example, compare with appropriate control, the part that gives effective dose can reduce or postpone the COPD paresthesia epilepsy.
In specific embodiments, described part or dAb monomer specificity are in conjunction with TNFR1 and comprise the aminoacid sequence (SEQ ID NO:31) of TAR2-10 or have the sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with it.
In specific embodiment, described part or dAb monomer specificity are in conjunction with TNFR1 and comprise the aminoacid sequence (SEQ ID NO:195) of TAR2-5 or have the sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with it.
In other embodiments, described part IncFlds antibody (dAb) monomer, described dAb monomer and Tumor Necrosis Factor Receptors 1 (TNFR1, p55, CD120a) the bonded K of specificity dBe 300nM～5pM, and the monomeric aminoacid sequence of described dAb be selected from following aminoacid sequence or dAb has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-12 (SEQ ID NO:32), TAR2h-13 (SEQ ID NO:33), TAR2h-14 (SEQ ID NO:34), TAR2h-16 (SEQ ID NO:35), TAR2h-17 (SEQ ID NO:36), TAR2h-18 (SEQ ID NO:37), TAR2h-19 (SEQ ID NO:38), TAR2h-20 (SEQ ID NO:39), TAR2h-21 (SEQ ID NO:40), TAR2h-22 (SEQ ID NO:41), TAR2h-23 (SEQ ID NO:42), TAR2h-24 (SEQ ID NO:43), TAR2h-25 (SEQ ID NO:44), TAR2h-26 (SEQ ID NO:45), TAR2h-27 (SEQ ID NO:46), TAR2h-29 (SEQ ID NO:47), TAR2h-30 (SEQ ID NO:48), TAR2h-32 (SEQ ID NO:49), TAR2h-33 (SEQ ID NO:50), TAR2h-10-1 (SEQ ID NO:51), TAR2h-10-2 (SEQ ID NO:52), TAR2h-10-3 (SEQ ID NO:53), TAR2h-10-4 (SEQ ID NO:54), TAR2h-10-5 (SEQ ID NO:55), TAR2h-10-6 (SEQ ID NO:56), TAR2h-10-7 (SEQ ID NO:57), TAR2h-10-8 (SEQ ID NO:58), TAR2h-10-9 (SEQ ID NO:59), TAR2h-10-10 (SEQ ID NO:60), TAR2h-10-11 (SEQ ID NO:61), TAR2h-10-12 (SEQ ID NO:62), TAR2h-10-13 (SEQ ID NO:63), TAR2h-10-14 (SEQ ID NO:64), TAR2h-10-15 (SEQ ID NO:65), TAR2h-10-16 (SEQ ID NO:66), TAR2h-10-17 (SEQ ID NO:67), TAR2h-10-18 (SEQ ID NO:68), TAR2h-10-19 (SEQ ID NO:69), TAR2h-10-20 (SEQ ID NO:70), TAR2h-10-21 (SEQ ID NO:71), TAR2h-10-22 (SEQ ID NO:72), TAR2h-10-27 (SEQ ID NO:73), TAR2h-10-29 (SEQ ID NO:74), TAR2h-10-31 (SEQ ID NO:75), TAR2h-10-35 (SEQ ID NO:76), TAR2h-10-36 (SEQ ID NO:77), TAR2h-10-37 (SEQ ID NO:78), TAR2h-10-38 (SEQ ID NO:79), TAR2h-10-45 (SEQ ID NO:80), TAR2h-10-47 (SEQ ID NO:81), TAR2h-10-48 (SEQ ID NO:82), TAR2h-10-57 (SEQ ID NO:83), TAR2h-10-56 (SEQ ID NO:84), TAR2h-10-58 (SEQ ID NO:85), TAR2h-10-66 (SEQ ID NO:86), TAR2h-10-64 (SEQ ID NO:87), TAR2h-10-65 (SEQ ID NO:88), TAR2h-10-68 (SEQ ID NO:89), TAR2h-10-69 (SEQ ID NO:90), TAR2h-10-67 (SEQ ID NO:91), TAR2h-10-61 (SEQ ID NO:92), TAR2h-10-62 (SEQ ID NO:93), TAR2h-10-63 (SEQ ID NO:94), TAR2h-10-60 (SEQ ID NO:95), TAR2h-10-55 (SEQ ID NO:96), TAR2h-10-59 (SEQ ID NO:97), TAR2h-10-70 (SEQ ID NO:98), TAR2h-34 (SEQ ID NO:373), TAR2h-35 (SEQ ID NO:374), TAR2h-36 (SEQ ID NO:375), TAR2h-37 (SEQ ID NO:376), TAR2h-38 (SEQ ID NO:377), TAR2h-39 (SEQ ID NO:378), TAR2h-40 (SEQID NO:379), TAR2h-41 (SEQ ID NO:380), TAR2h-42 (SEQ ID NO:381), TAR2h-43 (SEQ ID NO:382), TAR2h-44 (SEQ ID NO:383), TAR2h-45 (SEQ ID NO:384), TAR2h-47 (SEQ ID NO:385), TAR2h-48 (SEQ ID NO:386), TAR2h-50 (SEQ ID NO:387), TAR2h-51 (SEQID NO:388), TAR2h-66 (SEQ ID NO:389), TAR2h-67 (SEQ ID NO:390), TAR2h-68 (SEQ ID NO:391), TAR2h-70 (SEQ ID NO:392), TAR2h-71 (SEQ ID NO:393), TAR2h-72 (SEQ ID NO:394), TAR2h-73 (SEQ ID NO:395), TAR2h-74 (SEQ ID NO:396), TAR2h-75 (SEQID NO:397), TAR2h-76 (SEQ ID NO:398), TAR2h-77 (SEQ ID NO:399), TAR2h-78 (SEQ ID NO:400), TAR2h-79 (SEQ ID NO:401) and TAR2h-15 (SEQ ID NO:431).
In other embodiments, described part IncFlds antibody (dAb) monomer, described dAb monomer and Tumor Necrosis Factor Receptors 1 (TNFR1, p55, CD120a) the bonded K of specificity dBe 300nM～5pM, and the monomeric aminoacid sequence of described dAb be selected from following aminoacid sequence or dAb has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-131-8 (SEQ ID NO:433), TAR2h-131-24 (SEQ ID NO:434), TAR2h-15-8 (SEQ ID NO:435), TAR2h-15-8-1 SEQ ID NO:436), TAR2h-15-8-2 (SEQ ID NO:437), TAR2h-185-23 (SEQ ID NO:438), TAR2h-154-10-5 (SEQ ID NO:439), TAR2h-14-2 (SEQ ID NO:440), TAR2h-151-8 (SEQ ID NO:441), TAR2h-152-7 (SEQ ID NO:442), TAR2h-35-4 (SEQ ID NO:443), TAR2h-154-7 (SEQ ID NO:444), TAR2h-80 (SEQ ID NO:445), TAR2h-81 (SEQ ID NO:446), TAR2h-82 (SEQ ID NO:447), TAR2h-83 (SEQ ID NO:448), TAR2h-84 (SEQID NO:449), TAR2h-85 (SEQ ID NO:450), TAR2h-86 (SEQ ID NO:451), TAR2h-87 (SEQ ID NO:452), TAR2h-88 (SEQ ID NO:453), TAR2h-89 (SEQ ID NO:454), TAR2h-90 (SEQ ID NO:455), TAR2h-91 (SEQ ID NO:456), TAR2h-92 (SEQ ID NO:457), TAR2h-93 (SEQID NO:458), TAR2h-94 (SEQ ID NO:459), TAR2h-95 (SEQ ID NO:460), TAR2h-96 (SEQ ID NO:461), TAR2h-97 (SEQ ID NO:462), TAR2h-99 (SEQ ID NO:463), TAR2h-100 (SEQ ID NO:464), TAR2h-101 (SEQ ID NO:465), TAR2h-102 (SEQ ID NO:466), TAR2h-103 (SEQ ID NO:467), TAR2h-104 (SEQ ID NO:468), TAR2h-105 (SEQ ID NO:469), TAR2h-106 (SEQ ID NO:470), TAR2h-107 (SEQ ID NO:471), TAR2h-108 (SEQ ID NO:472), TAR2h-109 (SEQ ID NO:473), TAR2h-110 (SEQ ID NO:474), TAR2h-111 (SEQ ID NO:475), TAR2h-112 (SEQ ID NO:476), TAR2h-113 (SEQ ID NO:477), TAR2h-114 (SEQ ID NO:478), TAR2h-115 (SEQ ID NO:479), TAR2h-116 (SEQ ID NO:480), TAR2h-117 (SEQ ID NO:481), TAR2h-118 (SEQ ID NO:482), TAR2h-119 (SEQ ID NO:483), TAR2h-120 (SEQ ID NO:484), TAR2h-121 (SEQ ID NO:485), TAR2h-122 (SEQ ID NO:486), TAR2h-123 (SEQ ID NO:487), TAR2h-124 (SEQ ID NO:488), TAR2h-125 (SEQ ID NO:489), TAR2h-126 (SEQ ID NO:490), TAR2h-127 (SEQ ID NO:490), TAR2h-128 (SEQ ID NO:492), TAR2h-129 (SEQ ID NO:493), TAR2h-130 (SEQ ID NO:494), TAR2h-131 (SEQ ID NO:495), TAR2h-132 (SEQ ID NO:496), TAR2h-133 (SEQ ID NO:497), TAR2h-151 (SEQ ID NO:498), TAR2h-152 (SEQ ID NO:499), TAR2h-153 (SEQ ID NO:500), TAR2h-154 (SEQ ID NO:501), TAR2h-159 (SEQ ID NO:502), TAR2h-165 (SEQ ID NO:503), TAR2h-166 (SEQ ID NO:504), TAR2h-168 (SEQ ID NO:505), TAR2h-171 (SEQ ID NO:506), TAR2h-172 (SEQ ID NO:507), TAR2h-173 (SEQ ID NO:508), TAR2h-174 (SEQ ID NO:509), TAR2h-176 (SEQ ID NO:510), TAR2h-178 (SEQ ID NO:511), TAR2h-201 (SEQ ID NO:512), TAR2h-202 (SEQ ID NO:513), TAR2h-203 (SEQ ID NO:514), TAR2h-204 (SEQ ID NO:515), TAR2h-185-25 (SEQ ID NO:516), TAR2h-154-10 (SEQ ID NO:517) and TAR2h-205 (SEQ ID NO:627).
In preferred embodiments, the aminoacid sequence of described part or dAb has at least about 90% with the aminoacid sequence that is selected from following dAb, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology: TAR2h-10-27 (SEQ ID NO:73), TAR2h-10-57 (SEQ ID NO:83), TAR2h-10-56 (SEQ ID NO:84), TAR2h-10-58 (SEQ ID NO:85), TAR2h-10-66 (SEQ ID NO:86), TAR2h-10-64 (SEQ ID NO:87), TAR2h-10-65 (SEQ ID NO:88), TAR2h-10-68 (SEQ ID NO:89), TAR2h-10-69 (SEQ ID NO:90), TAR2h-10-67 (SEQ ID NO:91), TAR2h-10-61 (SEQ ID NO:92), TAR2h-10-62 (SEQ ID NO:93), TAR2h-10-63 (SEQ ID NO:94), TAR2h-10-60 (SEQ ID NO:95), TAR2h-10-55 (SEQ ID NO:96), TAR2h-10-59 (SEQ ID NO:97) and TAR2h-10-70 (SEQ ID NO:98).
The aminoacid sequence of the aminoacid sequence of particularly preferred part or dAb and SEQ ID NO:73 has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology.
In other embodiments, described part IncFlds antibody (dAb) monomer, described dAb monomer and Tumor Necrosis Factor Receptors 1 (TNFR1, p55, CD120a) the bonded K of specificity dBe 300nM～5pM, and the monomeric aminoacid sequence of described dAb be selected from following aminoacid sequence or dAb has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2m-14 (SEQ ID NO:167), TAR2m-15 (SEQ ID NO:168), TAR2m-19 (SEQ IDNO:169), TAR2m-20 (SEQ ID NO:170), TAR2m-21 (SEQ ID NO:171), TAR2m-24 (SEQ ID NO:172), TAR2m-21-23 (SEQ ID NO:173), TAR2m-21-07 (SEQ ID NO:174), TAR2m-21-43 (SEQ ID NO:175), TAR2m-21-48 (SEQ ID NO:176), TAR2m-21-10 (SEQ ID NO:177), TAR2m-21-06 (SEQ ID NO:178), TAR2m-21-17 (SEQ ID NO:179).
In certain embodiments, described part comprises the dAb monomer, and described dAb monomer can combine TNFR1 (for example mice TNFR1 and/or people TNFR1) in conjunction with TNFR1 and with any dAb competitiveness disclosed herein.
Preferred part or dAb monomer are when expressing in escherichia coli or Pichia sp. (for example pichia pastoris phaff), and its secretory volume is at least about 0.5mg/L.In other embodiment preferred, when expressing in escherichia coli or Pichia sp. (for example pichia pastoris phaff), the monomeric secretory volume of dAb is at least about 0.75mg/L, at least about 1mg/L, at least about 4mg/L, at least about 5mg/L, at least about 10mg/L, at least about 15mg/L, at least about 20mg/L, at least about 25mg/L, at least about 30mg/L, at least about 35mg/L, at least about 40mg/L, at least about 45mg/L, or at least about 50mg/L, or at least about 100mg/L, or at least about 200mg/L, or at least about 300mg/L, or at least about 400mg/L, or at least about 500mg/L, or at least about 600mg/L, or at least about 700mg/L, or at least about 800mg/L, at least about 900mg/L, or at least about 1g/L.In other embodiment preferred, when expressing in escherichia coli or Pichia sp. (for example pichia pastoris phaff), the monomeric secretory volume of dAb arrives at least about 1g/L at least about 1mg/L, arrive at least about 750mg/L at least about 1mg/L, arrive at least about 1g/L at least about 100mg/L, arrive at least about 1g/L at least about 200mg/L, arrive at least about 1g/L at least about 300mg/L, arrive at least about 1g/L at least about 400mg/L, arrive at least about 1g/L at least about 500mg/L, arrive at least about 1g/L at least about 600mg/L, arrive at least about 1g/L at least about 700mg/L, arrive at least about 1g/L at least about 800mg/L, or at least about 900mg/L at least about 1g/L.Although when in escherichia coli or Pichia sp. (for example pichia pastoris phaff), expressing, part as herein described and dAb monomer can be secreted, but they also can adopt any other appropriate method to prepare, for example do not need with the synthetic chemistry method of escherichia coli or Pichia sp. or biology preparation method.
The dAb monomer can comprise any suitable immune globulin variable region, preferably comprise people variable region or comprise the variable region of people's framework region.In certain embodiments, the dAb monomer comprises general framework as herein described.
General framework can be V LFramework (V λ or V κ), for example comprising by ethnic group is the framework of the coded framework aminoacid sequence of DPK1, DPK2, DPK3, DPK4, DPK5, DPK6, DPK7, DPK8, DPK9, DPK10, DPK12, DPK13, DPK15, DPK16, DPK18, DPK19, DPK20, DPK21, DPK22, DPK23, DPK24, DPK25, DPK26 or DPK28 immunoglobulin gene section.If necessary, V LIt is J κ 1, J κ 2, J κ 3, J κ 4 or the coded framework aminoacid sequence of J κ 5 immunoglobulin gene sections that framework also can comprise by ethnic group.
In other embodiments, general framework can be V HFramework, for example comprising by ethnic group is the framework of the coded framework aminoacid sequence of DP4, DP7, DP8, DP9, DP10, DP31, DP33, DP38, DP45, DP46, DP47, DP49, DP50, DP51, DP53, DP54, DP65, DP66, DP67, DP68 or DP69 immunoglobulin gene section.If necessary, V HIt is J that framework also can comprise by ethnic group H1, J H2, J H3, J H4, J H4b, J H5 and J HThe framework aminoacid sequence that 6 immunoglobulin gene sections are coded.
In specific embodiment, dAb monomer part comprises DPK9 V LFramework or be selected from the V of DP47, DP45 and DP38 HFramework.
The dAb monomer can comprise the binding site of general part, and described general part is protein A, albumen L and Protein G for example.
In certain embodiments, the dAb monomer comprises one or more framework regions, the aminoacid sequence in described framework district and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical, and perhaps to comprise maximum 5 jointly be the different aminoacid of aminoacid sequence of the coded described corresponding framework region of antibody gene section with ethnic group to the aminoacid sequence of one or more described framework regions.
In other embodiments, the aminoacid sequence of the monomeric FW1 of dAb, FW2, FW3 and FW4 and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical, and perhaps to comprise maximum 10 jointly be the different aminoacid of aminoacid sequence of the coded corresponding framework region of antibody gene section with described ethnic group to the aminoacid sequence of FW1, FW2, FW3 and FW4.
In other embodiments, the dAb monomer comprises FW1, FW2 and FW3 district, and the aminoacid sequence in described FW1, FW2 and FW3 district and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical.
In certain embodiments, the dAb monomer does not contain the camellid immune globulin variable region, and perhaps not containing the camellid kind is the peculiar one or more framework aminoacid of the coded immune globulin variable region of antibody gene section.
With bonded part of serum albumin and dAb monomer
The invention provides part or dAb monomer (the bispecific part that for example comprises such dAb), itself and the bonded K of serum albumin (SA) dBe 1nM～500 μ M (promptly 1 * 10 -9～5 * 10 -4), preferred 100nM～10 μ M.Preferably for containing the first anti-SA dAb and at the bispecific part of the 2nd dAb of another target, the 2nd dAb to the affinity of its target (for example through adopting BiaCore to carry out the K that surface plasma resonance is measured dAnd/or K Dissociate) be 1-100000 times (preferred 100-100000 times, more preferably 1000-100000 times or 10000-100000 times) of a dAb to the affinity of SA.For example, a dAb is about 10 μ M in conjunction with the affinity of SA, and the 2nd dAb is 100pM in conjunction with the affinity of its target.Preferred serum albumin is human serum albumin (HSA).In one embodiment, a dAb (or dAb monomer) and the bonded K of SA (for example HSA) dBe about 50nM, preferred 70nM, more preferably 100nM, 150nM or 200nM.
In certain embodiments, SA being had specific dAb monomer comprises the aminoacid sequence (SEQ ID NO:28) of MSA-16 or has the sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with it.
In other embodiments, SA being had specific dAb monomer comprises the aminoacid sequence (SEQ ID NO:30) of MSA-26 or has the sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with it.
In certain embodiments, can resist gathering, reversibly separate folding and/or comprise aforesaid framework region with the bonded dAb monomer of SA, this framework district is used for can be in conjunction with the dAb monomer of TNFR1.
Nucleic acid molecules, carrier and host cell
The present invention also provides the nucleic acid molecules that separates and/or recombinate, described molecule encoding part as herein described and dAb monomer.In certain embodiments, separate and/or the nucleic acid of reorganization comprises nucleotide sequence coded can specificity in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR), wherein said nucleotide sequence be selected from following nucleotide sequence and have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-12 (SEQ ID NO:32), TAR2h-13 (SEQ ID NO:33), TAR2h-14 (SEQ ID NO:34), TAR2h-16 (SEQ ID NO:35), TAR2h-17 (SEQ ID NO:36), TAR2h-18 (SEQ ID NO:37), TAR2h-19 (SEQ ID NO:38), TAR2h-20 (SEQ ID NO:39), TAR2h-21 (SEQ ID NO:40), TAR2h-22 (SEQ ID NO:41), TAR2h-23 (SEQ ID NO:42), TAR2h-24 (SEQ ID NO:43), TAR2h-25 (SEQ ID NO:44), TAR2h-26 (SEQ ID NO:45), TAR2h-27 (SEQ ID NO:46), TAR2h-29 (SEQ ID NO:47), TAR2h-30 (SEQ ID NO:48), TAR2h-32 (SEQ ID NO:49), TAR2h-33 (SEQ ID NO:50), TAR2h-10-1 (SEQ IDNO:51), TAR2h-10-2 (SEQ ID NO:52), TAR2h-10-3 (SEQ ID NO:53), TAR2h-10-4 (SEQ ID NO:54), TAR2h-10-5 (SEQ ID NO:55), TAR2h-10-6 (SEQ ID NO:56), TAR2h-10-7 (SEQ ID NO:57), TAR2h-10-8 (SEQ ID NO:58), TAR2h-10-9 (SEQ ID NO:59), TAR2h-10-10 (SEQ ID NO:60), TAR2h-10-11 (SEQ ID NO:61), TAR2h-10-12 (SEQ ID NO:62), TAR2h-10-13 (SEQ ID NO:63), TAR2h-10-14 (SEQ ID NO:64), TAR2h-10-15 (SEQ ID NO:65), TAR2h-10-16 (SEQ ID NO:66), TAR2h-10-17 (SEQ ID NO:67), TAR2h-10-18 (SEQ ID NO:68), TAR2h-10-19 (SEQ ID NO:69), TAR2h-10-20 (SEQ ID NO:70), TAR2h-10-21 (SEQ ID NO:71), TAR2h-10-22 (SEQ ID NO:72), TAR2h-10-27 (SEQ ID NO:73), TAR2h-10-29 (SEQ ID NO:74), TAR2h-10-31 (SEQ ID NO:75), TAR2h-10-35 (SEQ ID NO:76), TAR2h-10-36 (SEQ ID NO:77), TAR2h-10-37 (SEQ ID NO:78), TAR2h-10-38 (SEQ ID NO:79), TAR2h-10-45 (SEQ ID NO:80), TAR2h-10-47 (SEQ ID NO:81), TAR2h-10-48 (SEQ ID NO:82), TAR2h-10-57 (SEQ ID NO:83), TAR2h-10-56 (SEQ ID NO:84), TAR2h-10-58 (SEQ ID NO:85), TAR2h-10-66 (SEQ ID NO:86), TAR2h-10-64 (SEQ ID NO:87), TAR2h-10-65 (SEQ ID NO:88), TAR2h-10-68 (SEQ ID NO:89), TAR2h-10-69 (SEQ ID NO:90), TAR2h-10-67 (SEQ ID NO:91), TAR2h-10-61 (SEQ ID NO:92), TAR2h-10-62 (SEQ ID NO:93), TAR2h-10-63 (SEQ ID NO:94), TAR2h-10-60 (SEQ ID NO:95), TAR2h-10-55 (SEQ ID NO:96), TAR2h-10-59 (SEQ ID NO:97), TAR2h-10-70 (SEQ ID NO:98), TAR2h-34 (SEQ ID NO:402), TAR2h-35 (SEQ ID NO:403), TAR2h-36 (SEQ ID NO:404), TAR2h-37 (SEQ ID NO:405), TAR2h-38 (SEQ ID NO:406), TAR2h-39 (SEQID NO:407), TAR2h-40 (SEQ ID NO:408), TAR2h-41 (SEQ ID NO:409), TAR2h-42 (SEQ ID NO:410), TAR2h-43 (SEQ ID NO:411), TAR2h-44 (SEQ ID NO:412), TAR2h-45 (SEQ ID NO:413), TAR2h-47 (SEQ ID NO:414), TAR2h-48 (SEQ ID NO:415), TAR2h-50 (SEQID NO:416), TAR2h-51 (SEQ ID NO:417), TAR2h-66 (SEQ ID NO:418), TAR2h-67 (SEQ ID NO:419), TAR2h-68 (SEQ ID NO:420), TAR2h-70 (SEQ ID NO:421), TAR2h-71 (SEQ ID NO:422), TAR2h-72 (SEQ ID NO:423), TAR2h-73 (SEQ ID NO:424), TAR2h-74 (SEQID NO:425), TAR2h-75 (SEQ ID NO:426), TAR2h-76 (SEQ ID NO:427), TAR2h-77 (SEQ ID NO:428), TAR2h-78 (SEQ ID NO:429), TAR2h-79 (SEQ ID NO:430) and TAR2h-15 (SEQ ID NO:432).
In other embodiments, separate and/or the nucleic acid of reorganization comprises nucleotide sequence coded can specificity in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said nucleotide sequence be selected from following nucleotide sequence and have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2h-131-8 (SEQ ID NO:518), TAR2h-131-24 (SEQ ID NO:519), TAR2h-15-8 (SEQ ID NO:520), TAR2h-15-8-1SEQ ID NO:521), TAR2h-15-8-2 (SEQ ID NO:522), TAR2h-185-23 (SEQ ID NO:523), TAR2h-154-10-5 (SEQ ID NO:524), TAR2h-14-2 (SEQ ID NO:525), TAR2h-151-8 (SEQ ID NO:526), TAR2h-152-7 (SEQ ID NO:527), TAR2h-35-4 (SEQ ID NO:528), TAR2h-154-7 (SEQID NO:529), TAR2h-80 (SEQ ID NO:530), TAR2h-81 (SEQ ID NO:531), TAR2h-82 (SEQ ID NO:532), TAR2h-83 (SEQ ID NO:533), TAR2h-84 (SEQ ID NO:534), TAR2h-85 (SEQ ID NO:535), TAR2h-86 (SEQ ID NO:536), TAR2h-87 (SEQ ID NO:537), TAR2h-88 (SEQID NO:538), TAR2h-89 (SEQ ID NO:539), TAR2h-90 (SEQ ID NO:540), TAR2h-91 (SEQ ID NO:541), TAR2h-92 (SEQ ID NO:542), TAR2h-93 (SEQ ID NO:543), TAR2h-94 (SEQ ID NO:544), TAR2h-95 (SEQ ID NO:545), TAR2h-96 (SEQ ID NO:546), TAR2h-97 (SEQID NO:547), TAR2h-99 (SEQ ID NO:548), TAR2h-100 (SEQ ID NO:549), TAR2h-101 (SEQ ID NO:550), TAR2h-102 (SEQ ID NO:551), TAR2h-103 (SEQ ID NO:552), TAR2h-104 (SEQ ID NO:553), TAR2h-105 (SEQ ID NO:554), TAR2h-106 (SEQ ID NO:555), TAR2h-107 (SEQ ID NO:556), TAR2h-108 (SEQ ID NO:557), TAR2h-109 (SEQ ID NO:558), TAR2h-110 (SEQ ID NO:559), TAR2h-111 (SEQ ID NO:560), TAR2h-112 (SEQ ID NO:561), TAR2h-113 (SEQ ID NO:562), TAR2h-114 (SEQ ID NO:563), TAR2h-115 (SEQ ID NO:564), TAR2h-116 (SEQ ID NO:565), TAR2h-117 (SEQ ID NO:566), TAR2h-118 (SEQ ID NO:567), TAR2h-119 (SEQ ID NO:568), TAR2h-120 (SEQ ID NO:569), TAR2h-121 (SEQ ID NO:570), TAR2h-122 (SEQ ID NO:571), TAR2h-123 (SEQ ID NO:572), TAR2h-124 (SEQ ID NO:573), TAR2h-125 (SEQ ID NO:574), TAR2h-126 (SEQ ID NO:575), TAR2h-127 (SEQ ID NO:576), TAR2h-128 (SEQ ID NO:577), TAR2h-129 (SEQ ID NO:578), TAR2h-130 (SEQ ID NO:579), TAR2h-131 (SEQ ID NO:580), TAR2h-132 (SEQ ID NO:581), TAR2h-133 (SEQ ID NO:582), TAR2h-151 (SEQ ID NO:583), TAR2h-152 (SEQ ID NO:584), TAR2h-153 (SEQ ID NO:585), TAR2h-154 (SEQ ID NO:586), TAR2h-159 (SEQ ID NO:587), TAR2h-165 (SEQ ID NO:588), TAR2h-166 (SEQ ID NO:589), TAR2h-168 (SEQ ID NO:590), TAR2h-171 (SEQ ID NO:591), TAR2h-172 (SEQ ID NO:592), TAR2h-173 (SEQ ID NO:593), TAR2h-174 (SEQ ID NO:594), TAR2h-176 (SEQ ID NO:595), TAR2h-178 (SEQ ID NO:596), TAR2h-201 (SEQ ID NO:597), TAR2h-202 (SEQ ID NO:598), TAR2h-203 (SEQ ID NO:599), TAR2h-204 (SEQ ID NO:600), TAR2h-185-25 (SEQ ID NO:601), TAR2h-154-10 (SEQ ID NO:602) and TAR2h-205 (SEQ ID NO:628).
In a preferred embodiment, separate and/or nucleotide sequence that the nucleic acid of reorganization comprises be selected from following nucleotide and have at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2m-10-27 (SEQ ID NO:141), TAR2m-10-57 (SEQ ID NO:151), TAR2m-10-56 (SEQ ID NO:152), TAR2m-10-58 (SEQ ID NO:153), TAR2m-10-66 (SEQ ID NO:154), TAR2m-10-64 (SEQ ID NO:155), TAR2m-10-65 (SEQ ID NO:156), TAR2m-10-68 (SEQ ID NO:157), TAR2m-10-69 (SEQ ID NO:158), TAR2m-10-67 (SEQ ID NO:159), TAR2m-10-61 (SEQ ID NO:160), TAR2m-10-62 (SEQ ID NO:161), TAR2m-10-63 (SEQ ID NO:162), TAR2m-10-60 (SEQ ID NO:163), TAR2m-10-55 (SEQ ID NO:164), TAR2m-10-59 (SEQ ID NO:165) and TAR2m-10-70 (SEQ ID NO:166).
In other embodiments, separate and/or the nucleic acid of reorganization comprises nucleotide sequence coded can specificity in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said nucleotide sequence be selected from following nucleotide sequence and have at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology: TAR2m-14 (SEQ ID NO:180), TAR2m-15 (SEQID NO:181), TAR2m-19 (SEQ ID NO:182), TAR2m-20 (SEQ ID NO:183), TAR2m-21 (SEQ ID NO:184), TAR2m-24 (SEQ ID NO:185), TAR2m-21-23 (SEQ ID NO:186), TAR2m-21-07 (SEQ ID NO:187), TAR2m-21-43 (SEQ ID NO:188), TAR2m-21-48 (SEQ ID NO:189), TAR2m-21-10 (SEQ ID NO:190), TAR2m-21-06 (SEQ ID NO:191) and TAR2m-21-17 (SEQ ID NO:192) and TAR2m-21-23a (SEQ ID NO:626).
The present invention also provides the carrier that comprises recombinant nucleic acid molecules of the present invention.In certain embodiments, carrier is to comprise the expression control element that recombinant nucleic acid operability one or more and of the present invention is connected or the expression vector of sequence.Suitable carriers (for example plasmid, phasmid) and expression control element further describe as follows.
The present invention also provides recombinant host cell, and described cell comprises recombinant nucleic acid molecules of the present invention or carrier.The method that proper host cell and being used to prepares recombinant host cell of the present invention further describes as follows.
The present invention also provides the method for preparation part of the present invention (for example dAb monomer, bispecific part, polyspecific part), this method is included in and is suitable for keeping the recombinant host cell that comprises recombinant nucleic acid of the present invention under the condition that described recombinant nucleic acid expresses, thus express recombinant nucleic acid and produce part.In certain embodiments, this method also comprises and isolates part.
The part form
Part of the present invention can be made with extra care becomes monospecific or multi-specificity antibody or antibody fragment or monospecific or polyspecific NIg structure.Suitable form comprises any suitable polypeptide structure, and wherein antibody variable region or its CDR can combine, so that structurally give antigenic binding specificity.Various suitable antibody formations are known in the art, for example the homodimer of IgG sample form, chimeric antibody, humanized antibody, people's antibody, single-chain antibody, bi-specific antibody, heavy chain of antibody, light chain of antibody, heavy chain of antibody and/or light chain and heterodimer, any aforementioned Fab (for example Fv fragment (for example strand Fv (scFv), disulfide bond are connected Fv), Fab fragment, Fab ' fragment, F (ab ') 2Fragment), single variable region (V for example H, V L), dAb and any aforesaid modified forms (for example by covalently bound poly-alkane glycol (for example Polyethylene Glycol, polypropylene glycol, polytetramethylene glycol) or other suitable polymers) modify).Referring to PCT/GB03/002804 (application on June 30th, 2003, designated state is the U.S., and publication No. is WO 2004/081026) about the PEG of half-life, suitable PEG, preferred hydrodynamics size and the single variable region of PEGization and the dAb monomer and the polymer of preferred hydrodynamics size in the body of PEGization, its appropriate preparation method, the single variable region of PEGization and dAb monomer and the polymeric prolongation of single variable region and dAb.Whole disclosures of PCT/GB03/002804 (WO 2004/081026) comprise above mentioned part, all are attached to herein by reference.
In specific embodiment, described part (for example dAb monomer, dimer or polymer, bispecific form, polyspecific form) is the PEGization part, and can be in conjunction with territory 1 and the optional TNFR1 function that suppresses of TNFR1.Preferred PEGization part suppresses the signal transduction by TNFR1.Preferred PEGization part and the territory 1 bonded affinity of TNFR1 and the affinity of the identical ligands of PEGization is not identical.For example, in one embodiment, described part is the PEGization dAb monomer that can also suppress in conjunction with the territory 1 of TNFR1 by the signal transduction of TNFR1, wherein the difference between the affinity of the dAb of the territory 1 bonded affinity of PEGization dAb monomer and TNFR1 and non-PEGization form is no more than about 1000 times, preferably is no more than about 100 times, more preferably no more than about 10 times, perhaps the former affinity is constant substantially with respect to the affinity of PEGization form not.
In conjunction with the film of TNFR1 in conjunction with the territory 1 of (striding film) and soluble form but do not suppress TNF α and the bonded part of the present invention of receptor form, can be used on the barrier film bind receptor territory 1 (for example to suppress receptor clustering and/or to suppress signal transduction) and also in conjunction with soluble form, with prolong half-life, particularly when being connected with PEG or during according to above-mentioned other method.In a preferred embodiment, described part is not in conjunction with the film combination (striding film) of TNFR1 and the territory 2 of soluble form.In another embodiment preferred, described part is not in conjunction with the film combination (striding film) of TNFR1 and the territory 3 of soluble form.In another embodiment preferred, described part is not in conjunction with the film combination (striding film) of TNFR1 and the territory 2 or the territory 3 of soluble form.
In certain embodiments, described part is an IgG sample form.This class form has 4 chain structures (2 heavy chain and 2 light chains) of conventional IgG molecule, wherein one or more variable region (V HAnd/or V L) to have been had required specific dAb be that single variable region replaces.Preferred each variable region (2 V HDistrict and 2 V LThe district) be that single variable region replaces by dAb.The dAb that is included in the IgG sample form is that single variable region can have phase homospecificity or non-homospecificity.In certain embodiments, IgG sample form is quaternary, and can have 1,2,3 or 4 species specificity.For example, IgG sample form can be a monospecific, and comprises 4 and have mutually homospecific dAb; Can be bispecific, and comprise 3 and have mutually homospecific dAb and another and have not homospecific dAb; Can be bispecific, and comprise 2 and have that mutually homospecific dAb and two have jointly, but not homospecific dAb; Can be tri-specific, and comprise and have mutually homospecific first and second dAb to have not homospecific the 3rd dAb, have specific the 4th dAb that is different from first, second and the 3rd dAb; Perhaps can be four specific, and comprise 4 and have not homospecific dAb separately.Fab (for example Fab, the F (ab ') that can prepare IgG sample form 2, Fab ', Fv, scFy).Preferred IgG sample form or the not crosslinked TNFR1 of its Fab.
Part of the present invention comprises dAb monomer, dimer and trimer, can connect the antibody Fc district, and this district comprises one or two of CH2 and CH3 district, and optional hinge region.For example, coding can be used for preparing described polypeptide with the carrier of the part in mononucleotide sequence connection Fc district.
According to following aspect of the present invention, the present invention also provides the monomeric dimer of aforementioned dAb, trimer and polymer.
Part of the present invention can in conjunction with and/or make the form of NIg multiple ligand structure, forming the multivalence complex, it is in conjunction with having the target molecule of same antigen, thereby provides than high affinity.In certain embodiments, at least one variable region conjugated antigen is to prolong the polymeric half-life.For example natural bacteria receptor (for example SpA) is as the support of transplanting CDR, to produce the part of energy specificity in conjunction with one or more epi-positions.The details of this method is referring to US5, and 831,012.Other suitable support comprises the support based on fibronectin and affine body.The details of appropriate method is referring to WO 98/58965.Other suitable support comprises that lipocalin protein and CTLA4 are (referring to van den Beuken etc., J, Mol.Biol. (2001) 310,591-601) and for example be described in the support of WO00/69907 (Medical Research Council), described support is based on for example circulus or other chaperone polypeptide of antibacterial GroEL.
Protein scaffolds can make up; For example, CDR can be transplanted on the CTLA4 support and with immunoglobulin V HDistrict or V LThe district uses together, to constitute part.Equally, fibronectin, lipocalin protein and other support also can make up.
Bispecific part and polyspecific part
The inventor is at it simultaneously in the International Patent Application WO 03/002609 and the unpub UK patent application 0230203.2 of pending trial simultaneously of pending trial, described the double specificity immunoglobulin part, described part comprises and has not homospecific immunoglobulin list variable region separately.These domains can be each other competitive or antigen or the epi-position on the binding target molecule independently.
Bispecific part of the present invention preferably comprises the combination in heavy chain district and light chain district.For example, the bispecific part can comprise V HDistrict and V LThe district, this two district can link together and be the form of scFv.In addition, part can comprise one or more C HDistrict or C LThe district.For example, part can comprise C H1 district, C H2 districts or C H3 districts and/or C LDistrict, C μ 1, C μ 2, C μ 3 or C μ 4 districts or its any combination.Hinge region also can be included in wherein.Each district's combination like this can be for example to simulate natural antibody, for example IgG or IgM or its fragment, for example Fv, scFv, Fab or F (ab ') 2Molecule.Also consider other structure, for example comprised V H, V L, C H1 and C LThe single armed of the IgG molecule in district.
In an embodiment preferred of the present invention, the variable region is selected from single domain V gene bank.Usually, the single domain antibody storehouse all is illustrated on the filobactivirus surface.In a preferred embodiment, combine with antigenic, select each single domain antibody by phage library.
In an embodiment preferred of the present invention, when not existing in complementary variable region, can be by selecting each single variable region with combining of its target antigen or epi-position.In an alternate embodiment, when existing in complementary variable region, can be by selecting single variable region with combining of its target antigen or epi-position.Therefore, when existing, first single variable region can be selected, when existing, second variable region can be selected in the 4th complementary variable region in the 3rd complementary variable region.The the complementary the 3rd or the 4th variable region can be " dummy " variable region, the complementary district of natural related variable region (they have the specificity identical with single domain to be measured) or dereferenced-for example.
Preferred bispecific part of the present invention only comprises two variable regions, becomes same protein although several such part also can combine, and for example two such parts can be combined into the immunoglobulin of IgG or polymer form, for example IgM.Perhaps, in another embodiment, a plurality of bispecific parts are in conjunction with forming polymer.For example, two different bispecific parts combine, to produce four specific moleculars.
It will be understood by those skilled in the art that the variable region of light chain and the variable region of heavy chain of the bispecific part that the inventive method produces, can be on the same polypeptide chain or on different polypeptide chains.With regard to the variable region was on different polypeptide chains, they can connect by joint, normally flexible joint (for example polypeptide chain), cytotoxic compounds, perhaps connect by any other method known in the art.
In first general layout, the invention provides the further improvement of bispecific part, this improvement is by inventor's exploitation, and wherein part specificity is at protein that exists in the organism or polypeptide, and they are by combining the effect that prolongs the part half-life of playing with it.
Therefore, first aspect, the bispecific part is provided, described part comprises first antigen or epi-position is had the first immunoglobulin list variable region of binding specificity and second antigen or epi-position are had in conjunction with the active second complementary immunoglobulin list variable region, one or two of wherein said antigen or epi-position plays the effect that prolongs the half-life in the part body, and wherein said first and second districts lack and share mutually homospecific complementary district, and precondition is that described bispecific part be can't help anti-HSA V HDistrict and anti-beta galactosidase V κ district form.Preferred first or second variable region is not in conjunction with human serum albumin (HSA).
The antigen or the epi-position that prolong the half-life of part described herein preferably are positioned on the protein or polypeptide of organism existence.Example comprises the protein that exists in extracellular matrix protein, haemproteins and the organism different tissues.Protein for example by as extender (bulking agent), perhaps by part is anchored on the required action site, reduces the speed that part is removed and play from blood.The example of antigen/epi-position of half-life provides in following appendix 1 in the extension body.
The half-life that prolongs can be used for interior application of body of immunoglobulin, the especially antibody fragment of antibody, the most especially small size.This class fragment (Fv, Fab, scFv, dAb that Fv, disulfide bond connect) can be disposed in body fast; Therefore, although they can arrive most of position of health fast, and also can produce fast and operation easily, their short persistence have in vivo limited their application in vivo.The invention solves this problem, promptly, prolong the ligand function activity persistent period in vivo then by making part half-life prolongation in vivo.
Pharmacokinetic analysis method and part half life determination method are well known to those skilled in the art.Relevant details can be referring to Kenneth, A etc., Chemical Stability ofPharmaceuticals:A Handbook for Pharmacists and Peters etc., Pharmacokinetc analysis:A Practical Approach (1996).List of references also can be referring to " Pharmacokinetics ", M Gibaldi﹠amp; D Perron, Marcel Dekker publishes, and 2 NdRev.ex edition (1982), the document has been introduced pharmacokinetic parameter, for example t α and t β half-life and area under curve (AUC).
Half-life (t  α and t  β) and AUC can determine according to the curve of part serum-concentration and time.The WinNonlin analysis package (can derive from Pharsight Corp., Mountain View, CA94040 USA) can be used for for example curve modeling.In first phase (α phase), part mainly is to distribute in patient's body, and some eliminations are arranged simultaneously.Second phase (β phase) is whole last phase, when part has distributed and serum-concentration is eliminated in patient's body with part and descended.The t α half-life is the half-life of first phase, and the t β half-life then is the half-life of second phase.Therefore, preferably the invention provides part of the present invention or comprise the compositions of part of the present invention, the t α half-life scope of described part is 15 minutes or longer time.In one embodiment, the lower limit of scope is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.In addition, perhaps, the t α half-life scope of part of the present invention or compositions is maximum 12 hours and comprises 12 hours.In one embodiment, the upper limit of scope is 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours or 5 hours.The example of OK range is 1-6 hour, 2-5 hour or 3-4 hour.
Preferably the invention provides part of the present invention or comprise the compositions of part of the present invention, the t β half-life scope of described part is 2.5 hours or longer time.In one embodiment, the lower limit of scope is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours.In addition, perhaps, the t β half-life scope of part of the present invention or compositions is maximum 21 days and comprises 21 days.In one embodiment, the upper limit of scope is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days.The t β half-life scope of best part of the present invention or compositions is 12-60 hour.In another embodiment, scope is 12-48 hour.In an embodiment again, scope is 12-26 hour.
In addition, except above standard, the invention provides part or comprise the compositions of part of the present invention, its AUC value (area under curve) scope is more than the 1mg.min/ml.In one embodiment, the lower limit of scope is 5,10,15,20,30,100,200 or 300, and unit is mg.min/ml.In addition, perhaps, the AUC scope of part of the present invention or compositions is up to 600mg.min/ml.In one embodiment, the upper limit of scope is 500,400,300,200,150,100,75 or 50, and unit is mg.min/ml.Preferably the AUC scope of part of the present invention is selected from following: 15-150mg.min/ml, 15-100mg.min/ml, 15-75mg.min/ml and 15-50mg.min/ml.
In first embodiment, the bispecific part comprises two complementary variable regions, i.e. two variable regions, they under its natural surroundings, can combine become related to or associated group, even under situation of the present invention, they are respectively in conjunction with its related epi-position.For example, complementary variable region can be heavy chain immunoglobulin and variable region of light chain (V HAnd V L).V HDistrict and V LDistinguishing is preferably provided by scFv or Fab antibody fragment.The variable region can be joined together to form multivalent ligand, for example provides disulfide bond by providing between hinge region and the cysteine at hinge region at the C-in each V district end; Or being provided at the dAb that domain C-end has cysteine, these cysteine are to link together by disulfide bond; Perhaps by producing V-CH and V-CL, to produce the Fab form; Perhaps use peptide linker (Gly hereinafter described for example 4The Ser joint), to produce dimer, trimer and polymer.
The inventor knows, uses complementary variable region can allow two domain surfactant packages together and from solvent live in retirement (sequestered).And complementary district can stablize each other.In addition, it allows to produce bispecific IgG antibody, but does not have the shortcoming of the used hybrid hybridoma of prior art, perhaps need not transform heavy chain or light chain at the subunit interface.The bispecific part of first aspect present invention has at least one V H/ V LRight.Therefore, bispecific IgG of the present invention comprises two such pairings, respectively has a pair of on each arm of Y shape molecule.Therefore, be different from conventional bi-specific antibody or double-stranded antibody (wherein the ratio of used chain determines the whether difficulty put into practice of success and causing of its preparation), bispecific part of the present invention does not have the equilibrated problem of chain.Chain imbalance in the conventional bi-specific antibody is by two different V LChain and two different V HDue to chain connects, V wherein LChain 1 and V HChain 1 together can conjugated antigen or epi-position 1, V LChain 2 and V HChain 2 together can conjugated antigen or 2, two correct pairings of epi-position be connected to each other in some way.Therefore, in a molecule, only work as V LChain 1 and V HChain 1 pairing, V LChain 2 and V HDuring chain 2 pairings, just produce bispecific.Such bispecific molecule can produce by two kinds of different modes.First kind, they can be by two existing V that connect synantigen not or epi-position separately H/ V LMatch connection and produce (for example in bispecific IgG).In this case, V H/ V LPairing must all be a bispecific molecule colony so that produce all molecules with 1: 1 mixed together.This will never (even when complementary CH district by " pestle mortar structure (knobs into holes) " when engineering is strengthened) causes bispecific molecule and only can be in conjunction with an antigen or epi-position but not in conjunction with the mixture of the molecule of another antigen or epi-position.Second kind of mode that produces bi-specific antibody is by with two different V HChain and two different V LChain is simultaneously in conjunction with (for example at the double-stranded antibody of bispecific).In this case, although tend to preferentially allow V LChain 1 and V HChain 1 pairing, V LChain 2 and V H(this can pass through V in chain 2 pairings LAnd V H" the pestle mortar structure " in district transformed and strengthened), but this pairing can in all molecules, all do not obtained, cause mix preparation, incorrect pairing wherein takes place, make it can not conjugated antigen or epi-position.
The bi-specific antibody that makes up according to the bispecific part method of first aspect present invention has overcome all these problems, because be present in V with combining of antigen or epi-position 1 HDistrict or V LThe district is present in complementary V with combining of antigen or epi-position 2 LOr V HThe district.Because V HDistrict and V LMatch with 1: 1 ratio in the district, all V H/ V LPairing all is a bispecific, therefore adopts these V H/ V LThe form of ownership (Fv, scFv, Fab, miniantibody, IgG etc.) that pairing makes up all has 100% bispecific activity.
Under situation of the present invention, first and second " epi-positions " are considered to such epi-position: described epi-position difference, and not in conjunction with single monospecific part.In first general layout of the present invention, they are preferably in not on the synantigen, and one of them plays the effect that prolongs the half-life in the part body.Equally, first and second antigens are preferably different.
Bispecific part of the present invention does not comprise WO 02/02773 described part.Therefore, part of the present invention does not comprise complementary V H/ V LRight, it is collaborative in conjunction with any one or a plurality of antigen or epi-position.On the contrary, the part of first aspect present invention comprises V H/ V LComplementary pair, wherein the V district has not homospecificity.In addition, the part of first aspect present invention comprises V H/ V LComplementary pair, it has not homospecificity to non-structurally associated epi-position or antigen.The epi-position of structurally associated or antigen are such epi-position or antigen: it has enough structural similarities, can with conventional V H/ V LThe complementary pair combination, it is with cooperative mode conjugated antigen or epi-position; With regard to the epi-position of structurally associated, epi-position structurally is enough similar, makes them " be fit to " in V H/ V LThe same binding pocket (pocket) that forms on the dimeric antigen binding site.
Second aspect, the invention provides part, described part comprises first immune globulin variable region with first antigen or epi-position binding specificity and has second antigen or second immune globulin variable region of epi-position binding specificity, wherein prolong one or two conjugated antigen of described first and second variable regions of half-life in the part body, the variable region is not complementary each other.
In one embodiment, regulated the combination of part by the combination of a variable region by second variable region.
In this embodiment, the variable region can be V for example HThe district to or V LIt is right to distinguish.At the antigen in first site in conjunction with scalable, for example strengthen or be suppressed at the antigen combination in second site.For example, in the antigen combination that small part is suppressed at second site that is bonded in first site.In such embodiments, part can be for example by in patient's organism, being maintained with the protein bound that prolongs the part half-life, disintegrate down in conjunction with second target antigen and from the protein of prolong half-life up to it.
In these cases, reaching bonded adjusting is the antigen binding site approaching result of structure each other.The characteristic of structural constituent that can be by connecting two or more antigen binding sites, it is approaching to reach such structure, and for example by the part with relative stiffness structure is provided, such rigid structure can keep very approaching antigen binding site.Preferably two or more antigen binding sites are physically very approaching each other, by relating to the process of steric hindrance in the immunoglobulin molecules and/or conformation change, make a site can regulate the antigen combination in another site.
The covalently or non-covalently combination of first and second antigen binding domains.At domain is under the covalently bound situation, can or pass through for example (Gly of peptide linker by for example disulfide bond 4Ser) nAnd the mediation combination, n=1-8 wherein, for example 2,3,4,5 or 7.
Adopting display technique of bacteriophage for example as herein described to select from the V gene bank under the situation of variable region, these variable regions can comprise the general framework district, make them to be discerned by the general part of specificity as herein described.The use of relevant general framework, general part etc. is referring to WO 99/20749.In the present invention, relevant phage display comprises the use of phage and/or phasmid.
When using the V gene bank, the variation in the peptide sequence is preferably placed in the structure ring of variable region.The peptide sequence of each variable region can change by DNA reorganization or by sudden change, so that strengthen the interaction of each variable region and its complementary pair.DNA reorganization is known in the art, referring to for example Stemmer, and No. the 6th, 297,053, Nature 370:389-391 (1994) and United States Patent (USP), described document all is attached to herein by reference.Other method of mutagenesis is that those skilled in the art are well-known.
In a preferred embodiment of the invention, " bispecific part " is strand Fv fragment.In an alternate embodiment of the present invention, " bispecific part " is made up of the monoclonal antibody district.Term " Fab district " comprises Fab sample district, wherein uses two V HDistrict or two V LThe district.
The variable region can be from the antibody at target antigen or epi-position.Perhaps they can be from the single domain antibody storehouse, for example at those of filobactivirus surface expression.Can be according to hereinafter described selecting.
On the other hand, the invention provides one or more nucleic acid molecules, the described nucleic acid bispecific part as herein described of encoding at least.The bispecific part can be by a nucleic acid molecule encoding; Perhaps, each domain can be by nucleic acid molecule encoding separately.When part during by a nucleic acid molecule encoding, each domain can be expressed as the fused polypeptide of scFv molecular forms, perhaps can express respectively, links together subsequently again, for example uses chemical bridging agent.The expressed part of nucleic acid separately can link together by appropriate method.
Nucleic acid is the codified signal sequence also, is used for after expression polypeptide being exported from host cell, and also can merges with the particulate surface component of filobactivirus (or other component of selection display systems) after expression.
On the other hand, the invention provides carrier, described carrier comprises the nucleic acid of the bispecific part of the present invention of encoding.
Another aspect the invention provides the host cell with the carrier transfection, described vector encoded bispecific part of the present invention.
Express with such carrier, can for example produce the variable region, be used for selecting on the phage particle surface." bispecific part " can be selected with method of the present invention in the variable region that this allows selection to be showed therefore.
The present invention also provides test kit, and described test kit comprises bispecific part of the present invention at least.
The third aspect, the invention provides the method for preparing part, second single immunoglobulin list variable region that described part comprises the first immunoglobulin list variable region with first binding specificity and has second (difference) binding specificity, one or two of binding specificity has specificity to the antigen that prolongs the part half-life in vivo, and this method may further comprise the steps:
A) selecting can be in conjunction with first variable region of first epi-position,
B) selecting can be in conjunction with second variable region of second epi-position,
C) connect the variable region; With
D) selecting can be in conjunction with the part of described first epi-position and described second epi-position.
Part can be simultaneously in conjunction with first and second epi-positions, perhaps between in conjunction with the territory to epi-position when having competitiveness, the combination in a district can hinder another and distinguish combination with its related epi-position.Therefore in one embodiment, above step (d) needs simultaneously in conjunction with first and second (more if possible) epi-positions; In another embodiment, with the combining not simultaneously of first and second epi-positions.
Epi-position is preferably on antigen separately.
Part preferably comprises the V of aforesaid immune globulin variable region H/ V LCombination or V H/ V HOr V L/ V LCombination.Part also can comprise camellid V HHThe district, precondition is that the antigen that can prolong the part half-life is in vivo had specific V HHThe district is not in conjunction with hen egg-white lysozyme (HEL), Pancreas Sus domestica gland α-Dian Fenmei or NmC-A; RR6 azo dye or Streptococcus mutans (S.mutans) HG982 cell that hcg, BSA-connect, referring to (2001) JBC 276:7346-7350 and WO99/23221 such as Conrath, these two pieces of documents all do not have to introduce at the antigenic specificity purposes that can prolong the part half-life in vivo.
In one embodiment, under the non-existent situation in complementary variable region, select described first variable region to be used in conjunction with described first epi-position.In another embodiment, under the situation of the 3rd variable region existence, select described first variable region to be used in conjunction with described first epi-position/antigen, wherein said the 3rd variable region is different from described second variable region and complementary with first district.
Equally, can under existence of complementary variable region or non-existent situation, select second district.
Except the protein of prolong half-life, the antigen or the epi-position of part of the present invention institute targeting can be any antigen or epi-position, but preferably have the antigen or the epi-position of treatment benefit.The invention provides part, comprise open conformation, closed conformation and isolating dAb monomer part, they have specificity to those targets that any described target, especially this paper further identify.Described target can be natural existence or synthetic polypeptide, protein or nucleic acid or its part.Aspect this, part of the present invention can and play antagonist or the effect of agonist (for example EPO receptor stimulating agent) in conjunction with epi-position or antigen.It will be understood to those of skill in the art that it is huge and different selecting.They can be that the cofactor or the DNA of for example human or animal's albumen, cytokine, cytokine receptor, enzyme is conjugated protein.Suitable cytokine and somatomedin include but not limited to: ApoE, Apo-SAA, BDNF, myocardial nutrition albumen-1, EGF, the EGF receptor, ENA-78, eotaxin (eotaxin), eotaxin-2, Exodus-2, EpoR, FGF (acidity), FGF (alkalescence), fibroblast growth factor-10, the FLT3 part, CXXXC chemotactic factor (CX3C), GDNF, G-CSF, GM-CSF, GF-β 1, insulin, IFN-γ, IGF-I, IGF-II, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72a.a.), IL-8 (77a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), inhibin α, inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, leptin, LIF, lymphocyte chemotactic factor (LCF), Miller pipe (Mullerian) inhibiting substances, the mononuclear cell colony inhibition factor, monocyte chemoattractant protein, M-CSF, MDC (67a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67a.a.), MDC (69a.a.), MIG, MIP-1a, MIP-1 β, MIP-3 α, MIP-3 β, MDP-4, marrow progenitor inhibitory factor-1 (MPIF-1), NAP-2, neurotrophic factor, nerve growth factor, β-NGF, NT-3, NT-4, tumour inhibitor M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1 α, SDF1 β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β 2, TGF-β 3, tumor necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, the TNF receptor II, TNIL-1, TPO, VEGF, vegf receptor 1, vegf receptor 2, vegf receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1,1-309, HER1, HER2, HER3 and HER4.Cytokine receptor comprises the receptor of aforementioned cytokine.Be appreciated that this list is not is detailed and exhaustively.
In one embodiment of the invention, the variable region is from the corresponding antibodies at antigen or epi-position.In a preferred embodiment, the variable region is from storehouse, antibody list variable region.
In second general layout, the invention provides the polyspecific part.According to the present invention, term " polyspecific part " is meant the part with more than epi-position binding specificity defined herein.Usually, the polyspecific part comprises two or more epi-positions in conjunction with the territory.
Epi-position of the present invention comprises protein scaffolds and epi-position interaction sites (they are preferably in the protein scaffolds surface) in conjunction with the territory.According to the present invention, preferably each epi-position has different epi-position binding specificities in conjunction with the territory.
Under situation of the present invention, think that first and second " epi-positions " are different epi-positions and not in conjunction with the epi-position of a monospecific part.They can be on synantigen not, or on same antigen, but have enough distances to separate, make them can not form such entity: a monospecific V of described entity and conventional antibody H/ V LIn conjunction with to combination.From experimentally, if two (domain antibodies or dAb) of the single variable region in the single-chain antibody form are separately by the monospecific V at two epi-positions H/ V LThe part competition, then the distance between these two epi-positions is far away inadequately, can not think isolating epi-position of the present invention.
According to the present invention, advantageously, each epi-position all comprises immune globulin variable region in conjunction with the territory.More advantageously, each immunoglobulin all the variable region can be variable region of light chain (VL) or variable region of heavy chain V HIn second general layout of the present invention, immunoglobulin domains is non-complementary in being present in part of the present invention the time, that is to say, they are in conjunction with forming V H/ V LAntigen binding site.Therefore, the polyspecific part that defines in the present invention's second general layout comprises the immunoglobulin domains of identical hypotype, and it can be variable region of light chain (V L) or variable region of heavy chain (V H).In addition, when part of the present invention was closed conformation, immunoglobulin domains can be camellid V HHType.
In an alternate embodiment, part of the present invention does not comprise camellid V HHThe district.More particularly, with people V HThe district compares, and part of the present invention does not contain camellid V HHThe district has specific one or more amino acid residue.
Preferably single variable region is from antibody, and described antibody is by at choosing in conjunction with active of synantigen or epi-position not.For example, can pass through people's immunity, to small part separation variable region.Alternative method is known in the art, comprises from the human antibodies library separating and the synthesizing of artificial antibody's gene.
Variable region best incorporated superantigen, for example protein A or albumen L.With combining of superantigen is the characteristic of the correct antibody variable region that folds, and allows such domain to separate from the library of for example reorganization or saltation zone.
Epi-position also can be based on the protein scaffolds or the skeleton that are not immunoglobulin domains in conjunction with the territory.For example natural bacteria receptor (for example SpA) is as the support of transplanting CDR, to produce the part of energy specificity in conjunction with one or more epi-positions.The details of this method is referring to US5, and 831,012.Other suitable support comprises the support based on fibronectin and affine body.The details of appropriate method is referring to WO 98/58965.Other suitable support comprises that lipocalin protein and CTLA4 are (referring to van den Beuken etc., J.Mol.Biol. (2001) 310,591-601) and for example be described in the support of WO0069907 (Medical Research Council), described support is based on for example circulus or other polypeptide chaperone of antibacterial GroEL.
Protein scaffolds can make up; For example, CDR can be transplanted on the CTLA4 support and with immunoglobulin V HDistrict or V LThe district uses together, to constitute multivalent ligand.Equally, fibronectin, lipocalin protein and other support also can make up.
In one embodiment, the polyspecific part comprises epi-position in conjunction with the territory, and it comprises the CDR in conjunction with the immunoglobulin single domain (dAb) of TNFR1 as herein described, and described CDR is transplanted on the suitable protein scaffolds or skeleton.
The epi-position that it will be understood by those skilled in the art that the closed conformation polyspecific part that the inventive method produces can be on the identical polypeptide chain or on different polypeptide chains in conjunction with the territory.With regard to the variable region was on different polypeptide chains, they can connect by joint, preferably flexible joint (for example polypeptide chain), cytotoxic compounds, perhaps connect by any other method known in the art.
First and second epi-positions are in conjunction with covalently or non-covalently combination of territory.At domain is under the covalently bound situation, can be by for example disulfide bond mediation combination.
In some embodiment of second general layout of the present invention, epi-position can substitute and combination each other.For example, first epi-position may reside on the antigen, described antigen related with it first in conjunction with the territory in conjunction with the time, cause second steric hindrance or its conformation change in conjunction with the territory, combine the bonded epi-position in territory with second thereby substituted.
Best incorporated descend reach 25% or more, preferably 40%, 50%, 60%, 70%, 80%, 90% or more, preferably up to 100% or near 100%, feasible combination is suppressed fully.Can by conventional antigen in conjunction with measure (for example ELISA), based on the technology (comprising FRET) of fluorescence or measure the combination of epi-position by the technology such as surface plasma resonance of for example determining molecular weight.
In addition, the invention provides closed conformation polyspecific part, described part comprise first epi-position with first epi-position binding specificity in conjunction with territory and non-complementary second epi-position with second epi-position binding specificity in conjunction with the territory, wherein first and second binding specificities competition epi-position combination makes closed conformation polyspecific part not simultaneously in conjunction with these two epi-positions.
Closed conformation polyspecific part of the present invention does not comprise WO 02/02773 described part.Therefore, part of the present invention does not comprise complementary V H/ V LRight, it is collaborative in conjunction with any one or a plurality of antigen or epi-position.On the contrary, part of the present invention preferably comprises non-complementary V H-V HOr V L-V LRight.Best each V H-V HOr V L-V LEach V of centering HDistrict or V LThe district has different epi-position binding specificities, and the epi-position binding site is to arrange like this: make and compete with the combining of epi-position on another site in the combination of the epi-position on the site.
Best closed conformation polyspecific part can comprise can binding target molecule first district and can be in conjunction with molecule that prolongs the part half-life or second district of group.For example, molecule or group can be extender (bulky agent), for example HSA or cellular matrix albumen.Term used herein " prolongs molecule or the group of part half-life " and is meant such molecule or chemical group: when giving animal, with respect to the part of binding molecule or group not, when described molecule or chemical group during in conjunction with bispecific part as herein described, they prolong the half-life in the body of described bispecific part.The molecule or the examples of groups that prolong the part half-life are described below.In a preferred embodiment, only when the half-life prolonged molecule or group and replaced, closed conformation polyspecific part can binding target molecule.Therefore, for example, closed conformation polyspecific part can be kept in the blood samples of patients circulation by the molecule (for example HSA) of large volume.When running into target molecule, the competition in conjunction with between the territory of closed conformation polyspecific part causes the combination of substituting of HAS and target.
In an embodiment preferred of the present invention's second general layout, epi-position is immune globulin variable region in conjunction with the territory and is selected from single domain V gene bank.Usually, the single domain antibody storehouse all is illustrated on the filobactivirus surface.In a preferred embodiment, combine with antigenic, select each single domain antibody by phage library.In an embodiment preferred of second general layout of the present invention, epi-position is immune globulin variable region in conjunction with the territory and is selected from single domain V gene bank.Usually, the single domain antibody storehouse all is illustrated on the filobactivirus surface.In a preferred embodiment, combine with antigenic, select each single domain antibody by phage library.
On the one hand, the polyspecific part comprises at least two incomplementarity variable regions.For example, part can comprise a pair of V HDistrict or a pair of V LThe district.Preferably these domains are not the camellid source; Preferably they are people's domains or comprise people's framework region (FW) and one or more allos CDR.CDR and framework region are the domains of immune globulin variable region, and be defined as the Sequences of Protein of Immunological Interest data base of Kabat.
Preferred people's framework region is that kind is constant gene segment C DP47 and the coded domain of DPK9.Best V HDistrict or V LFW1, the FW2 in district and FW3 have FW1, FW2 or the FW3 sequence from DP47 or DPK9.People's framework can be chosen wantonly and contain sudden change, for example contains about at most 5 aminoacid variation or about at the most 10 aminoacid jointly and change in the used people's framework of part of the present invention.
According to second general layout of the present invention, the variable region in the polyspecific part can be arranged in open or closed conformation; That is to say that they can be arranged like this: make the variable region can be independently with simultaneously in conjunction with its related part, make that perhaps only a variable region can be at any time in conjunction with its related part.
The inventor knows, under some structural condition, non-complementary variable region (for example two variable region of light chains or two variable region of heavy chaines) may reside in the part, make combining of win epi-position and first variable region suppress combining of second epi-position and second variable region, even do not work in such incomplementarity district to one as association.
Preferably part comprises two pairs or more to the variable region; That is to say that it comprises at least 4 variable regions.Preferably these 4 variable regions all comprise people source framework.
In a preferred embodiment, people's framework and ethnic group are that the framework of sequence is identical.
The inventor thinks that such antibody can be used in particular for part in conjunction with mensuration, is used for the treatment of purposes and other purposes.
In an embodiment of second general layout of the present invention, the variable region is from the antibody at first and/or second antigen or epi-position.In a preferred embodiment, the variable region is from storehouse, antibody list variable region.In an example, the storehouse is the storehouse that can not produce in animal or synthetic storehouse.In another example, (to small part) do not separated by animal immune in single variable region.Therefore, single domain can be separated from natural library.
On the other hand, second general layout of the present invention provides the polyspecific part, and it comprises first epi-position with first epi-position binding specificity in conjunction with the territory with have incomplementarity second epi-position of the second epi-position binding specificity in conjunction with the territory.First and second binding specificities can be identical or different.
On the other hand, the invention provides closed conformation polyspecific part, it comprises first epi-position with first epi-position binding specificity in conjunction with the territory with have incomplementarity second epi-position of the second epi-position binding specificity in conjunction with the territory, wherein first and second binding specificities can be competed the epi-position combination, make that closed conformation polyspecific part can not be simultaneously in conjunction with two epi-positions.
Another aspect the invention provides and comprises the open conformation part of incomplementarity in conjunction with the territory, and wherein said domain has specificity to the different epi-positions on the same target.Described part has the affinity of increase with combining of target.Equally, the invention provides and comprise the multivalent ligand of incomplementarity in conjunction with the territory, described have specificity to identical epi-position and at the multicopy target that comprises described epi-position in conjunction with the territory, described epi-position is IL-5, PDGF-AA, PDGF-BB, TGF β, TGF β 2, TGF β 3 and TNFe α for example, for example people TNF receptor 1 and human TNF alpha.
In similar aspect, part of the present invention can low-affinity in conjunction with single epi-position, make and do not treat meaning with combining of single epi-position; But the affinity that combines with two epi-positions and cause can provide the treatment benefit.In an instantiation, can be at separately existing in the normal cell type but be present in epi-position in unusual or the diseased cells (for example tumor cell) together.Under these circumstances, only unusual or diseased cells is effective target of bispecific part of the present invention.
Identical epi-position or adjacent epi-position to multicopy on the same target have specific part (being called chelating dAb), also can be the parts of trimer or polymer (tetramer or higher aggregation), its comprise 3,4 or more a plurality of incomplementarity in conjunction with the territory.For example, can make up and comprise 3 or 4 V HDistrict or V LThe part in district.
In addition, providing can be in conjunction with the part of a plurality of subunit targets, and wherein each has specificity in conjunction with the territory to described target subunit.Part can be dimer, trimer or polymer.
The polyspecific part of the preferred above aspect of the present invention is that the method by first aspect present invention obtains.
The above aspect of second general layout according to the present invention, best first epi-position is incomplementarity immune globulin variable region as herein described in conjunction with the territory and second epi-position in conjunction with the territory.That is to say, can be V H-V HOr V L-V LThe variable region.
Specifically, can prepare chelating dAb according to the preferred aspect of the present invention, promptly adopt grappling dAb, wherein adopt carrier to make up the library of dimer, trimer or polymer dAb, described carrier comprises the constant dAb in joint sequence upstream or downstream, and it has the storehouse of second, third and the Geng Duo dAb that are inserted into the joint opposite side.For example, grappling or guiding dAb can be TAR1-5 (V κ), TAR1-27 (V κ), TAR2h-5 (VH) or TAR2h-6 (V κ).
In alternative method, can avoid adopting joint, for example by using in conjunction with territory V for example HAnd non-covalent bond between the V κ or natural affinity.Therefore, the invention provides the method for preparing chelating polymer part, this method may further comprise the steps:
(a) provide carrier, this carrier comprises coding first epi-position on the target is had the nucleotide sequence that specific unijunction closes the territory;
(b) provide carrier, this vector encoded comprises second epi-position on the described target is had specific second storehouse in conjunction with the territory, and described epi-position can be identical or different with first epi-position, and described second epi-position is adjacent with described first epi-position; With
(c) express described first and second in conjunction with the territory; With
(d) separating and combining first and second combinations in conjunction with the territory together obtain the dimer in conjunction with target.
First and second epi-positions are adjacent, make that the polymer part can be simultaneously in conjunction with these two epi-positions.This provide in conjunction with the time have a part that increases the affinity advantage.When epi-position is identical, can be by the multicopy epi-position that exists on the target, and the affinity that obtains increasing allows simultaneously in conjunction with at least two copies, so that the affinity effect increases.
Can and adopt joint and combination by Several Methods in conjunction with the territory.For example, can comprise cys residue, avidin and streptavidin group or other in conjunction with the territory and after synthetic, be used for non-covalent ways of connecting; Effectively these combinations in conjunction with target can be separated.Perhaps, joint can be present in first and second in conjunction with between the territory, and they can be used as a polypeptide and express from a carrier, for example, as mentioned above, this polypeptide comprise first in conjunction with territory, joint and second in conjunction with the storehouse, territory.
One preferred aspect, when combining with antigen, first and second link together with regard to natural in conjunction with the territory; For example, V HDistrict and V κ district when in conjunction with adjacent epi-position, with three kinds of natural connections of interaction mode, form and stablize dimer.Such connection albumen can be separated in measuring at target.An advantage of this method is exactly: only with correct conformation in conjunction with just can the connecting of tight adjacent epi-position in conjunction with the territory, and can separate because of its affinity to target increases.
In an alternate embodiment aspect second general layout of the present invention above, at least one epi-position comprises NIg as herein described " protein scaffolds " or " protein skeleton " in conjunction with the territory.Suitable NIg protein scaffolds includes but not limited to be selected from following support any listed above: SpA, fibronectin, GroEL and other chaperone, lipocalin protein, CCTLA4 and affine body.
According to the above aspect of second general layout of the present invention, preferably epi-position is in conjunction with territory conjugated protein skeleton.Best protein skeleton of the present invention is the immunoglobulin skeleton.
According to the present invention, term " immunoglobulin skeleton " is meant and aforesaidly comprises at least one immunoglobulin folding and play the protein of one or more epi-positions in conjunction with the territory central role.
Preferred immunoglobulin skeleton as herein described comprises any one or a plurality ofly is selected from following skeleton: comprise the immunoglobulin molecules with the lower part at least: (i) CL of antibody (κ or λ subclass) district; Or the (ii) CH1 district of heavy chain of antibody; The immunoglobulin molecules that comprises heavy chain of antibody CH1 district and CH2 district; The immunoglobulin molecules that comprises heavy chain of antibody CH1 district, CH2 district and CH3 district; Or the CL of any (ii) subclass and antibody (κ or λ subclass) district.Hinge region also can be included in wherein.Each district's combination like this can be for example to simulate natural antibody, for example IgG or IgM or its fragment, for example Fv, scFv, Fab or F (ab ') 2Molecule.One skilled in the art will appreciate that this list is not is detailed and exhaustively.
Skeleton as herein described can obtain on the polypeptide level in conjunction with being connected of territory with epi-position, is after coding skeleton and/or the expression of nucleic acid of epi-position in conjunction with the territory.Perhaps, Connection Step can carry out on nucleic acid level.Protein skeleton of the present invention combines the territory with one or more epi-positions method of attachment comprises employing protein chemistry and/or Protocols in Molecular Biology, and these technology are well known to those skilled in the art and description is arranged in this article.
Best closed conformation polyspecific part can comprise can binding target molecule first district and can be in conjunction with molecule that prolongs the part half-life or second district of group.For example, molecule or group can be extender (bulky agent), for example HSA or cellular matrix albumen.Term used herein " prolongs molecule or the group of part half-life " and is meant such molecule or chemical group: when giving animal, with respect to the part of binding molecule or group not, when described molecule or chemical group during in conjunction with bispecific part as herein described, they prolong the half-life in the body of described bispecific part.The molecule or the group that prolong the part half-life are described below.In a preferred embodiment, only when the half-life prolonged molecule or group and substitutes, closed conformation polyspecific part can binding target molecule.Therefore, for example, closed conformation polyspecific part can be kept in the blood samples of patients circulation by the molecule (for example HSA) of large volume.When running into target molecule, the competition in conjunction with between the territory of closed conformation polyspecific part causes the combination of substituting of HAS and target.
In second general layout of the present invention on the other hand, the invention provides one or more nucleic acid molecules, the described nucleic acid polyspecific part as herein described of encoding at least.In one embodiment, described part is closed conformation part.In another embodiment, it is open conformation part.The polyspecific part can be by a nucleic acid molecule encoding; Perhaps, each epi-position can be by nucleic acid molecule encoding separately in conjunction with the territory.When part during by a nucleic acid molecule encoding, each district can be expressed as fused polypeptide, perhaps can express respectively, links together subsequently again, for example uses chemical bridging agent.The expressed part of nucleic acid separately can link together by appropriate method.
Nucleic acid is the codified signal sequence also, is used for after expression polypeptide being exported from host cell, and also can merges with the particulate surface component of filobactivirus (or other component of selection display systems) after expression.The targeting sequencing that can be used for the displaying of bacterial expression and/or phage or phasmid comprises pelB, stII, ompA, phoA, bla and pelA.
In second general layout of the present invention on the other hand, the invention provides the carrier that comprises nucleic acid of the present invention.
Again on the one hand, the invention provides host cell with carrier transfection of the present invention.
From such vector expression, can for example produce epi-position in conjunction with the territory on the phage particle surface, be used for selecting.Therefore the domain that this allows selection to be showed selects " polyspecific part " with method of the present invention.
The present invention also provides test kit, and described test kit comprises polyspecific part of the present invention at least, and described part can be open conformation or closed conformation part.Test kit of the present invention can be the detection kit of for example diagnostic kit, treatment usefulness test kit, chemistry or biological sample etc.
The invention provides the method for preparing the polyspecific part, this method may further comprise the steps:
A) selecting can be in conjunction with first epi-position of first epi-position in conjunction with the territory,
B) selecting can be in conjunction with second epi-position of second epi-position in conjunction with the territory,
C) connect epi-position in conjunction with the territory; With
D) selecting can be in conjunction with the closed conformation polyspecific part of described the one the second epi-positions and described second epi-position.
In second general layout on the other hand, the invention provides the method for the closed conformation polyspecific part of preparation, described part comprises first epi-position with first epi-position binding specificity in conjunction with the territory with have incomplementarity second epi-position of the second epi-position binding specificity in conjunction with the territory, wherein first and second binding specificities competition epi-position combination makes closed conformation polyspecific part not simultaneously in conjunction with these two epi-positions; This method may further comprise the steps:
A) selecting can be in conjunction with first epi-position of first epi-position in conjunction with the territory,
B) selecting can be in conjunction with second epi-position of second epi-position in conjunction with the territory,
C) connect epi-position in conjunction with the territory, make each domain be closed conformation; With
D) selecting can be in conjunction with described the one the second epi-positions and described second epi-position but not simultaneously in conjunction with the closed conformation polyspecific part of described first and second epi-positions.
An alternate embodiment of the above aspect of second general layout of the present invention is optional also comprise step (b1), this step comprise select the 3rd or more multi-epitope in conjunction with the territory.Like this, the polyspecific part that is produced (no matter being open or closed conformation) comprises more than two epi-position binding specificities.Aspect second general layout of the present invention preferred, when the polyspecific part comprised more than two epi-positions in conjunction with the territory, at least two described domains were closed conformation and compete combination; Other district can compete combination or can be independent of its related epi-position and connection arbitrarily.
In second general layout of the present invention, preferred first and second epi-positions are different.They can be natural existence or synthetic polypeptide, protein or nucleic acid or its part.Aspect this, part of the present invention can and play antagonist or the effect of agonist (for example EPO receptor stimulating agent) in conjunction with epi-position or antigen.In one embodiment, the epi-position of described part has identical epitope specificity in conjunction with the territory, and can be for example simultaneously in conjunction with its epi-position, when the epi-position of multicopy is present on the same antigen.In another embodiment, do not providing these epi-positions on the synantigen, making part and to connect antigen in conjunction with epi-position.It will be understood to those of skill in the art that epi-position is huge and different with antigenic selection.They can be that the cofactor or the DNA of for example human or animal's albumen, cytokine, cytokine receptor, enzyme is conjugated protein.Suitable cytokine and somatomedin include but not limited to: ApoE, Apo-SAA, BDNF, myocardial nutrition albumen-1, EGF, the EGF receptor, ENA-78, eotaxin, eotaxin-2, Exodus-2, EpoR, FGF (acidity), FGF (alkalescence), fibroblast growth factor-10, the FLT3 part, CXXXC chemotactic factor (CX3C), GDNF, G-CSF, GM-CSF, GF-β 1, insulin, IFN-γ, IGF-I, IGF-II, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72a.a.), IL-8 (77a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), inhibin α, inhibin β, EP-10, keratinocyte growth factor-2 (KGF-2), KGF, leptin, LIF, lymphocyte chemotactic factor (LCF), Miller pipe (Mullerian) inhibiting substances, the mononuclear cell colony inhibition factor, monocyte chemoattractant protein, M-CSF, MDC (67a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67a.a.), MDC (69a.a.), MIG, MIP-1 α, MIP-1 β, MIP-3 α, MIP-3 β, MIP-4, marrow progenitor inhibitory factor-1 (MPIF-1), NAP-2, neurotrophic factor, nerve growth factor, β-NGF, NT-3, NT-4, tumour inhibitor M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1 α, SDF1 β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β 2, TGF-β 3, tumor necrosis factor (TNF), TNF-α, TNF-β, TNF receptor I, the TNF receptor II, TNIL-1, TPO, VEGF, vegf receptor 1, vegf receptor 2, vegf receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1,1-309, HER1, HER2, HER3, HER4, the TACE recognition site, TNF BP-I and TNF BP-II, and any target that is disclosed in appendix 2 or appendix 3, no matter be combination to list in the appendix, the various combination form still is independent form.Cytokine receptor comprises the receptor of aforementioned cytokine, for example IL-1R1; IL-6R; IL-10R; IL-18R, and disclosed receptor in the receptor of the cytokine of listing in appendix 2 or the appendix 3 and appendix 2 and the appendix 3.Be appreciated that this list is not is detailed and exhaustively.When polyspecific part during in conjunction with two epi-positions (on identical or different antigen), antigen can be selected from this list.In specific embodiment, described part comprises can be in conjunction with the dAb of TNFR1 and can be in conjunction with arbitrary so antigenic the 2nd dAb or epi-position in conjunction with the territory.In such embodiments, the polyspecific part can comprise any combination (V for example of immune globulin variable region HV H, V HV L, V LV L).
Preparation based on the polyspecific part of immunoglobulin
Bispecific part of the present invention, on the conformation of general layout required for the present invention, no matter be that open or closed, can prepare according to the technology of the previous foundation that is used for the antibody engineering field, be used to prepare the antibody molecule of scFv, " phage " antibody and other transformation.The technology of preparing of antibody, especially bi-specific antibody for example reaches the list of references of wherein being quoted referring to following summary: Winter and Milstein, (1991) Nature 349:293-299; Pluckthun (1992) Immunological Reviews 130:151-188; Wright etc. (1992) Crti.Rev.Immunol., 12:125-168; Holliger, P. and Winter, G. (1993) Curr.Op.Biotechn., 4,446-449; Carter etc. (1995) J.Hernatother., 4,463-470; Chester, K.A. and Hawkins, R.E. (1995) Trends Biotechn., 13,294-300; Hoogenboom, H.R. (1997) Nature Biotechnol., 15,125-126; Fearon, D. (1997) Nature Biotechnol., 15,618-619; Pl ü ckthun, A. and Pack, P. (1997) Immunotechnology3,83-105; Carter, P. and Merchant, A.M. (1997) Curr.Opin.Biotechnol., 8,449-454; Holliger, P. and Winter, G. (1997) CancerImmunol.Immunother., 45,128-130.
The invention provides be used at two not the variable region of synantigen or epi-position select and subsequently variable region combination.
What used technology employing was selected in the variable region is library known in the art and system of selection.Employing is from natural library (Marks etc. (1991) J.Mol.Biol., the 222:581 of the rearrangement V gene of human B cell results; Vaughan etc. (1996) Nature Biotech. is that those skilled in the art are well-known 14:309).Synthetic library (Hoogenboom and Winter (1992) J.Mol.Biol., 227:381; Barbas etc. (1992) Proc.Natl.Acad.Sci.U.S.A., 89:4457; Nissim etc. (1994) EMBO J., 13:692; Griffiths etc. (1994) EMBO J., 13:3245; (1995) J.Mol.Biol. such as De Kruif, 248:97) then adopt usually PCR, prepare by clone immunoglobulin V gene.The mistake that occurs in the PCR process can cause height randomization.Can select V at target antigen or epi-position respectively HAnd/or V LThe single domain combination is directly selected or select together in the library in this case.
The preferred for preparation method of bispecific part of the present invention comprises the employing selective system, wherein selects the storehouse, variable region in conjunction with first antigen or epi-position, and selects the storehouse, variable region in conjunction with second antigen or epi-position.Again selected first and second variable regions are combined and select bispecific part in conjunction with first and second antigens or epi-position.Select separately but not simultaneously in conjunction with the closed conformation part of first and second antigens or epi-position.
A. carrier library system
Various selective systems are known in the art, are applicable to the present invention.The example of these systems is as described below.
Can directly screen the plaque of phage expression system or the bacterium colony of lysogen, the two had before all had description (Huse etc. (1989) Science, 246:1275; Caton and Koprowski (1990) Proc.Natl.Acad.Sci.U.S.A., 87; Mullinax etc. (1990) Proc.Natl.Acad.Sci.U.S.A., 87:8095; Persson etc. (1991) Proc.Natl.Acad.Sci.U.S.A., 88:2432) and be used for the present invention.The expression system of even now can be used for screening and reach 10 6Individual different library member, but they are not suitable for the bigger quantity of screening (greater than 10 6Individual member).
Useful especially in the library construction is to select display systems, and described system is connected on its expressed polypeptide nucleic acid.Selection display systems used herein is the system that permission is selected in conjunction with the suitable methods of exhibiting of general part and/or target ligands by single library member.
The selection scheme of isolating required member in big library is known in the art, is representative with the display technique of bacteriophage.This (Scott and Smith (1990) Science of system that diversified peptide sequence is illustrated in the filobactivirus surface, 249:386), verifiedly can be used for producing antibody fragment (and encode their nucleotide sequence) library, be used for external selection and in conjunction with the segmental amplification of the specific antibody of target antigen (McCafferty etc., WO 92/01047).Coding V HDistrict and V LThe nucleotide sequence in district is connected on the genetic fragment of coding targeting signal, such signal can instruct them to arrive the periplasmic space of Bacillus coli cells, the gained antibody fragment can be illustrated in phage surface as a result, normally merges with bacteriophage coat protein (for example pIII or pVIII).Perhaps, antibody fragment outwards is illustrated on the bacteriophage lambda capsid (phage).An advantage based on the display systems of phage is exactly: because they are biosystems, thus the phage that contains selected library member can be cultivated in bacterial cell, and the selected library member that simply increases.In addition, because coded polypeptide library member's nucleotide sequence is comprised by phage or phasmid carrier, so check order, express relative direct with genetic manipulation thereafter.
The construction method of phage antibody display libraries and bacteriophage lambda expression library is (McCafferty etc. (1990) Nature, 348:552 well-known in the art; Kang etc. (1991) Proc.Natl.Acad.Sci.U.S.A., 88:4363; Clackson etc. (1991) Nature, 352:624; Lowman etc. (1991) Biochemistry, 30:10832; Burton etc. (1991) Proc.Natl.Acad.Sci.U.S.A., 88:10134; Hoogenboom etc. (1991) Nucleic Acids Res., 19:4133; Chang etc. (1991) J.Immunol., 147:3610; Breitling etc. (1991) Gene, 104:147; Marks etc. (1991), ibid; Barbas etc. (1992), ibid; Hawkins and Winter (1992) J.Immunol., 22:867; Marks etc., 1992, J.Biol.Chem., 267:16007; Lerner etc. (1992) Science, 258:1313, described document is attached to herein by reference).
A method that has advantage especially is to use scFv phage-library (Huston etc., 1988, Proc.Natl.Acad.Sci.U.S.A., 85:5879-5883; Chaudhary etc. (1990) Proc.Natl.Acad.Sci.U.S.A., 87:1066-1070; McCafferty etc. (1990), ibid; Clackson etc. (1991) Nature, 352:624; Marks etc. (1991) J.Mol.Biol., 222:581; Chiswell etc. (1992) Trends Biotech., 10:80; Marks etc. (1992) J.Biol.Chem., 267).The various embodiments in the scFv library of showing on bacteriophage coat protein are existing to be described.The improvement of phage display method also is known, and for example referring to WO96/06213 and WO92/01047 (Medical Research Council etc.) and WO97/08320 (Morphosys), described document is attached to herein by reference.
Other system that produces polypeptide libraries comprises the acellular enzymatic mechanism of use, is used for external synthetic library member.In a method, select RNA molecule (Tuerk and Go1d (1990) Science, 249:505 by many wheels selections and pcr amplification at target ligands; Ellington and Szostak (1990) Nature, 346:818).A similar techniques can be used for identifying DNA sequence (Thiesen and Bach (1990) Nucleic Acids Res., the 18:3203 of the predetermined human transcriptionfactor hTF of combination; Beaudry and Joyce (1992) Science, 257:635; WO92/05258 and WO92/14843).In a similar manner, external translation can be used for synthetic polypeptide, and this is as the method that produces big library.These methods that generally include stable polyribosome camplex are further referring to WO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058 and WO92/02536.Be not alternative display systems based on phage for example those are disclosed in the system of WO95/22625 and WO95/11922 (Affymax), adopt polysome to come displayed polypeptides, be used for selecting.
Also have a technology to comprise selection to the storehouse in the artificial compartment, this permission combines gene and its gene outcome.For example, by the selective system of the nucleic acid of the required gene outcome of selection coding in the microcapsule that forms at water in oil emulsion, referring to WO99/02671, WO00/40712; Tawfik and Griffiths (1998) Nature Biotechnol 16 (7), 652-6.With genetic elements compartmentation in microcapsule that coding has required active gene outcome, in microcapsule, transcribe then and/or translate to produce their corresponding gene outcomes (RNA or protein).Sorting produces the genetic elements with required active gene outcome more subsequently.This method detects required activity and the select target gene outcome by the whole bag of tricks.
B. library construction
Can adopt technology known in the art, make up library for example set forth above and be used for selecting, perhaps buy from commercial source.Can be used for library of the present invention referring to for example WO99/20749.In case select carrier system and the nucleotide sequence of one or more coding target polypeptides be cloned in the carrier library,, just can in cloning molecular, produce multiformity by before expression, carrying out mutation; Perhaps, can before selecting to carry out, aforesaid mutation and many wheels express and select coded protein.According to the standard molecule method, the nucleotide sequence of coding structure being optimized polypeptide carries out mutation.Useful especially is that the polymerase chain reaction is PCR (described document is attached to herein by reference for Mullis and Faloona (1987) Methods Enzymol, 155:335).PCR is well-known in the art, and it is to adopt by the catalytic many wheel dna replication dnas of heat-staple DNA dependent dna-polymerases, with amplification target sequence.The structure of various antibody libraries is referring to (1994) Ann.Rev.Immunology such as Winter, and 12,433-55 and the list of references of wherein quoting.
With template DNA (1fg at least; That more useful is 1-1000ng) and 25pmol oligonucleotide primers at least, carry out PCR; As primer storehouse (primer pool) very during heterogeneity, preferably adopt relatively large primer, because each sequence only has a small amount of primer storehouse molecule as representative, and quantity becomes restriction in amplification cycles subsequently.Usually reactant mixture comprises: 2 μ l DNA, 25pmol oligonucleotide primers, 2.5 μ l 10X PCR buffer, 1 (Perkin-Elmer, FosterCity, CA), 0.4 μ l 1.25 μ M dNTP, 0.15 μ l (or 2.5 units) Taq archaeal dna polymerase (Perkin Elmer, Foster City, CA) adding to cumulative volume with deionized water is 25 μ l.Cover mineral oil, carry out PCR, with the programmed heating instrument that circulates.Length and the temperature and the cycle-index of each step are adjusted according to the stringency that the result is required in the PCR circulation.Annealing temperature and time are estimated to decide with the efficient of template annealing and the mispairing degree that can tolerate by primer; Obviously, when nucleic acid molecules increases and mutation simultaneously, mispairing is essential, at least in the synthetic first round.The ability of optimizing the stringency of primer annealing condition is within those of ordinary skills' the ken.The annealing temperature that adopts is between 30 ℃ and 72 ℃.Template molecule begins degeneration and usually occurs in and reach 4 minutes between 92 ℃ and 99 ℃, is 20-40 circulation again, and circulation is (according to said temperature by degeneration (94-99 ℃ 15 seconds to 1 minute), annealing; 1-2 minute) and extend (72 ℃ 1-5 minute, depend on the length of amplified production) and form.Final extend common 72 ℃ 4 minutes, then be uncertain (0-24 hour) step again, at 4 ℃.
C. in conjunction with single variable region
The domain that the present invention is used can connect by the whole bag of tricks known in the art in case just select, and comprises covalency and non-covalent method.
Preferable methods comprises the aforesaid peptide linker of employing, for example connects scFv molecule (Bird etc. (1988) Science 242; 423-426).The discussion of relevant suitable joint is referring to Bird etc., and Science 242,423-426; Hudson etc., Journal Immunol Methods 231 (1999) 177-189; Hudson etc., Proc.Nat.Acad.Sci.USA, 85,5879-5883.Joint is preferably flexible, allows two single domains interactions.The example of a joint is (Gly 4Ser) nJoint, n=1-8 wherein, for example 2,3,4,5 or 7.Also can adopt the joint (its flexibility is relatively poor) (Holliger etc. (1993) PNAS (USA) 90:6444-6448) that uses in the double-stranded antibody.
In one embodiment, used joint is not an immunoglobulin hinge region.
Combination can be adopted without the method for joint in the variable region.For example, can develop the purposes (described disulphide bridges is to provide by cysteine residues naturally occurring or that transform) of disulphide bridges, so that stablize V H-V H, V L-V LOr V H-V LDimer (Reiter etc. (1994) ProtemEng., 7:697-704) or the reconstruct by interface between the variable region improving " fitness (fit) ", thereby stable phase mutual effect (Ridgeway etc. (1996) Protein Eng.7:617-621; Zhu etc. (1997) Protein Science 6:781-788).
If suitable, can use to be used for connecting or stabilizing immunoglobulin variable region, especially antibody V HOther technology in district.
According to the present invention, the bispecific part can be " closure " conformation in solution." closure " configuration is meant: two districts (V for example wherein HAnd V L) there is the V of Lian Jieing for example with type of attachment H-V LTo forming antibody combining site.For example, scFv can be closed conformation, and this depends on and is used to connect V HDistrict and V LThe arrangement of the joint in district.Allow this two district connect if it has enough flexibilities, perhaps hold them in rigidly on the link position, then this two district may take closed conformation.
Equally, V HThe district to and V LThe district is to existing closed conformation.Usually, this will become by tight connection of for example rigid joint with two districts in the ligand molecular.The combination simultaneously of part in the closed conformation prolongs the molecule and second target molecule of the half-life of part.Therefore, part is usually only in conjunction with second target molecule that disintegrates down from the molecule that prolongs the part half-life.
In addition, need not the V of joint H/ V H, V L/ V LOr V H/ V LDimeric being configured between two districts brought competition.
And part of the present invention also can be open conformation.In such conformation, the combination simultaneously of described part prolongs the molecule and second target molecule of the half-life of part.Usually, the variable region in the open configuration is (with regard to V H-V LFor) keeping distance enough far away with following domain: described domain can not interact and form antibody combining site nor competitive their epi-positions separately that combine of meeting.With regard to V H/ V HOr V L/ V LDimer, these domains do not combine by rigid joint.Certainly, such domain pairing is not competed the antigen combination yet or is formed antibody combining site.
Fab fragment and complete antibody will mainly exist with closed conformation, may exist with various equilibriums under different situations although be appreciated that opening and closed bispecific part.Part may change the isostatic balance of open configuration with combining of target.Therefore, can there be two kinds of conformations in some part of the present invention in solution, and one (opening mode) can be independently in conjunction with two antigens or epi-position, and another conformation (closed form) only can be in conjunction with antigen or epi-position simultaneously; Therefore, in this conformation, the competitive binding partner of antigen or epi-position.
Although therefore the opening mode of bispecific part exists the equilibrium with closed form in solution, consider that equilibrium will help closed form; In addition, opening mode can be sheltered by the combination of the closed conformation of targeting.Therefore, there are two balances between (open and closed) conformation in some bispecific part preferred of the present invention.
Can modify bispecific part of the present invention, so that help open or closed conformation.For example, V H-V LMutual relation stablize because of disulfide bond, thereby stablized closed conformation.In addition, can make up and be used for (comprising V in conjunction with each district HDistrict and V LThe district to) joint, to help opening mode; For example, joint can spatially hinder the combination in each district, for example by on correct position in conjunction with big amino acid residue, perhaps design can physical property separates the suitable rigid structure in each district.
D. the sign of bispecific part
The bispecific part can be measured with method well known to those skilled in the art with combining of its specific antigen or epi-position, and this method comprises ELISA.In an embodiment preferred of the present invention, adopt monoclonal phage E LISA to measure combination.
Can be according to any appropriate method, carry out phage E LISA: exemplary arrangement is as follows.
Can by ELISA screen every take turns produce in the selection, with selected antigen or the bonded phage of epi-position colony, to identify " polyclone " phage antibody.Then can be by the phage of ELISA screening, to identify " monoclonal " phage antibody from these colonies of single infected bacterial clump.Preferably also screen the soluble antibody fragment of conjugated antigen or epi-position, this also can carry out at reagent such as C-end or N-end labellings by ELISA, employing, and (referring to (1994) Ann.Rev.Immunology 12 such as for example Winter, 433-55 reaches the list of references of wherein quoting.
(Marks etc. 1991, and ibid for gel electrophoresis that can be by the PCR product; Nissim etc. 1994, and ibid), survey (Tomlinson etc., 1992) J.Mol.Biol.227,776) or, estimate the multiformity of selected phage monoclonal antibody by the carrier DNA order-checking.
E. the structure of bispecific part
As mentioned above, in this article, antibody is defined as antibody (for example IgG, IgM, IgA, IgA, IgE) or its fragment (Fv, scFv, double-stranded antibody that Fab, Fv, disulfide bond connect), and it comprises at least one heavy chain and variable region of light chain, at least two variable region of heavy chaines or at least two variable region of light chains.It can derive from the antibody of the natural generation of any species to small part, or the antibody that produces by recombinant DNA technology; No matter be isolating from following which kind of sample: serum, B cell, hybridoma, transfectoma, yeast or antibacterial.
In a preferred embodiment of the invention, the bispecific part comprises single variable region of light chain or two the substance chain variable regions or the variable region of light chain of at least one the substance chain variable region of antibody and antibody.For example, part can comprise a pair of V H/ V L, a pair of V HDistrict or a pair of V LThe district.
First and second variable regions of described part can be on same polypeptide chain.Perhaps they can be on different polypeptide chains.With regard to they on same polypeptide chain with regard to, they can connect by joint, described joint is preferably aforesaid peptide sequence.
The covalently or non-covalently combination of first and second variable regions.At them is under the covalently bound situation, and covalent bond can be a disulfide bond.
Adopting display technique of bacteriophage for example as herein described to select from the V gene bank under the situation of variable region, these variable regions can comprise the general framework district, make them to be discerned by the general part of specificity as herein described.The purposes of relevant general framework, general part etc. is referring to WO 99/20749.
When using the V gene bank, the variation in the peptide sequence is preferably placed in the structure ring of variable region.The peptide sequence of each variable region can change by DNA reorganization or by sudden change, so that strengthen the interaction of each variable region and its complementary pair.DNA reorganization is known in the art, referring to for example Stemmer, and No. the 6th, 297,053, Nature 370:389-391 (1994) and United States Patent (USP), described document all is attached to herein by reference.Other method of mutagenesis is that those skilled in the art are well-known.
In a preferred embodiment of the invention, " bispecific part " is strand Fv fragment.In an alternate embodiment of the present invention, " bispecific part " is made up of the Fab form.
On the other hand, the invention provides nucleic acid molecules, described nucleic acid is encoded at least " bispecific part " as herein described.
It should be appreciated by those skilled in the art that this aspect according to the present invention, antigen or epi-position can be simultaneously in conjunction with same antibody molecules.Perhaps, they can be competitive in conjunction with same antibody molecule.For example, when two epi-positions simultaneously in conjunction with the time, two variable regions of bispecific part can independent target epi-positions in conjunction with them.When the competition of these domains, variable region can be in conjunction with its target, but not in other variable region in conjunction with it in related target; Perhaps first variable region can be in conjunction with its target, but not in second variable region in conjunction with it in related target.
The variable region can be from the antibody at target antigen or epi-position.Perhaps, they can be from the single domain antibody storehouse, for example at those of filobactivirus surface expression.Can as described belowly select.
Generally speaking, can be according to for example method of following standard laboratory handbook, make up and operate realization nucleic acid molecules and vector construction body: Sambrook etc. (1989) Molecular Cloning:A Laboratory Manual required for the present invention, Cold Spring Harbor, USA.
The operation of the nucleic acid that the present invention is used is carried out in recombinant vector usually.
Therefore, on the other hand, the invention provides carrier, described carrier comprises the nucleic acid of encode at least " bispecific part " as herein described.
Carrier used herein is meant so isolating element: described element is used for allogeneic dna sequence DNA is introduced cell, is used for expressing and/or duplicating allogeneic dna sequence DNA.The selection of described carrier or structure and using method subsequently are that those skilled in the art are well-known.A large amount of carriers are public Ke De, comprise bacterial plasmid, phage, artificial chromosome and episomal vector.This class carrier can be used for simple clone and mutation; Perhaps adopt expression vector.Can select the used carrier of the present invention, adapting to the polypeptid coding sequence of required size, length be generally 0.25 kilobase to (kb) to 40kb or bigger.After the body outer clone operation, transform proper host cell with carrier.Each carrier contains different functional assemblies, generally includes clone's (or " polylinker ") site, origin of replication and at least one selectable marker gene.If given carrier is an expression vector, it also can have one or more following elements: enhancer element, promoter, tanscription termination and signal sequence, each all is positioned near the cloning site, makes them effectively be connected with the gene of code book invention part.
Cloning vehicle and expression vector generally all contain makes carrier reproducible nucleotide sequence in one or more selected host cells.Usually in cloning vehicle, this sequence makes carrier be independent of host chromosome DNA and duplicates, and comprises origin of replication or autonomous replication sequence.This class sequence can be used for diversified antibacterial, yeast and virus as everyone knows.The origin of replication of plasmid pBR322 is suitable for most of gram negative bacterias, and 2 μ plasmid starting points are suitable for yeast, and various viral starting point (for example SV40, adenovirus) can be used for the cloning vehicle in the mammalian cell.Usually, origin of replication is unwanted for mammalian expression vector, unless these starting points are used for the mammalian cell of the high-level repetition DNA of energy, for example COS cell.
Preferably cloning vehicle or expression vector can contain the selection gene, are also referred to as selected marker.This gene code transformed host cell is the survival or the required protein of growing in selecting culture medium.Therefore, not contained the carrier transformed host cells of selecting gene can not survive in culture medium.The typical such protein of gene code of selecting: described protein is given antibiotic resistance (for example ampicillin, neomycin, methotrexate or tetracycline) and other toxin resistance, is replenished unexistent auxotrophy or additional key nutrient in the growth medium.
Because the carrier of code book invention part duplicate in escherichia coli conventional carrying out, so adopt the escherichia coli selected marker, for example give the beta-lactamase gene of antibiotic amicillin resistance.These can derive from escherichia coli plasmid, for example pBR322 or pUC plasmid, for example pUC18 or pUC19.
Expression vector contains the discernible promoter of host living beings usually, and this promoter is connected with the target code series of operations.Such promoter can be induction type or composing type.Term " operability connection " is meant and the mode of putting makes the relation of described assembly allow them to work in set mode.Control sequence and coded sequence " operability is connected " are meant that connected mode makes and can carry out the expression of coded sequence in the compatible condition of control sequence.
The promoter that is applicable to prokaryotic hosts comprises for example beta-lactamase and lactose promoter systems, alkali phosphatase, tryptophan (trp) promoter systems and hybrid promoter (for example tac promoter).The promoter that is used for bacterial system generally also contains the Shine-Delgarno sequence that is connected with the coded sequence operability.
Preferred carrier is the expression vector that can express corresponding to polypeptide libraries member's nucleotide sequence.Therefore, can pass through express polypeptide library member's monoclonal independent propagation and expression or pass through any selection display systems of use, select with first and/or second antigen or epi-position.As mentioned above, preferably selecting display systems is phage display.Therefore, can use phage or phasmid carrier, for example pIT1 or pIT2.Be used for targeting sequencing of the present invention and comprise pelB, stII, ompA, phoA, bla and pelA.An example is the phasmid carrier with escherichia coli origin of replication (being used for two strands duplicates) and phage replication starting point (being used to produce single stranded DNA).The operation of this class carrier and expression be well-known in the art (Hoogenboom and Winter (1992), ibid; Nissim etc. (1994), ibid).In brief, carrier contains the lac promoter of beta-lactamase gene (so that on phasmid that imparts selective) and expression cassette upstream, and the latter is by form (N end → C end) with the lower part: pelB targeting sequencing (instructing express polypeptide to periplasmic space), multiple clone site (the clone library member who is used for the nucleotide form), optional one or more peptide-labeled (being used for detecting), optional one or more TAG termination codoies and phage albumen pIII.Therefore, use colibacillary different bacterial strain and the non-inhibition bacterial strain of suppressing, and add glucose, isopropylthio-(IPTG) or helper phage (for example VCS M13), carrier can duplicate as plasmid, but do not express, only produce a large amount of polypeptide libraries members or produce phage, some in them contain the polypeptide-pIII fusant of at least one copy on its surface.
The structure of the carrier of code book invention part adopts conventional interconnection technique.Isolated vectors or dna fragmentation are cut, cut out and connect into again the ideal form that produces required carrier.If necessary, can analyze in a known manner, confirm to have correct sequence in the constructed carrier.The used appropriate method of analysis that be used for construction of expression vector, prepares the in vitro transcription thing, DNA is introduced host cell and carries out evaluation expression and function is well known by persons skilled in the art.Pass through conventional method, the gene order that exists in the test sample or its amplification and/or expression, described conventional method be the sequencing analysis of southern blotting technique analysis or rna blot analysis, Western blotting, DNA, RNA or plaque of protein dot blotting, in situ hybridization, immunocytochemistry or nucleic acid or protein molecule for example.If necessary, those skilled in the art can easily know these methods of how improving.
The structure of closed conformation polyspecific part
The one side of second general layout according to the present invention links together two or more incomplementarity epi-positions in conjunction with the territory, make them present closed conformation as herein described.Preferably except joint as herein described, they can also connect skeleton, so that form and/or keep epi-position binding site closed conformation each other.
(I) skeleton
Skeleton can or can be the NIg of originating as mentioned above based on immunoglobulin molecules.Preferred immunoglobulin skeleton as herein described comprises any one or a plurality ofly is selected from following skeleton: comprise the immunoglobulin molecules with the lower part at least: (i) CL of antibody (κ or λ subclass) district; Or the (ii) CH1 district of heavy chain of antibody; The immunoglobulin molecules that comprises heavy chain of antibody CH1 district and CH2 district; The immunoglobulin molecules that comprises heavy chain of antibody CH1 district, CH2 district and CH3 district; Or the CL of any (ii) subclass and antibody (κ or λ subclass) district.Also can comprise hinge area.Each district's combination like this can be for example to simulate natural antibody, for example IgG or IgM or its fragment, for example Fv, scFv, Fab or F (ab ') 2Molecule.One skilled in the art will appreciate that this list is not is detailed and exhaustively.
(II) protein scaffolds
Each epi-position comprises protein scaffolds and one or more CDR in conjunction with the territory, and the specificity of their participation structure territories and one or more epi-positions interacts.Best epi-position of the present invention comprises 3 CDR in conjunction with the territory.Suitable protein scaffolds comprises and is selected from following any support: based on the support of immunoglobulin domains, based on the support of fibronectin, based on the support of affine body, based on the support of CTLA4, based on chaperone for example GroEL support, based on the support of lipocalin protein with based on the support of antibacterial Fc receptor SpA and SpD.It will be understood by those skilled in the art that this list is not is detailed and exhaustively.
F: the support that is used to make up the bispecific part
I. the selection of main chain conformation
The immunoglobulin superfamily member shares the similar folding of its polypeptide chain.For example, although antibody has the height multiformity on its primary sequence, but sequence comparison and crystal structure show, against one's expectation, there be 5 (H1, H2, L1, L2, L3) to adopt limited main chain conformation or canonical structure (Chothia and Lesk (1987) J.Mol.Biol., the 196:901 of number in 6 antigen coupling collars of antibody; Chothia etc. (1989) Nature, 342:877).Therefore, ring length and Key residues are analyzed, can be predicted main chain conformation (Chothia etc. (1992) J.Mol.Biol., the 227:799 of H1, the H2, L1, L2 and the L3 that in most of people's antibody, exist; Tomlinson etc. (1995) EMBO J., 14:4628; Williams etc. (1996) J.Mol.Biol., 264:220).Although the H3 district has more multiformity (because using the D section) on sequence, length and structure, but it also constitutes the main chain conformation of the limited becate length of number, this depends on the length of specific residue on the ring and the key position of antibody framework and whether exists, or residue kind (Martin etc. (1996) J.Mol.Biol., 263:800; Shirai etc. (1996) FEBS Letters, 399:1).
Bispecific part of the present invention preferably assembles from domain libraries, for example V HLibrary, district and/or V LThe library, district.In addition, bispecific part of the present invention itself can provide with the library form.In one aspect of the invention, design bispecific part and/or domain libraries wherein can be selected some ring length and Key residues, are known with the main chain conformation that guarantees each member.As mentioned above, preferably these are true conformations of naturally occurring immunoglobulin superfamily molecule, to reduce to the functional chance of their right and wrong minimum.Planting is the effect that the V constant gene segment C plays a suitable basic boom, is used to make up antibody or TXi Baoshouti library; Also can use other sequence.Can low frequency morph, make a small amount of functional member have the main chain conformation of change, such change does not influence its function.
The canonical structure theory also is used to estimate the number of the coded different main chain conformations of part, also selects to be used for diversified residue with prediction based on the ligand sequence of main chain conformation, does not influence canonical structure simultaneously again.Know that in people V κ district, the L1 ring can adopt one of 4 kinds of canonical structures, the L2 ring has a kind of canonical structure, and one of the employing 4 of the L3 in 90% people V κ district ring or 5 kind of canonical structure (Tomlinson etc. (1995), ibid); Therefore, only in V κ district, the different main chain conformations that the generation capable of being combined of different canonical structures is a large amount of.Suppose that V λ district is the various different canonical structures of L1, L2 and L3 ring coding, V κ district and V λ district can with any V HDistrict's (it can be H1 and several canonical structures of H2 ring coding) pairing, then the number of combinations of these 5 the viewed canonical structures of ring will be very huge.This shows that the multifarious generation of main chain conformation is absolutely necessary concerning producing various binding specificities.Yet, by making up a kind of known main chain conformation, have been found that to against one's expectation based on antibody library, the multiformity of main chain conformation be not produce enough necessary at nearly all antigenic multiformity.Even it is shocking that more a kind of main chain conformation not necessarily apokoinou construction-a kind of naturally occurring conformation can be as the basis in whole library.Therefore, aspect preferred, bispecific part of the present invention has a kind of known main chain conformation.
Selected a kind of main chain conformation is common in the molecule of described immunoglobulin superfamily type preferably.When observing a large amount of naturally occurring molecules and adopt this conformation, this conformation is common.Therefore, of the present invention preferred aspect, consider the natural incidence rate of different main chain conformations of each coupling collar of immunoglobulin domains separately, select to have the naturally occurring variable region of the required combination of main chain conformation of different rings then.If do not have availablely, just can select immediate equivalent.The required combination of the main chain conformation of preferred different rings is that to be chosen seeds by institute be constant gene segment C (its required main chain conformation of encoding) generation.More preferably choosing seeds is that constant gene segment C is frequently expressed at nature, and most preferably they are to be the most frequent expression in the constant gene segment C in all natural kinds.
In design bispecific part or its library process, can consider the incidence rate of the different main chain conformations of each in 6 antigen coupling collars separately.For H1, H2, L1, L2 and L3, select the given conformation that is adopted by 20% and 100% natural molecule antigen coupling collar.Usually, viewed its incidence rate (promptly between 35% and 100%) more than 35%, better be more than 50% or even more than 65%.Because most H3 rings do not have canonical structure, so preferentially be chosen in main chain conformation common in the ring that can show canonical structure.Therefore, for each ring, select modal conformation in the natural storehouse.In people's antibody, it is as follows that each encircles modal canonical structure (CS): the L1-CS 2 (39%) of H1-CS 1 (79% expression library), H2-CS 3 (46%), V κ, (ratio of calculation assumption κ: λ is 70: 30 to the L3-CS 1 (36%) of L2-CS 1 (100%), V κ, Hood etc. (1967) Cold Spring Harbor Symp.Quant.Biol., 48:133).For H3 ring with canonical structure, it seems that the CDR3 length (Kabat etc. (1991) Sequences of Proteins of immunological interest, U.S. HHS) that has from residue 94 to residue 7 residues of 101 salt bridge be modal.There are at least 16 human sequence antibodies to have required H3 length and Key residues in the EMBL data library, constitute this conformation, and in Protein Data Bank, have at least two crystal structures to can be used as the basis of antibody modeling (2cgr and 1tet).The kind of normal expression is that the canonical structure combination of constant gene segment C is V HSection 3-23 (DP-47), J HSection J H4b, V κ section O2/O12 (DPK9) and J κ section J κ 1.V HSection DP45 and DP38 also are suitable.Therefore, these sections can be used for combination, have the basis in the library of required single main chain conformation as structure.
Perhaps, be not to select single main chain conformation, but the natural incidence rate that adopts the combination of main chain conformation is as the basis of selecting single main chain conformation according to the natural incidence rate of the different main chain conformations of isolating each coupling collar.With regard to antibody, for example, can determine any 2,3,4,5 or the natural incidence rate of the canonical structure of all 6 antigen coupling collars combination.At this, preferred selected conformation is common in naturally occurring antibody, and most preferably it is viewed modal in the natural storehouse.Therefore, in people's antibody, for example when considering the natural combination of H1, H2, L1, L2 and five antigen coupling collars of L3, determine the combination of modal canonical structure, then in conjunction with modal H3 ring conformation, as the basis of selecting single main chain conformation.
Ii. the variation of regular sequence
If have selected several known main chain conformation or preferred single known main chain conformation, can be by changing the binding site of molecule, make up bispecific part of the present invention or the used library of the present invention, have structure and/or functional multifarious storehouse so that produce.This shows, produces variant, makes them have enough multiformity on its structure and/or function, makes them that various activity can be provided.
Required multiformity normally produces by the selected molecule of change on one or more positions.Can be at random or the preferred position that changed selected.Then, can reach variation in the following manner:, produce the variant of very large amount therebetween by randomization (remaining aminoacid is replaced by any aminoacid or its analog (natural or synthetic)); Perhaps, produce the more limited variant of number by replacing remaining aminoacid with one or more amino acid whose qualification subclass.
Report the whole bag of tricks, be used to introduce such multiformity.Fallibility PCR (Hawkins etc. (1992) J.Mol.Biol., 226:889), chemomorphosis (Deng etc. (1994) J.Biol.Chem., 269:9533) or the mutant bacteria bacterial strain (Low etc. (1996) J.Mol.Biol. 260:359) can be used for random mutation is introduced in the gene of coding molecule.The method that is undergone mutation in the selected location also is well-known in the art, and it comprises uses mispairing oligonucleotide or degenerate oligonucleotide, uses or do not use PCR.For example, by the orthomutation of antigen coupling collar, some synthetic antibody libraries have been produced.Make the H3 district randomization of people's tetanus toxoid in conjunction with Fab, so as to produce various new binding specificities (Barbas etc. (1992) Proc.Natl.Acad.Sci.U.S.A., 89:4457).At random or partly to have appended to kind be on the V constant gene segment C H3 and L3 district at random, produces big library (Hoogenboom and Winter (1992) J.Mol.Biol., 227:381 with the framework region that do not suddenly change; Barbas etc. (1992) Proc.Natl.Acad.Sci.U.S.A., 89:4457; Nissim etc. (1994) EMBO J., 13:692; Griffiths etc. (1994) EMBO J., 13:3245; (1995) J.Mol.Biol. such as De Kruif, 248:97).Such variation can extend to and comprise some or all other antigen coupling collar (Crameri etc. (1996) Nature Med., 2:100; Riechmann etc. (1995) Bio/Technology, 13:475; Morphosys, WO97/08320, ibid).
Because only just having generation to H3, the ring randomization surpasses 10 approximately 15The potentiality of kind of structure, and other 5 rings are also had the potentiality that produce similarly big meristic variation body, thus can not by use existing transformation technology or use cell free system to produce represent the library that might make up.For example, in one of constructed up to now maximum library, produce 6 * 10 10Individual different antibodies, this only is the potential multifarious part (Griffiths etc. (1994), ibid) in this design library.
In a preferred embodiment, only participate in the residue of generation or decorating molecule required function directly, just by variation.For many molecules, function is in conjunction with target, and therefore, multiformity should concentrate in the target binding site, and avoids changing those concerning the molecule overall package or keep requisite residue the selected main chain conformation.
The variation of regular sequence is when it is used for the antibody district
With regard to antibody bispecific part, the target binding site is modal antigen binding site.Therefore, aspect highly preferred, the invention provides the library of antibody bispecific part or be used to assemble their library, wherein only just change at the residue of antigen binding site.In people's antibody library, the extreme multiformity of these residues, known its contact high-resolution antibody/antigen complex that allows.For example, in L2, known location 50 is different with 53 in naturally occurring antibody, and observes them and contact with antigen.By contrast, conventional method has changed seven residues of diversified two certain that compare in the used library of all residues in the respective complementary determining area (CDR1) (definition is referring to Kabat etc., 1991, ibid), the present invention.This has shown the remarkable improvement that produces the required functional diversity of various antigen-binding specificities.
At nature, antibody diversity is the result of two processes: kind is the somatic cell reorganization of V, D and J constant gene segment C, produces to be used for experiment (naive) elementary storehouse (so-called kind system and junctional diversity) first; And gained is reset the somatic hypermutation of V gene.Human sequence antibody be the analysis showed that, multiformity in the elementary storehouse concentrates on the center of antigen binding site, somatic hypermutation then is diffused into multiformity the antigen binding site peripheral region, and these the district in elementary storehouse be high conservative (referring to (1996) J.Mol.Biol. such as Tomlinson, 256:813).This complementarity may become the spatial available strategy of retrieve sequence gradually, and although obviously be that antibody is peculiar, it also can easily be used for other peptide library.The residue that changes is the subclass of residue that constitutes the binding site of target.If necessary, the different times during selecting can change difference (the comprising overlapping) subclass of residue in the target binding site.
With regard to antibody library, produce initial " naivety " storehouse (initial " naive " repertoire), wherein change some of antigen binding site rather than whole residues.In this case, term used herein " inmature (naive) " is meant there is not pre-determined target target antibody molecule.These molecules are similar to those the coded molecules of immunoglobulin gene that do not experience immune diversified individuality (for example under the situation of fetus and neonate individuality), and the immune system of described individuality is not subjected to the attack that various antigenicities stimulate as yet.Then, select this storehouse at various antigens or epi-position.If necessary, also can outside the domain of initial storehouse change, introduce more multiformity.Can select to have this sophisticated storehouse of rhetorical function, specificity or affinity.
The invention provides two different naiveties in conjunction with the storehouse, territory, be used to make up the inmature library of bispecific part or bispecific part, wherein changed some or all residue in the antigen binding site.Natural elementary storehouse is simulated in " elementary " library, and its multiformity is limited in the residue at antigen binding site center, and they are to have multiformity (kind is a multiformity) in the V constant gene segment C in kind, perhaps are endowed multiformity (junctional diversity) in regrouping process.These diversified residues include but not limited to H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96.In " somatic cell " library, residue that multiformity is confined to change in the regrouping process (junctional diversity) or height somatic mutation).These diversified residues include but not limited to H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96.Known allowing is suitable for multifarious all residues listed above in these libraries and contacts one or more antibody-antigenic compounds.Because in these two kinds of libraries, be not that all residues all change in the antigen binding site, in selection, by changing the residue residue in conjunction with other multiformity, if desired like this.Any subclass that it should be apparent to those skilled in the art that any of these residue (other residue that perhaps comprises antigen binding site) may be used to the initial of antigen binding site and/or variation afterwards.
Be used for library construction of the present invention, the variation of selected location is normally carried out on nucleic acid level, promptly by changing the coded sequence of specifying peptide sequence, makes many possible aminoacid (all 20 kinds or its subclass) can be combined in this position.Adopt the IUPAC nomenclature, the most general codon is NNK, its encode all aminoacid and TAG termination codon.The NNK codon is preferred for introducing required multiformity.Also can use other codon that reaches same end, comprise the NNN codon, this codon causes producing extra termination codon TGA and TAA.
The multifarious feature of side chain obviously has bias to some amino acid residue in people's antibody antigen-binding site.If add up each V H, 10 positions the most changeable in V κ and the V λ district the words formed of aminoacid, surpass 76% side chain multiformity from 7 different residues only, they are serine (24%), tyrosine (14%), agedoite (11%), glycine (9%), alanine (7%), aspartic acid (6%) and threonine (6%).This tending to can provide the hydrophilic residue of main chain flexibility and the bias of little residue, may react surperficial evolution, and this evolution is tended in conjunction with various antigens or epi-position, and helps to explain that the required of antibody mixes in the elementary storehouse.
Distribute because preferably simulate such aminoacid, so distribute preferred simulation at seen those of the antigen binding site of antibody remaining to be changed locational aminoacid.The bias in the aminoacid replacement of some polypeptide (being not only antibody polypeptides) is selected in this permission at various target antigens, be easy to be used for any peptide library.The whole bag of tricks is arranged, be used for making on the position that remains to be changed aminoacid to distribute bias (comprise and use trinucleotide mutation, referring to WO97/08320) takes place, wherein preferable methods owing to synthetic easily, is to adopt conventional degenerate codon.By relatively by the coded aminoacid profile of all combinations of degenerate codon (on each position, have equal proportion single, double, three and the quadruple degeneracy) with the use of natural amino acid, can calculate the most representative codon.Codon (AGT) is (AGC) C and (AGT) (AGC) (CT) of T, (AGT) (AGC), that is to say, be exactly the codon of approaching amino acid needed profile with DVT, the DVC of IUPAC nomenclature name and DVY-respectively: their encode 22% serine and 11% tyrosine, agedoite, glycine, alanine, aspartic acid, threonine and cysteine.Therefore, preferred library makes up with DVT, DVC or DVY codon on each diversified position.
G: the antigen that can prolong the part half-life
Bispecific part of the present invention (general layout that presents it) can be in conjunction with one or more molecules that prolong the part half-life in vivo.Usually, this quasi-molecule is a naturally occurring polypeptide in the body, and its opposing is by degraded and/or removing due to the endogenous mechanism, and described mechanism can be removed undesired material from organism.For example, can from following material, select the molecule that prolongs the organism half-life:
Protein from extracellular matrix; For example collagen, laminin, integrin and fibronectin.Collagen is the main protein of extracellular matrix.About 15 kinds of tropocollagen molecules have been known at present, different parts at health is found, for example type i collagen (account for the total collagen of health 90%) is found in bone, skin, tendon, ligament, cornea, internal organs, and the II Collagen Type VI is found in the vitreous body of cartilage, intervertebral disc (invertebral disc), notochord, eyes.
The protein of finding in the blood comprises:
Plasma protein, for example fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid A protein, globin, Profilin, ubiquitin, uteroglobin and beta-2-microglobulin;
Enzyme and inhibitor, for example plasminogen, lysozyme, cysteine proteinase inhibitor C, α-1-antitrypsin and pancreatic trypsin inhibitor.Plasminogen is the inactive precursor of trypsin-like serine protease fibrinolysin.It exists in the blood flow circulation usually.When plasminogen activates, just change into fibrinolysin, it opens potent enzymatic district, snarls the fibrin fibril of hemocyte in the dissolved blood clot.This is called fibrinolysis.
Immune system protein matter, for example IgE, IgG, IgM.
Transport protein, for example retinol binding protein, α-1 microglobulin.
Sozin, for example beta-defensin 1, neutrophil cell sozin 1,2 and 3.
The protein of finding in blood brain barrier or the nervous tissue, for example melanocortin receptor, myelin, ascorbic acid transport protein.
TfR ligands specific-neurologic agent (neuropharmaceutical agent) fusion rotein (referring to US5977307);
Brain capillary endothelial cell receptor, transferrins, TfR, insulin, type-1 insulin like growth factor (IGF1) receptor, insulin like growth factor 2 (IGF2) receptor, Insulin receptor INSR.
Be confined to the protein in the kidney, for example many capsules albumen, IV Collagen Type VI, organic anion transport protein K1, Heymann antigen.
Be confined to the protein in the liver, for example alcoholdehydrogenase, G250.
The blood coagulation X factor
Alpha1 Anti-trypsin
HNF1α
Be confined to the protein of pulmonary, for example secretory component (in conjunction with IgA).
Be confined to the protein in the heart, for example HSP27.It is relevant with dilated cardiomyopathy.
Be confined to the protein of skin, for example keratin.
The bone specific proteins, bone morphogenetic protein (BMP) for example, they are the transforming growth factor subclass that show osteogenic activity (osteogenic activity).Example comprises that BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 (are also referred to as BMP (OP-1) and BMP-8 (OP-2).
Tumour-specific albumen comprises for example cathepsin B's (finding) of people's TA, herceptin receptor, estrogen receptor, cathepsin in liver and spleen.
Disease specific albumen, the antigen of for example only on activating T cell, expressing: comprise
LAG-3 (lymphocyte activation gene), protect bone protein (osteoprotegerin) part (OPGL), referring to Nature 402,304-309; 1999, OX40 (TNF receptor family member expresses on activating T cell, known have only common stimulation T cellular elements in human T-cell leukemia virus I type (HTLV-I) produces cell, to be raised) by specificity.Referring to J.Immunol.2000 July 1; 165 (1): 263-70; Metalloproteases (with arthritis/related to cancer) comprises CG6512 fruit bat (Drosophila), people's paraplegia albumen, people FtsH, people AFG3L2, Mus ftsH; Angiogenesis growth factor comprises acid fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), VEGF/vascular permeability factor (VE-GF/VPF), transforminggrowthfactor-(TGF α), tumor necrosis factor-alpha (TNF-α), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derived endothelial cell growth factor (ECGF) (PD-ECGF), placental growth factor (PlGF), the factor in mid-term (midkine) platelet derived growth factor-BB (PDGF), CXXXC chemotactic factor.
Stress protein (heatshock protein)
HSP finds in born of the same parents usually.When outside born of the same parents, finding them, just show that cell is dead and overflow its content.Non-procedural cell death (necrosis) so only when as wound, i or I as a result the time just take place, therefore, in vivo, the outer HSP of born of the same parents triggers the reaction of immune system to infection and disease.Dual specificity protein in conjunction with the outer HSP of born of the same parents can be confined to disease location.
Participate in the protein of Fc transhipment
Brambell receptor (being also referred to as FcRB)
This Fc receptor has two functions, and the two can potentially be used to send.
Its function is
Cross over Placenta Hominis IgG is transitted to child from mother
Protection IgG exempts from degraded, thereby prolongs the serum half-life of IgG.
Think receptor recirculation IgG from endosome.
Referring to Holliger etc., Nat Biotechnol in July, 1997; 15 (7): 632-6.
Can design part of the present invention, make it have specificity, and do not need the half-life in the extension body above target.For example, part of the present invention can have specificity to being selected from aforesaid tissue specificity target, therefore make the bispecific part or the dAb monomer of the relevant target of conjunctive tissue specific treatment have tissue specificity, no matter how the half-life prolongs, although this can cause the prolongation of half-life.In addition, when part or dAb monomer targeting kidney or liver, this can make part or dAb monomer change and substitute removing approach (for example, part can change the kidney removing into from hepatic clearance) in the body.
H: the purposes of the polyspecific part of the present invention's second general layout
The polyspecific part of the present invention's second general layout can be used for interior therapeutic and preventive use, external and in-vivo diagnostic purposes, external test and reagent purposes etc.For example, according to method known to those skilled in the art, antibody molecule can be used for the determination techniques based on antibody, for example elisa technique.
As mentioned above, polyspecific part of the present invention can be used for diagnosis, prevention and Therapeutic Method.Multi-specificity antibody of the present invention can be used for western blot analysis and undertaken by standard immunoassay histochemical method in diagnosis original position protein detection; For these purposes, can adopt technology known in the art, part is carried out labelling.In addition, be used for to this antibody-like polypeptide preparation property the affinity chromatography method, when with chromatograph holder for example during resin-bonded.All these methods all are that those skilled in the art are well-known.
The diagnostic application of closed conformation polyspecific part of the present invention comprises homogenizing mensuration, be used to measure such analyte: described analyte can utilize closed conformation polyspecific part and two competitive bonded abilities of target, make that two targets can not be simultaneously in conjunction with (closed conformation), perhaps they can be simultaneously in conjunction with two targets (open conformation).
Aspect the present invention's second general layout another, the invention provides the homogenizing immunoassay, adopt part of the present invention.
Diagnostic reagent and research are measured system manufacturer always earnestly seeking real homogenizing immunoassay form, are used for drug discovery and exploitation.Main diagnostic reagent market is included in the human detection of carrying out in hospital, doctor's office and clinic, commercial reference laboratory, blood bank and the family, non-human diagnosis (for example food test, water quality test, environmental monitoring, biological control (bio-defence) and veterinary inspection), and final research (comprises drug development; Basic research and academic research).
At present, immunoassay system is all adopted in all these markets, and these are all set up around chemiluminescence, ELISA, fluorescence or (under situation seldom) ria-determination technology.These measure forms each all need separating step (conjugate and conjugate are not separated).In some cases, need several separating steps.Adding these additional steps has increased reagent and self-reacting device, spended time and has influenced final measurement result.In human diagnosis, separating step can be an automatization, and this has covered problem but has not eliminated problem.Robotics, extra reagent, extra incubation time or the like have all increased sizable cost and complexity.At drug development for example in the high flux screening,, increase extra separating step and can eliminate the ability of screening when detecting millions of duplicate samples and testing molecule level simultaneously when very low.Yet, avoid separating when reading, producing too many noise.Therefore, need real homogenizing form, described form provides sensitivity in existing test format gained scope.Preferably measure to have and completely quantitatively read, and have high sensitivity and big dynamic range.Sensitivity is important requirement, because reduced required sample size.These features all are that homogeneous system provides.In the medical test (care testing) and drug development that sample is of great rarity, this point is extremely important.The Heterogeneous systems that adopt at present this area needs a large amount of samples and expensive reagent.
The application that homogenizing is measured comprises the cancer inspection, and wherein Zui Da mensuration is prostate specific antigen, is used to screen man's carcinoma of prostate.Other application comprises fertility inspection, and it provides a series of inspections for thinking conceived women, comprises α-hcg, is used for gestation.The detection of infectious disease comprises hepatitis, HIV, rubella and other virus and microorganism and sexually transmitted disease (STD).Detection can be used for blood bank, particularly detects HIV, hepatitis A, hepatitis B, hepatitis C, non-A non-B hepatitis.The medicine monitoring test comprises the monitoring prescription drugs in the intravital level of patient, so that reach effect and avoid toxicity, for example digoxin is to ARR effect and the intravital phenobarbital level of psychotic; Theophylline is to the effect of asthma.Diagnostic assay also can be used for drug dependence to be measured, and for example measures ***e, Fructus Cannabis etc.Metabolic determination is used to measure thyroid function, anemia and other physiological obstacle and function.
Homogenizing immunoassay form also is used for the manufacturing of standard clinical chemical assay.Immunoassay and chemical assay are all carried out on same instrument, are very favorable to diagnostic assay.Suitable chemical assay comprises mensuration such as glucose, cholesterol, potassium.
Another main uses of homogenizing immunoassay is exactly drug discovery and exploitation: high flux screening comprises with ultra-large volume, measures the combinatorial chemistry library at target.Detection signal is littler group with positive component again, finally in cell, measure in animal body then.Homogenizing is measured the mensuration that can be used for all these types.In drug development, especially zooscopy and clinical trial, adopt immunoassay in a large number.Homogenizing is measured and is greatly promoted and simplified these methods.Other is used and comprises that also food and drink detects: the escherichia coli in detection meat and other food, Salmonella (salmonella) etc.; Water quality test is included in water factory and detects all types of pollutant, comprises escherichia coli; And veterinary inspector.
At one widely in the embodiment, the invention provides comprise detectable in conjunction with measuring, described reagent is in conjunction with closed conformation polyspecific part of the present invention, it detects characteristic and changes with combining of described closed conformation polyspecific part because of analyte.Such mensuration can be carried out by several different modes, and every kind of mode adopts the above characteristic of closed conformation polyspecific part.
Mensuration depends on reagent analyte replacement directly or indirectly, causes the detection characteristic of reagent to change.For example, when reagent be can catalytic reaction (described reaction has end point detectable) enzyme, and part can be in conjunction with described enzyme, hindering for example its active site, thereby makes enzyme deactivation.Closed conformation polyspecific part also can bound analyte, with substituted enzyme, makes its activation by discharging active site.Then, the enzyme-to-substrate reaction causes the incident that can detect.In an alternate embodiment, described part can combine with enzyme outside active site, influences the conformation of enzyme, thereby changes its activity.For example, the structure of active site can be restricted because of binding partner, perhaps can stop the combination of active necessary cofactor.
The practical operation of measuring can be adopted any form known in this area.For example, can on test strip, provide closed conformation polyspecific part/multienzyme complex; Can provide substrate in the not same district of test strip, and the solvent that contains analyte is provided, allow analyte to move by part/multienzyme complex, substituted enzyme, and carry its arrival substrate zone, produce signal.Perhaps, can on test bar or other solid phase, provide part/multienzyme complex, be dipped into again in analyte/substrate solution, enzyme is discharged in the solution, the analyte that exists with response.
Because enzyme molecule of the potential release of each analyte molecule is quantitative so measure, its signal intensity that produces in preset time depends on analyte concentration in the solution.
Other general layout that adopts the analyte of closed conformation also is possible.For example, closed conformation polyspecific part can be at allosteric site desmoenzyme, activating enzymes thus.In such embodiments, when analyte did not exist, enzyme was activated.Add the analyte substituted enzyme, remove the allosteric activation effect, thereby make enzyme deactivation.
Just use enzymatic activity as with regard to the metric above embodiment of analyte concentration, the activation of enzyme or inactivation are meant the increase or the reduction of enzymatic activity, are to decide with the ability of the reaction of enzyme catalysis generation signal.For example, but but the substrate conversion that enzyme catalysis can't detect becomes its test format.For example, horseradish peroxidase is widely used in this area with chromogenic substrate or chemical luminous substrate, their all commercially available getting.The increase of enzymatic activity or decline level can be between 10% and 100%, and for example 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; Under the situation that activity increases, increase can surpass 100%, promptly 200%, 300%, more than 500%, perhaps, if the baseline activity of inhibitory enzyme can't detect, just can not measure its increase percentage ratio.
In another general layout, the substrate rather than the enzyme of closed conformation polyspecific part energy desmoenzyme/substrate centering.Therefore, substrate can not utilize enzyme, up to by the analyte combination enzyme being discharged from closed conformation polyspecific part.The enforcement of this general layout is the same with the general layout of desmoenzyme.
In addition, can make and measure the combined with fluorescent molecule, for example fluorescein or other fluorogen, its conformation makes that fluorescence is by quencher behind binding partner.In this case, analyte will replace fluorescence molecule with combining of part, thereby produce signal.The substitute of the fluorescence molecule that the present invention is used comprises cold light reagent (for example luciferin/luciferase) and colour reagent (comprising reagent commonly used in the immunoassay, for example HRP).
Treatment and diagnosis composition and uses thereof
The treatment and the diagnostic method that the invention provides compositions and use part of the present invention or compositions, described compositions comprises TNFR1 antagonist of the present invention (for example part) (for example bispecific part, polyspecific part, dAb monomer) and pharmaceutically acceptable carrier, diluent or excipient.Antagonist of the inventive method and part (for example bispecific part, polyspecific part, dAb monomer) can be used for interior therapeutic and preventive use, in-vivo diagnostic purposes etc.
The treatment of antagonist of the present invention and part (for example polyspecific part, bispecific part, dAb monomer) and preventive use comprise and give mammalian receptors with antagonist of the present invention and/or part, for example the people.Bispecific and polyspecific part (for example bi-specific antibody form) with high-affinity in conjunction with polymer antigen.Bispecific or polyspecific part can make two antigen crosslinkings, for example in the cytotoxic T cell of raising, thus the killing and wounding of mediation tumor cell line.
Preferably part or its conjugated protein (for example dAb monomer) with the basic purification of 90-95% homogeneity at least (homogeneity) gives mammal, the above homogeneity hyoscine of 98-99% most preferably, especially when mammal be man-hour.In case partial purification or when being purified to required homogeneity, part can be used for diagnosis or treatment (comprising stripped) or is used for exploitation and implements (Lefkovite and Pernis such as assay method, immunofluorescence dyeing, (1979 and 1981) ImmunologicalMethods, I and II volume, Academic Press, NY).
For example, part of the present invention or its are conjugated protein, and for example the dAb monomer can be used for prevention usually, suppresses or the treatment inflammatory states, comprises acute and chronic inflammatory disease.For example, can give antagonist and/or part, with treatment, suppress or the prevention chronic inflammatory disease, the allergia allergy, cancer, bacillary or viral infection, autoimmune disease (includes but not limited to type i diabetes, asthma, multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis arthropathica, spondyloarthropathy (spondylarthropathy) (for example ankylosing spondylitis), systemic lupus erythematosus (sle), inflammatory bowel (Crohn disease (Crohn ' s disease) for example, ulcerative colitis), myasthenia gravis and Behcet Cotard), psoriasis, endometriosis and abdominal part adhesion (for example abdominal postoperative).
Can the antagonist of the present invention (for example part) (for example bispecific part, polyspecific part, dAb monomer) of target (for example clathrin (Clathrin)) can be by endocytosis outward in conjunction with the born of the same parents that participate in endocytosis, make them can be near target in the born of the same parents.In addition, bispecific or polyspecific part can provide such method: by this method specificity is delivered to environment in the born of the same parents in conjunction with born of the same parents' internal target target in conjunction with territory (for example dAb monomer).This strategy needs for example such bispecific part: its physical characteristic can allow its reservation function in cell.Perhaps, if the intracellular region chamber of final destination is an oxidation state, good folding part may not need free disulphide.
In an application of the invention, term " prevention " be included in disease bring out before granting asylum property compositions." inhibition " is meant after inducing incident and occurs clinically giving compositions before the disease." treatment " be included in disease symptoms occur after granting asylum property compositions.
Preferably bispecific or polyspecific part can be used at treating synergistic cytokine and other molecule in the environment in vivo.Therefore, the invention provides collaborative can be in conjunction with two or more of cytokine or other molecule in conjunction with the active method in territory (for example dAb), this method comprises that give can be in conjunction with the bispecific or the polyspecific part of described two or more molecules (for example cytokine).In this one side of the present invention, bispecific or polyspecific part can be any bispecific or polyspecific part, comprise the part of being made up of complementation and/or incomplementarity district, the part of open conformation and the part of closed conformation.For example, of the present invention this relates to V on the one hand HDistrict and V LThe combination in district, only V arranged HThe district combination and V is only arranged LThe combination in district.
In treatment, can reach collaborative in many ways.For example, only when part during simultaneously at two targets, the target combination just has therapeutic activity, only then is of no curative effect at a target.In another embodiment, only a target can provide low slightly or minimum curative effect, but has increased curative effect with the combination concertedness ground of second target.Preferably the present invention this on the one hand bispecific or the polyspecific part bonded cytokine be selected from the tabulation shown in the appendix 2.
In addition, bispecific or polyspecific part can be used for oncology's purposes, and a specificity of described part is at CD89 (it is to be expressed by cytotoxic cell), and other specificity then is a tumour-specific.At the example of tumor antigen see appendix 3.
Can adopt animal model system, be used for exempting from the effectiveness of disease or treatment disease screening TNFR1 antagonist (for example part, antibody or its conjugated protein) at the protection body.The method of detection system lupus erythematosus (SLE) is (Knight etc. (1978) J.Exp.Med., 147:1653 known in the art in the susceptible mice; Reinersten etc. (1978) New Eng.J.Med., 299:515).In the SJL/J female mice, induce an illness by using solubility AchR albumen from another species, detection myasthenia gravis (MG) (Lindstrom etc. (1988) Adv.Immunol., 42:233).In the mouse species of susceptible, by injection II Collagen Type VI bring out arthritis (Stuart etc. (1984) Ann.Rev.Immunol., 42:233).In the susceptible rat by injection mycobacteria heatshock protein bring out adjuvant arthritis existing description of model ((1988) Nature such as Van Eden, 331:171).Described by giving Elityran, in mice, bring out thyroid (Maron etc. (1980) J.Exp.Med., 152:1115).In the mice of some strain, insulin dependent diabetes mellitus (IDDM) (IDDM) but spontaneous generation or can bring out generation, described mice is (1984) Diabetologia such as Kanasawa for example, those that 27:113 describes.The EAE of mice and rat can be used as model, is used for human MS.In this model, demyelination can bring out by giving myelin basic protein (referring to Paterson (1986) Textbook of Immunopathology, Mischer etc. (chief editor), Grune and Stratton, NewYork, 179-213 page or leaf; McFarlin etc. (1973) Science, 179:478; Satoh etc. (1987) J.Immunol., 138:179).
Usually, the antagonist of the present invention of purified form (for example part) can use with the last suitable carriers of pharmacology.Usually, these carriers comprise aqueous or alcohol/aqueous pharmaceutical, Emulsion or suspensoid, comprise saline and/or buffer medium arbitrarily.The parenteral solvent comprises sodium chloride solution, woods Ge Shi glucose, glucose and sodium chloride and lactic acid Ringer's solution.Can select suitable physiologically acceptable adjuvant from thickening agent, keep polypeptide complex if desired in suspensoid, these thickening agents are carboxymethyl cellulose, polyvinylpyrrolidone, gelatin and alginate for example.
The intravenous solvent comprises liquid and supplementary and electrolyte replenisher, for example based on those of woods Ge Shi glucose.Also can contain antiseptic and other additive, for example antimicrobial, antioxidant, chelating agen and noble gas (Mack (1982) Remington ' sPharmaceutical Sciences, the 16th edition).Various appropriate formulation all can adopt, and comprise prolonging the preparation that discharges.
The composition forms that can give separately uses antagonist of the present invention (for example part), perhaps with itself and other medicines coupling.These medicines can comprise various immunization therapy medicines, for example cyclosporin, methotrexate, amycin or cisplatin and immunotoxin.Pharmaceutical composition can comprise various cytotoxic agents or other medicines and antagonist of the present invention (for example part) or or even have " mixture " of the combination of not homospecific part of the present invention (for example using the selected part of different target antigens or epi-position), no matter whether they mixed before administration.
The route of administration of pharmaceutical composition of the present invention can be any approach known to a person of ordinary skill in the art.For treatment (including but not limited to immunization therapy), can adopt standard technique, give any patient with the selected part of the present invention.Can give by any suitable method, comprise parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, by pulmonary route, perhaps suitable is, by the direct infusion of conduit.The dosage and the frequency will depend on other parameter that patient age, sex and situation, the other medicines that give simultaneously, antagonism indication (counterindication) and clinician will consider.As indicated, can topical (topical administration pulmonary for example, by pulmonary administration, intranasal administration for example) or be administered systemically.
Can the present invention's ligand freeze dried back storage is standby, reprovision is in suitable carrier before using.Known this technology is effectively to the routine immunization globulin, can adopt lyophilizing known in the art and reprovision technology.It will be understood by those skilled in the art that lyophilizing and reprovision can cause antibody activity forfeiture (for example for the routine immunization globulin, IgM antibody is higher than the loss of IgG antibody activity) in various degree, so usage level should be heightened so that compensate.
Can contain the compositions of antagonist of the present invention (for example part) or its mixture, be used to the treatment of preventing and/or treating property.In some treatment is used, suppress, contain, regulate, kill and wound or enough consumptions of the selected cell colony of some other detected parameters but reach to small part, be defined as " treatment effective dose ".Although reach the general state that the required consumption of this dosage will depend on disease severity and patient's autoimmune system, but usually scope is every kg body weight 0.005-5.0mg part (for example antibody, receptor (for example TXi Baoshouti) or its conjugated protein), and dosage more commonly used is 0.05-2.0mg/kg/ dosage.For prophylactic applications, also can be similar or low dosage contains part of the present invention or its mixture slightly compositions, with prevention, suppress or postpone seizure of disease (for example keep and alleviate or resting state, perhaps prophylaxis of acute phase).Skilled clinician can determine suitable dosing interval, so that treatment, inhibition or prevent disease.When giving TNFR1 antagonist (for example part) with treatment, when inhibition or prevention chronic inflammatory disease, can give maximum 4 times/day, twice weekly, once in a week, whenever biweekly, every month once, whenever bimonthly, its dosage for example is that about 10 μ g/kg are to about 80mg/kg, about 100 μ g/kg are to about 80mg/kg, about 1mg/kg is to about 80mg/kg, about 1mg/kg is to about 70mg/kg, about 1mg/kg is to about 60mg/kg, about 1mg/kg is to about 50mg/kg, about 1mg/kg is to about 40mg/kg, about 1mg/kg is to about 30mg/kg, about 1mg/kg is to about 20mg/kg, about 1mg/kg is to about 10mg/kg, about 10 μ g/kg are to about 10mg/kg, about 10 μ g/kg are to about 5mg/kg, about 10 μ g/kg are to about 2.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg or about 10mg/kg.In specific embodiment, give TNFR1 antagonist (for example part) with treatment, inhibition or prevention chronic inflammatory disease, whenever biweekly or every month once, dosage is about 10 μ g/kg to about 10mg/kg (for example about 10 μ g/kg, about 100 μ g/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg or about 10mg/kg).
With respect to the described symptom that had before the treatment or for the described symptom of the individuality (people or animal pattern) of not treating with described compositions or other appropriate control, when one or more symptoms reduce (for example reach at least 10% or reach at least one point) in the clinical evaluation scale, just think that the treatment of carrying out with compositions as herein described is " effectively ".Although symptom is obviously different, depend at disease or obstacle, can have the clinician of technology or technical staff to measure by common.These symptoms can be measured by for example following method: one or more biochemical indicator levels by monitoring of diseases or obstacle (for example enzyme of disease association or metabolite level, the cell quantity etc. of getting involved), by monitoring physical manifestations (inflammation for example, tumor size etc.), perhaps (for example enlarge disabled state scale (Expanded Disability Status Scale) (being used for multiple sclerosis) by acceptable clinical evaluation scale, Irvine inflammatory bowel questionnaire (32 evaluation tables, estimate relevant intestinal function, General Symptoms, social functions and affective state the quality of life-the scoring scope is 32-224, mark highly more to show that quality of life is good more), rheumatoid arthritis quality of life scale, or other acceptable clinical evaluation scale known in the art.Disease or obstacle symptom continue (for example more than one day, preferably above one day) and descend reach at least 10% or descend and reach a point or a plurality of point on given clinical scale, just show it is that " effectively " treated.Equally, with respect to for the described symptom of the similar individuality (human or animal's model) of combination treatment, when the outbreak or the order of severity of one or more symptoms is delayed, when reducing or eliminating, prevents " effectively " with compositions as herein described.
The compositions that contains antagonist of the present invention (for example part) or its mixture can be used for prevention and therapy equipment, to help change, passivation, to kill and wound or remove mammiferous selected targeted cell population.In addition, selected peptide library as herein described can be used for exsomatizing or external selective killing, exhaust or effectively remove targeted cell population from foreign cell colony.Can be according to standard technique, will under the situation of exsomatizing, mix from mammiferous blood with part (for example antibody, cell surface receptor or its conjugated protein), kill and wound thus or from blood, remove undesired cell, feed back in the mammalian body again.
The compositions that contains antagonist of the present invention (for example part) can be used for prevention and therapy equipment, to help change, passivation, to kill and wound or remove mammiferous selected target cell.
Can give TNFR1 antagonist (for example part, dAb monomer) and/or prepare with one or more other curatives or active drug.When TNFR1 antagonist (for example part, dAb monomer) when giving with other curative, the TNFR1 antagonist can be before giving other medicines, simultaneously or give afterwards.Usually, the TNFR1 antagonist (for example part, dAb monomer) and the mode that gives of other medicines can provide overlapping curative effect.
In one embodiment, the present invention is treatment, suppress or the method for prevention chronic inflammatory disease, and this method comprises that the mammal that needs are arranged treats the TNFR1 antagonist of effective dose (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1).
In one embodiment, the present invention is treatment, suppress or the method for prevention arthritis (for example rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, psoriasis arthropathica), and this method comprises that the mammal that needs are arranged treats the TNFR1 antagonist of effective dose (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1).
In another embodiment, the present invention is treatment, suppresses or the mammal that prevents psoriatic method, this method to comprise needs are arranged is treated the TNFR1 antagonist (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1) of effective dose.
In another embodiment, the present invention is the method for treatment, inhibition or prophylaxis of inflammatory bowel disease (for example Crohn disease, ulcerative colitis), and this method comprises the TNFR1 antagonist (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1) of the mammal treatment effective dose that needs are arranged.
In another embodiment, the present invention is treatment, suppress or the method for prevention chronic obstructive pulmonary disease (for example chronic bronchitis, chronic obstructive bronchitis, emphysema), and this method comprises that the mammal that needs are arranged treats the TNFR1 antagonist of effective dose (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1).
In another embodiment, the present invention is treatment, suppress or prevention pneumonia (bacterial pneumonia for example, staphylococcus pneumonia for example) method, this method comprise the TNFR1 antagonist (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1) of the mammal treatment effective dose that needs are arranged.
The invention provides treatment, suppress or prevention except chronic obstructive pulmonary disease other pneumonopathy and the method for pneumonia.Other pneumonopathy that can treat, suppress or prevent according to the present invention comprises for example cystic fibrosis and asthma (for example steroidal opposing type asthma).Therefore, in another embodiment, the present invention is treatment, suppress or the method for prevention pneumonopathy (for example cystic fibrosis, asthma), and this method comprises that the mammal that needs are arranged treats the TNFR1 antagonist of effective dose (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1).
In specific embodiment, the TNFR1 antagonist is by pulmonary administration, for example by suck (for example in the bronchus, intranasal or the oral cavity sucks, the intranasal drop) or give (for example parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous) by system.
In another embodiment, the present invention is treatment, suppress or the method for prevention septic shock, and this method comprises that the mammal that needs are arranged treats the TNFR1 antagonist of effective dose (for example comprise can in conjunction with the monomeric part of the dAb of TNFR1).
One side again in second general layout of the present invention the invention provides compositions, and described compositions comprises closed conformation polyspecific part and pharmaceutically acceptable carrier, diluent or excipient, and described part obtains by method of the present invention.
In addition, the invention provides the method for usefulness " closed conformation polyspecific part " or composition therapeuticing disease of the present invention.
In a preferred embodiment of the invention, described disease is cancer or inflammatory diseases, for example rheumatoid arthritis, asthma or Crohn disease.
In second general layout of the present invention on the other hand, the invention provides diagnostic method, comprise with closed conformation polyspecific part of the present invention or compositions diagnosing the illness.Therefore, generally speaking, that can utilize analyte and closed conformation polyspecific part combines to substitute certain reagent (agent), and described reagent causes signal to produce when alternative.For example, analyte (second antigen) in conjunction with alternative enzyme (first antigen) binding antibody, provide the basis of immunoassay, especially when enzyme keeps antibody by its active site.
Therefore, the invention provides and detect the method that target molecule exists, this method comprises:
(a) provide closed conformation polyspecific part in conjunction with certain reagent, described part has specificity to target molecule and this reagent, wherein causes the generation of detectable signal with the bonded reagent of part, when substituting part;
(b) give target molecule with closed conformation polyspecific ligand exposed; With
(c) detectable substitutes and the signal of generation.
According to the above aspect of second general layout of the present invention, preferably reagent is enzyme, and described enzyme is a non-activity when combining with closed conformation polyspecific part.Perhaps, reagent can be to be selected from following any or multiple material: the substrate of enzyme and fluorescence molecule, light emitting molecule or colour developing molecule, described molecule when combine with part be non-activity or by quencher.
In addition, selected peptide library as herein described can be used for exsomatizing or external selective killing, exhaust or effectively remove targeted cell population from foreign cell colony.Can be according to standard technique, will under the situation of exsomatizing, mix from mammiferous blood with part (for example antibody, cell surface receptor or its conjugated protein), kill and wound thus or from blood, remove undesired cell, feed back in the mammalian body again.
Embodiment
The following examples have further described the present invention, and these embodiment only are used for illustration purpose.In order to name to dAb, human TNF alpha used herein is called TAR1, and human TNF alpha receptor 1 (p55 receptor) is called TAR2.
Embodiment 1. is at (the selection of the bispecific scFv antibody (K8) of β-gal) of human serum albumin (HSA) and beta galactosidase
This embodiment has illustrated the method for preparation at the bi-specific antibody of β-gal and HAS, has wherein selected and kind be (dummy) V HThe storehouse, V κ variable region that the district connects is used in conjunction with β-gal, and has selected and kind be the V that (dummy) V κ district is connected HThe storehouse, variable region is used in conjunction with HAS.Then with selected variable V HHSA and V κ β-gal district combine, and select the antibody in conjunction with β-gal and HSA.HAS prolongs albumen the half-life that exists in the human blood.
4 human phage antibody libraries have been used in this experiment.
Library 1: kind is V κ/DVT V H8.46 * 10 7
Library 2: kind is V κ/NNK V H9.64 * 10 7
Library 3: kind is V H/ DVT V κ 1.47 * 10 8
Library 4: kind is V H/ NNK V κ 1.45 * 10 8
All libraries are all based on V H(V3-23/DP47 and J H4b) and single people's framework of V κ (O12/O2/DPK9 and J κ 1), wherein the side chain multiformity is attached in the complementary determining region (CDR2 and CDR3).
Dummy V κ sequence is contained in library 1 and library 2, and V HSequence has multiformity (Fig. 1) on position H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97 and H98 (DVT or NNK encode respectively).Dummy V is contained in library 3 and library 4 HSequence, V κ sequence has multiformity (Fig. 1) on position L50, L53, L91, L92, L93, L94 and L96 (DVT or NNK encode respectively).The library presents phasmid pIT2/ScFv form (Fig. 2) and has selected the associativity of described library to protein A and the general part of albumen L in advance, makes that the major part clone in the non-selective library is functional.More than shown in the size of library size after corresponding to preliminary election.Before selecting at antigen, library 1 and library 2 are mixed, obtain single V HLibrary 3 and library 4 are mixed in/dummy V κ library, form single V κ/dummy V HThe library.
β-gal is carried out 3 take turns selection, use V κ/Dummy V HThe library, and HSA is carried out 3 take turns selection, V used H/ dummy V κ library.With regard to β-gal, phage titer is from 1.1 * 10 of the first round 6To 2.0 * 10 of third round 8With regard to HAS, phage titer is from 2 * 10 of the first round 4To 1.4 * 10 of third round 9Described according to (1993) such as Griffith, select, tryptic PBS carries out the eluting with 1mg/ml except using KM13 helper phage (it contains the pIII albumen of protease cutting site in D2 and D3 district) and phage.Add trypsin, by cutting at the c-myc labelling, cutting is from the pIII albumen of helper phage (rather than from phasmid), elution of bound scFv-phage fusant (Fig. 2), thereby further enrichment the functional scFvs of phage expression, and corresponding background (Kristensen and Winter, the Folding﹠amp of having lowered; Design 3:321-328, on July 9th, 1998).The immunity pipe (Immunotube) of the HSA of working concentration 100 μ g/ml or β-gal bag quilt is selected.
In order to check combination, with 24 bacterium colonies of monoclonal phage E LISA screening from the third round of each selection.According to (Methods Enzymol.1996 such as Harrison; 267:83-109) described, produce phage particle.96 hole elisa plates are spent the night at 4 ℃ of bags with the PBS of 100 μ l, 10 μ g/ml HSA or β-gal.According to standard ELISA scheme (Hoogenboom etc., 1991), adopt detection in conjunction with phage and anti-M13-HRP conjugate.The clone is selected, obtain ELISA signal in the 50 μ l supernatant greater than 1.0.
Then, adopt QIAprep Spin Miniprep test kit (Qiagen), use the V that selects at HAS H/ dummy V κ library and the V κ/dummy V that selects at β-gal HThe library prepares the DNA prepared product.In order to obtain most of multiformity, take turns preparation DNA prepared product from 3 each of taking turns selection, lump together again and be used for every kind of antigen.Again the DNA prepared product is spent the night 37 ℃ of digestion with SalI/NotI.Then fragment is carried out gel-purified, will be from the V κ/dummy V that selects at β-gal HThe V κ chain in library is connected the V that selects at HAS HOn the position of the dummy V κ chain in/dummy V κ library, obtain 3.3 * 10 9Individual clone's library.
Then, HSA/ β-gal selection is carried out in this library, perhaps β-gal/HSA selection is carried out in this library at β-gal (first round) and HSA (second takes turns) at HSA (first round) and β-gal (second takes turns).Select as mentioned above.Under second every kind of situation after taking turns,, measure combining of 48 clones and HSA and β-gal by monoclonal phage E LISA (as mentioned above) and by the segmental ELISA of solubility scFv.According to (1996) and standard ELISA schemes such as Harrison (Hoogenboom etc. (1991) Nucleic Acids Res., 19:4133) described, produce the soluble antibody fragment, just adopt 2% tween (Tween)/PBS as the sealing buffer, and detect in conjunction with scFv with albumen L-HRP.3 clones (E4, E5 and E8) that select from HSA/ β-gal and can be in conjunction with these two kinds of antigens from 2 clones (K8 and K10) of β-gal/HSA selection.According to (1999) J.Mol.Biol. such as Ignatovich, on November 26th, 1999; 294 (2): 457-65 is described, uses primer LMB3 and pHENseq, and the scFv from these clones is carried out pcr amplification and order-checking.Sequence analysis shows that all clones are identical.Therefore, only select the clone (K8) of a coding bi-specific antibody to carry out further work (Fig. 3).
The sign of embodiment 2.K8 antibodies characteristic
At first, the binding characteristic of K8 antibody characterizes by monoclonal phage E LISA.96 orifice plates are spent the night at 4 ℃ of bags with the PBS of 100 μ l, 10 μ g/ml HSA and β-gal and alkali phosphatase (APS), bovine serum albumin (BSA), peanut agglatinin, lysozyme and cytochrome c (to check cross reaction).Described according to (1996) such as Harrison, will save with KM13 from K8 clone's phasmid, the supernatant (50 μ l) that contains phage is directly measured.According to standard ELISA scheme (Hoogenboom etc., 1991), detect in conjunction with phage with anti-M13-HRP conjugate.When at phage display, find bispecific K8 antibody capable in conjunction with HSA and β-gal, its absorption signal is greater than 1.0 (Fig. 4).Also observe strong combination the (Fig. 4) with BSA.Because HSA and BSA have 76% homology on amino acid levels, so not strange, the K8 antibody capable is discerned the protein of these structurally associateds.Do not detect and other proteinic cross reactivity (Fig. 4).
Secondly, in solubility scFv ELISA, detected the binding characteristic of K8 antibody.According to (1996) such as Harrison, solubility scFv fragment is induced generation by IPTG.In order to measure the expression of K8 scFv, according to Harlow and Lane (Antibodies:aLaboratory Manual, (1988) Cold Spring Harbor) described, with protein A-agarose Seph post, from the inductive supernatant of 50ml, be purified into the soluble antibody fragment.Described according to (1989) such as Sambrook, measure OD 280And calculating protein concentration.The K8 scFv that produces in the supernatant is 19mg/L.
The K8 antibody fragment of reuse concentration known carries out solubility scFv ELISA.96 orifice plates are with 100 μ l HSA, BSA and β-gal 1 μ g/ml concentration bag quilt with 10 μ g/ml and 100 μ l protein As.Adopt the serial dilutions of 50 μ l K8 scFv, the binding antibody fragment detects with albumen L-HRP.ELISA result confirms the bispecific (Fig. 5) of K8 antibody.
In order to confirm with combining of β-gal is to be determined by V κ district, is V by K8 scFv antibody with combining of HSA/BSA HThe district determines, through SalI/NotI digestion, V κ district from K8 scFvDNA cutting-out, and is connected to through what SalI/NotI digested and contains dummy V HIn the pIT2 carrier of chain (Fig. 1 and 2).The DCRP K8 V κ of institute/dummy V HAnalyze by solubility scFv ELISA in conjunction with characterizing.According to (1996) such as Harrison, solubility scFv fragment is induced generation by IPTG, and the supernatant (50 μ) that contains scFv is directly measured.Described according to embodiment 1, carry out solubility scFv ELISA, measure with albumen L-HRP in conjunction with scFv.ELISA result shows, this clone still can be in conjunction with β-gal, and is eliminated (Fig. 6) with combining of BSA.
Embodiment 3. is at the V of antigen A and B HThe selection of single domain antibody, and at the selection of the V κ single domain antibody of antigens c and D
This embodiment has described the V at antigen A and B HSingle domain antibody, and at the preparation method of the V κ single domain antibody of antigens c and D promptly when not existing in complementary variable region, selects storehouse, virgin (virgin) monoclonal antibody body variable region to these antigenic combinations.
(, PCT/GB02/003014), carry out selection and sign according to preceding method in conjunction with the clone referring to embodiment 5.Select 4 clones to carry out further work:
The anti-A V of VH1- H
The anti-B V of VH2- H
The anti-C V of VK1-κ
The anti-D V of VK2-κ
Can adopt the described method of above embodiment 1-3, according to described similar fashion, generation comprises V HDistrict's combination (is V H-V HPart) and V LDistrict's combination (V L-V LPart) dimer molecule.
Embodiment 4. bispecific ScFv production of antibodies and sign (at the VH1/VH2 of antigen A and B and at the VK1/VK2 of antigens c and D)
This embodiment proves, can be by will be at the V κ and the V that select of antigen separately in the ScFv carrier HSingle domain combines, and produces bispecific ScFv antibody (at the VH1/VH2 of antigen A and B and at the VK1/VK2 of antigens c and D).
In order to produce bi-specific antibody VH1/VH2,, downcut VH1 single domain (Fig. 7) and be connected on the variable region carrier 2 of NcoI/XhoI digestion (Fig. 7) generation VH1/ variable region carrier 2 from variable region carrier 1 through NcoI/XhoI digestion.By PCR, use primer, from the single district of variable region carrier 1 amplification VH2, the SalI restriction site is introduced 5 ' end and the NotI restriction site is introduced 3 ' end.Again with the PCR product with SalI/NotI digestion and be connected on the VH1/ variable region carrier 2 of SalI/NotI digestion, produce VH1/VH2/ variable region carrier 2.
VK1/VK2/ variable region carrier 2 produces in a similar manner.(, PCT/GB02/003014), measure the VH1/VH2 ScFV that produced and the bispecific of VK1/VK2 ScFv according to the method for aforementioned solubility ScFvELISA referring to embodiment 6.According to preceding method (referring to embodiment 8, PCT/GB02/003014), being at war with property ELISA.
Possible outcome:
-VH1/VH2 ScFv is antigen A and B simultaneously
-VK1/VK2 ScFv is antigens c and D simultaneously
-VH1/VH2 ScFV is in conjunction with being emulative (when conjugated antigen A, VH1/VH2ScFv can not conjugated antigen B)
-VK1/VK2 ScFv is in conjunction with being emulative (when conjugated antigen C, VK1/VK2ScFv can not conjugated antigen D).
Embodiment 5. bispecific VH1/VH2 Fab and the structure of VK1/VK2 Fab and the analysis of binding characteristic thereof
In order to produce VH1/VH2 Fab, the VH1 single domain is connected on the CH carrier of NcoI/XhoI digestion (Fig. 8), produce VH1/CH, again the VH2 single domain is connected on the CK carrier of SalI/NotI digestion (Fig. 9), produce VH2/CK.(, PCT/GB02/003014), use plasmid DNA to be used for cotransformation competence Bacillus coli cells according to preceding method from VH1/CH and VH2/CK referring to embodiment 8.
(referring to embodiment 8, PCT/GB02/003014), the clone's reuse IPTG that contains VH1/CH and VH2/CK plasmid induces, and produces solubility VH1/VH2 Fab according to preceding method.
VK1/VK2 Fab produces by similar approach.
(, PCT/GB02/003014), measure the binding characteristic of the Fab that is produced according to aforesaid competitive ELISA referring to embodiment 8.
Possible outcome:
-VH1/VH2 Fab is antigen A and B simultaneously
-VK1/VK2 Fab is antigens c and D simultaneously
-VH1/VH2 Fab is in conjunction with being emulative (when conjugated antigen A, VH1/VH2 Fab can not conjugated antigen B)
-VK1/VK2 Fab is in conjunction with being emulative (when conjugated antigen C, VK1/VK2 Fab can not conjugated antigen D).
Embodiment 6. makes dAb dimer chelating
General introduction
Use flexible peptide linker, produce the VH and the VK homodimer of dAb-joint-dAb form.Produce the carrier of dAb joint-dAb form, it contains the glycine-serine joint 3U:(Gly of different length 4Ser) 3(SEQ ID NO:199), 5U:(Gly 4Ser) 5(SEQ ID NO:629), 7U:(Gly 4Ser) 7(SEQ TD NO:630).Library with corresponding the 2nd dAb after the directed dAb upstream of joint and the joint produces the dimer library, and described joint is: TAR1-5 (VK), TAR1-27 (VK), TAR2-5 (VH) or TAR2-6 (VK).Adopt this method, select new dimer dAb.By ELISA and BIAcore research, in cell neutralization and receptors bind mensuration, detect dimerization to the bonded influence of antigen.The dimerization of TAR1-5 and TAR1-27 causes binding affinity and neutralization levels to improve.
1.0 method
1.1 the generation in library
1.1.1 carrier
Digestion pEDA3U, pEDA5U and pEDA7U carrier are introduced the different joint length compatible with dAb-joint-dAb form.For pEDA3U, make 73 base pair oligomerization joint annealing of justice and antisense, adopted slow cycle of annealing (95 ℃-5 minutes, 80 ℃-10 minutes, 70 ℃-15 minutes, 56 ℃-15 minutes, 42 ℃ up to using), in the buffer that contains 0.1M NaCl, 10mMTris-HCl (pH7.4), clone with XhoI and NotI restriction site.Joint comprises 3 (Gly 4Ser) unit and fill area, it is (scheme 1) between SalI and NotI cloning site.In order to reduce the probability that monomer dAb is selected by phage display, the fill area of design comprises 3 termination codoies, SacI restriction site and frameshift mutation, so that this district is placed on outside the frame, when the 2nd dAb does not exist.For pEDA5U and pEDA7U, because required joint length digests overlapping oligomerization joint, be used for each carrier, annealing is also extended with Klenow.The purification fragment digests with suitable enzymes then, reuse XhoI and NotI restriction site clone.
Figure A20058004204201541
Scheme 1
1.1.2 the preparation in library
With NcoI and XhoI restriction site, will arrive the joint upstream corresponding to the N-end V gene clone of directed dAb.The VH gene has ready-made compatibility site, yet clone VK gene need be introduced suitable restriction site.By using the PCR primer (VK-DLIBF:5 ' cggccatggcgtcaacggacat (SEQ ID NO:377) that modifies; VK XholR:5 ' atgtgcgctcgagcgtttgattt 3 ' (SEQ ID NO:378)), in 30 circulation pcr amplifications, use 2: 1 mixture of SuperTaq (HTBiotechnology Ltd) and pfu turbo (Stratagene), reach this point.This keeps the NcoI site at 5 ' end, destroys adjacent S alI site simultaneously and introduces the XhoI site at 3 ' end.5 directed dAb are cloned in each of 3 dimer carriers: TAR1-5 (VK), TAR1-27 (VK), TAR2-5 (VH), TAR2-6 (VK) and TAR2-7 (VK).All constructs are all confirmed through sequence analysis.
Each carrier ((pEDA3U, pEDA 5U and pEDA 7U): TAR1-5 (VK), TAR1-27 (VK), TAR2-5 (VH) or TAR2-6 (VK)) all has the directed dAb of clone of joint upstream, with the library clone of corresponding the 2nd dAb after joint.In order to reach this point, through PCR, complementary (complimentary) dAb of phage amplification library of reclaiming, described phage from selecting through the first round can be when TAR1-5 or TAR1-27 are directed dAb, at the first round in the VK library of human TNF alpha select and the phage of recovery (after the first round about 1 * 10 6Multiformity), or when TAR2-5 or TAR2-6 are directed dAb respectively, at the first round in the VH of people p55 TNF receptor or VK library, select and the phage that reclaims (all is about 1 * 10 after the first round 5Multiformity).For the VK library, use primer, in 30 circulation pcr amplifications, use 2: 1 mixture of SuperTaq and pfu turbo, carry out pcr amplification.Use primer, pcr amplification is carried out in the VH library, so that introduce the SalI restriction site at 5 of gene ' end.DAb library PCR digests with suitable restriction endonuclease, is connected to respective carrier joint downstream, uses the SalI/NotI restriction site, and enters in the competence TG1 cell of prepared fresh through electroporation.
Each library gained is tired as follows:
TAR1-5：pEDA3U＝4×10 8，pEDA5U＝8×10 7，pEDA7U＝1×10 8
TAR1-27：pEDA3U＝6.2×10 8，pEDA5U＝1×10 8，pEDA7U＝1×10 9
TAR2h-5：pEDA3U＝4×10 7，pEDA5U＝2×10 8，pEDA7U＝8×10 7
TAR2h-6：pEDA3U＝7.4×10 8，pEDA5U＝1.2×10 8，pEDA7U＝2.2×10 8
1.2 select
1.2.1 TNFα
With the passive human TNF alpha that is coated on the immune pipe, select.In brief, required antigen coated the spending the night of immune effective 1-4ml.Immunity pipe reuse PBS washing 3 times was sealed 1-2 hour with 2% milk powder/PBS, and then is washed 3 times with PBS.Phage solution was at room temperature hatched 2 hours with 2% milk powder/PBS dilution.These pipe reuse PBS washing, phage 1mg/ml trypsin-PBS eluting.For TAR1-5 dimer library, 3 kinds of selection strategyes have been studied.The first round is chosen in the immune pipe carries out, with the human TNF alpha bag quilt of 1 μ g/ml or 20 μ g/ml, and reuse PBS 0.1% tween washing 20 times.The TG1 cell is with through the phage-infect of eluting, measures and tire that (on December 5th, 1991 for Marks etc. for example, J.Mol.Biol.; 222 (3): 581-97, Richmann etc., Biochemistry, on August 31st, 1993; 32 (34): 8848-55).
Reclaim tire for:
pEDA3U＝2.8×10 7(1μg/ml?TNF)，1.5×10 8(20μg/ml?TNF)，
pEDA5U＝1.8×10 7(1μg/ml?TNF)，1.6×10 8(20μg/ml?TNF)，
pEDA7U＝8×10 6(1μg/ml?TNF)，7×10 7(20μg/ml?TNF)。
Carry out second with following three kinds of diverse ways and take turns selection.
1. in immune pipe, 20 washings and night incubation then are 10 washings again.
2. in immune pipe, 20 washings and at room temperature, hatched 1 hour with (1 μ g/mlTNF α) in lavation buffer solution then are to wash for 10 times again.
3. on the streptavidin pearl, use 33pmole biotinylation human TNF alpha to select (Henderikx etc., 2002, Selection of antibodies against biotinylatedantigens.Antibody Phage Display:Methods and protocols, O ' Brien and Atkin (chief editor), Humana Press).Picking is inoculated in 96 orifice plates from the second single clone who takes turns selection, prepares thick supernatant prepared product with the form of 2ml 96 orifice plates.
Table 1.
First round human TNF alpha immunity pipe bag is by concentration Second takes turns system of selection 1 Second takes turns system of selection 2 Second takes turns system of selection 3
?pEDA3U ?1μg/ml ?1×10 9 1.8×10 9 2.4×10 10
?pEDA3U ?20μg/ml ?6×10 9 1.8×10 10 8.5×10 10
?pEDA5U ?1μg/ml ?9×10 8 1.4×10 9 2.8×10 10
?pEDA5U ?20μg/ml ?9.5×10 9 8.5×10 9 2.8×10 10
?pEDA7U ?1μg/ml ?7.8×10 8 1.6×10 8 4×10 10
?pEDA7U ?20μg/ml ?1×10 10 8×10 9 1.5×10 10
For TAR1-27,, select according to preceding method and through following improvement.With 1 μ g/ml or 20 μ g/ml human TNF alpha bags by, and immune pipe with PBS 0.1% tween washing 20 times in, carry out the first round and select.In the immune pipe of 20 washings, night incubation and 20 washings once more, carry out second and take turns selection.Picking is inoculated into 96 orifice plates from the second single clone who takes turns selection, prepares thick supernatant prepared product with the form of 2ml 96 orifice plates.
TAR1-27 tires as follows:
Table 2.
Human TNF alpha immunity pipe bag is by concentration The first round Second takes turns
pEDA3U ?1μg/ml ?4×10 9 6×10 9
pEDA3U ?20μg/ml ?5×10 9 4.4×10 10
pEDA5U ?1μg/ml ?1.5×10 9 1.9×10 10
pEDA5U ?20μg/ml ?3.4×10 9 3.5×10 10
pEDA7U ?1μg/ml ?2.6×10 9 5×10 9
pEDA7U ?20μg/ml ?7×10 9 1.4×10 10
1.2.2 TNF receptor 1 (p55 receptor; TAR2)
Only, select according to preceding method for the TAR2h-5 library.With 1 μ g/ml people p55 TNF receptor or 10 μ g/ml people p55 TNF receptors, and immune pipe with PBS 0.1% tween washing 20 times, night incubation and 20 washings once more in, carry out third round and select.Picking is taken turns the single clone who selects with third round from second, is inoculated into 96 orifice plates, prepares thick supernatant prepared product with the form of 2ml 96 orifice plates.
TAR2h-5 tires as follows:
Table 3.
First round people p55 TNF receptor immunity pipe bag is by concentration The first round Second takes turns Third round
pEDA3U ?1μg/ml ?2.4×10 6 1.2×10 7 1.9×10 9
pEDA3U ?10μg/ml ?3.1×10 7 7×10 7 1×10 9
pEDA5U ?1μg/ml ?2.5×10 6 1.1×10 7 5.7×10 8
pEDA5U ?10μg/ml ?3.7×10 7 2.3×10 8 2.9×10 9
pEDA7U ?1μg/ml ?1.3×10 6 1.3×10 7 1.4×10 9
pEDA7U ?10μg/ml ?1.6×10 7 1.9×10 7 3×10 10
1.3 screening
From each 3U, 5U and 7U library, take turns from second with different choice method picking in the time of suitably or the single clone of third round selection.At 37 ℃, the 2xTY overnight incubation that contains 100 μ g/ml ampicillin and 1% glucose will be cloned in.1/100 diluent of this culture is inoculated into 2ml and contains in the 2ml96 well plate format of 2xTY of 100 μ g/ml ampicillin and 0.1% glucose, 37 ℃ of shaken cultivation up to OD 600Reach about 0.9.Then, culture is induced at 30 ℃ with 1mM IPTG and is spent the night.In the board-like centrifuge of sorval centrifugal 15 minutes, make the supernatant clarification with 4000rpm.The supernatant prepared product is used for Preliminary screening.
1.3.1 ELISA
By protein A/L ELISA or antigen ELISA, the dimer recombinant protein is compared with the monomeric activity that combines.In brief, 96 orifice plates are spent the night at 4 ℃ of bags with antigen or protein A/L.This plate sealed 2 hours with 2% tween-PBS with 0.05% tween-PBS washing.Sample is added on this plate, at room temperature hatched 1 hour.Wash this plate, at room temperature hatched 1 hour with second reagent.Wash this plate, develop the color with tmb substrate.Protein A/L-HRP or India-HRP are as second reagent.For antigen ELISA, used antigen concentration is the PBS of 1 μ g/ml, is used for human TNF alpha and people THF receptor 1.Because in most cases there is directed dAb, dimer provides positive ELISA signal, so dissociation rate detects through BIAcore.
1.3.2 BIAcore
TAR1-5 and TAR2h-5 clone are carried out the BIAcore analysis.For screening, human TNF alpha is coupled on the CM5 chip with high density (about 10000RU).50 μ l human TNF alphas (50 μ g/ml) are coupled on the chip with 5 μ l minutes acetate buffer (pH5.5).Chip after the analysis can not be regenerated with standard method, because the human TNF alpha instability, therefore, after each analytic sample, chip washed 10 minutes with buffer.For TAR1-5, screen from second clone's supernatant of taking turns selection with BIAcore.
Adopt following system of selection, from each 3U of gained, 5U and 7U library, filter out 48 clones:
R1:1 μ g/ml human TNF alpha immunity pipe, R2:1 μ g/ml human TNF alpha immunity pipe, the washing of spending the night.
R1:20 μ g/ml human TNF alpha immunity pipe, R2:20/xg/ml human TNF alpha immunity pipe, the washing of spending the night.
R1:1 μ g/ml human TNF alpha immunity pipe, R2:33pmoles biotinylation human TNF alpha is on pearl.
R1:20 μ g/ml people TNFa immunity pipe, R2:33pmoles biotinylation human TNF alpha is on pearl.
For screening, people p55 TNF receptor is coupled on the CM5 chip with high density (about 4000RU).100 μ l people p55 TNF receptors (10 μ g/ml) are coupled on the chip with 5 μ l/ minutes acetate buffer (pH5.5).Check criteria regeneration condition (glycine pH2 or pH3), but in each case, antigen is removed from chip surface with TNF α, and after therefore each analytic sample, chip washed 10 minutes with buffer.
For TAR2-5, screened clone's supernatant of taking turns selection from second.
Adopt following system of selection, from each 3U, 5U and 7U library, filter out 48 clones:
R1:1 μ g/ml people p55 TNF receptor immunity pipe, R2:1 μ g/ml people p55 TNF receptor immunity pipe, the washing of spending the night.
R1:10 μ g/ml people p55 TNF receptor immunity pipe, R2:10 μ g/ml people p55 TNF receptor immunity pipe, the washing of spending the night.
1.3.3 receptor and raji cell assay Raji
In receptor determination, the mensuration of dimer neutralising capacity is following carries out:
Receptors bind
Measure anti-TNF dAb and suppress TNF and the bonded ability of reorganization TNF receptor 1 (p55).In brief, the anti-people Fc of Maxisorp plate and 30mg/ml mouse monoclonal antibody (Zymed, SanFrancisco, USA) overnight incubation together.Wash with the phosphate buffered saline(PBS) (PBS) that contains 0.05% tween 20 in each hole, and reuse 1%BSA/PBS sealing is then with 100ng/ml TNF receptor 1Fc fusion rotein (R﹠amp; D system, Minneapolis USA) is hatched together.Anti-TNFdAb and TNF (it joins in the washing hole, and final concentration is 10ng/ml) mix.The bonded mensuration of TNF is as follows: with 0.2mg/ml biotinylation anti-TNF antibodies (HyCult biotechnology, Uben, Netherlands), follow streptavidin (Amersham Biosciences with the horseradish peroxidase-labeled of dilution in 1: 500, UK), (KPL, Gaithersburg USA) are hatched together with tmb substrate again.Add the HCl cessation reaction, read absorbance at 450nm.Anti-TNF dAb activity causes the bonded reduction of TNF, therefore, and only compares with TNF contrast, causes absorbance to descend.
The L929 cytotoxic assay
On mice L929 fibroblast, also measured among the anti-TNF dAb and the ability of TNF cytotoxic activity (Evans, T. (2000) Molecular Biotechnology 15,243-248).In brief, L929 cell on the microtitration plate and anti-TNF dAb, 100pg/ml TNF and 1mg/ml actinomycin D (Sigma, Poole, UK) overnight incubation together will be seeded in.Cell survival rate is measured by reading absorbance at 490nm, then with [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxyl (carbboxy) methoxyphenyl)-2-(4-sulfo group phenyl)-(the 2H-tetrazolium together USA) is hatched by Promega, Madison.Anti-TNF dAb activity causes the Cytotoxic reduction of TNF, therefore, and only compares with TNF contrast, causes absorbance to increase.
In Preliminary screening, as mentioned above,, BIAcore also can be used for receptor determination for analyzing the supernatant for preparing.In receptor and raji cell assay Raji, use protein purification, also can carry out selected dimeric further analysis.
HeLa IL-8 measures
In the HeLa cell, measure among anti-TNFR1 or the anti-TNF alpha dAb and the excretory inducing action of IL-8 (is adopted Akeson with TNF, L. etc. (1996) Journal of BiologicalChemistry 271, improving one's methods of the described method of 30517-30523, described document have been introduced in HUVEC IL-1 to the inducing action of IL-8; At this, we have observed the inducing action of human TNF alpha, and we adopt HeLa cell replacement HUVEC cell line).In brief, be seeded in HeLa cell on the microtitration plate with dAb and 300pg/ml TNF overnight incubation.After hatching, the sucking-off supernatant is by sandwich ELISA (R﹠amp; D system) measures cell and IL-8 concentration.With only compare with TNF contrast, anti-TNFR1 dAb activity causes that the IL-8 secretion reduces in the supernatant.
L929 measures and is used for following experiment; Yet, preferably adopt HeLa IL-8 to measure to detect anti-TNF receptor 1 (p55) part; In L929 measured, the existence of mice p55 had some restriction to its use.
1.4 sequence analysis
The dimer that has target property for proof in BIAcore and receptor determination screening checks order.Sequence sees sequence table for details.
1.5 structure forms (formatting)
1.5.1 TAR1-5-19 dimer
In cell and receptor determination, make with extra care and analyze in having well again and the TAR1-5 dimer of characteristic.The directed dab of TAR1-5 is replaced by affinity maturation clone TAR1-5-19.In order to reach this point, this TAR1-5 is cloned (cloneout) from single dimer centering, and use TAR1-5-19 to replace through pcr amplification.In addition, in 3U, 5U and TU carrier, make up the TAR1-5-19 homodimer.The N end copy of gene is also cloned as mentioned above through pcr amplification, clones the C-end group because of fragment with existing SalI and NotI restriction site.
1.5.2 mutation
By direct mutagenesis, the succinum termination codon (one of them is at the dAb of TAR1-5 dimer to the C end) that exists among the dAb2 is mutated into glutamine.
1.5.3 Fab
The dimer that will contain TAR1-5 or TAR1-5-19 is refined in the Fab expression vector again.With SfiI and NotI restriction site, dAb is cloned into contains in CK gene or the CH expression carrier, confirmed by sequence analysis.The CK carrier derives from the amicillin resistance carrier based on pUC, and the CH carrier derives from pACYC chlorampenicol resistant carrier.Express for Fab,, in the 2xTY that contains 0.1% glucose, 100 μ g/ml ampicillin and 10 μ g/ml chloromycetin, cultivate dAb-CH and dAb-CK construct cotransformation HB2151 cell.
1.5.3 hinge dimerization
Checked by forming the dAb dimerization of cystine linkage.With the short sequence (a kind of human IgG Cl hinge of modified forms) of aminoacid EPKSGDKTHTCPPCP (SEQ ID NO:379), through transforming the C petiolarea that is connected dAb.As mentioned above, the oligomerization joint of this sequence of composite coding and annealing.Joint is cloned in the pEDA carrier that contains TAR1-5-19, with XhoI and NotI restriction site.Dimerization original position in pericentral siphon takes place.
1.6 express and purification
1.6.1 express
As mentioned above, with 2ml, 96 well plate format prepare supernatant, are used for Preliminary screening.After the Preliminary screening process, selected dimer is further analyzed.The dimer construct is expressed in TOP10F ' or HB2151 cell conditioned medium liquid.In brief, will from single bacterium colony of fresh streak plate 37 ℃, containing overnight incubation among the 2xTY of 100 μ g/ml ampicillin and 1% glucose.This culture of 1/100 dilution is inoculated among the 2xTY that contains 100 μ g/ml ampicillin and 0.1% glucose, 37 ℃ of shaken cultivation, up to OD 600Reach about 0.9.Culture reuse 1mM IPTG induces at 30 ℃ and spends the night.The centrifugal cell of removing, supernatant is with protein A or L agarose purification.
In the HB2152 cell, Fab and cysteine hinge dimer are expressed as periplasm protein.The overnight culture of 1/100 dilution is inoculated into and contains among 0.1% glucose and the suitable antibiotic 2xTY, 30 ℃ of shaken cultivation, up to OD 600Reach about 0.9.Culture reuse 1mMIPTG induced 3-4 hour at 25 ℃.Centrifugal cell harvesting, precipitation is resuspended in pericentral siphon and prepares in the buffer (30mM Tris-HCl (pH8.0), 1mM EDTA, 20% sucrose).After centrifugal, keep supernatant, precipitation is resuspended in 5mM MgSO 4By centrifugal results supernatant, merge, then purification once more.
1.6.2 protein A/L purification
From albumen L agarose (Affitech, Norway) or the protein A agarose (Sigma optimizes the purification dimer protein in UK), and it is detected.Protein is eluting in batches, perhaps passes through the post eluting with peristaltic pump.Check 3 kinds of buffer: 0.1M phosphoric acid-citrate buffer solution (pH2.6), 0.2M glycine (pH2.5) and 0.1M glycine (pH2.5).Determine the optimal conditions under the peristaltic pump condition, adopt 0.1M glycine pH2.5, surpass 10 times of column volumes.From the protein A purification, the peristaltic pump condition adopts 0.1M glycine pH2.5.
1.6.3 FPLC purification
Analyze by FPLC, at the enterprising single step purification of advancing of AKTA Explorer 100 systems (AmershamBiosciences Ltd).TAR1-5 is by cation-exchange chromatography (1ml Resource S-Amersham BiosciencesLtd) with TAR1-5-19 dimeric separation, with 0-1M NaCl gradient/50mM acetate buffer (pH4) eluting.The dimeric purification of hinge is by ion exchange (1ml Resource Q Amersham BiosciencesLtd), with 0-1M NaCl gradient/25mM Tris HCl (pH8.0) eluting.The purification of Fab is by size exclusion chromatography, with superose 12 (Amersham Biosciences Ltd) post, flow velocity 0.5ml/ minute PBS (containing 0.05% tween).Behind the purification, adopt vivaspin 5K by concentrator (Vivascience Ltd) concentrating sample.
2.0 result
2.1 TAR1-5 dimer
Take turns 6 * 96 clones of picking the selection from second, comprise all libraries and alternative condition.Preparation supernatant prepared product is analyzed with antigen and albumen L ELISA, BIAcore and receptor determination.In ELISA, from each system of selection, identify positive combination clone, they are distributed in 3U, 5U and the 7U library.Yet,,, therefore carry out BIAcore and analyze so can not distinguish high and low-affinity conjugate by this method because directed dAb always exists.
Carrying out BIAcore with the 2ml supernatant analyzes.BIAcore the analysis showed that TAR1-5 compares with monomer, and dimer K dissociation rate is improved.Monomer K dissociation rate scope is 10 -1M, dimer K dissociation rate scope is 10 -3-10 -4M.Option table reveals 16 very slow clones of the speed of dissociating, and they are from 3U, 5U and TU library, and it is checked order.In addition, in receptor determination, analytically in the clear liquid and the ability of human TNF alpha.
6 the guide clones (to call d1-d6 in the following text) that have neutralising capacity in measuring at these are checked order.The result shows among 6 clones of gained that only 3 have different the 2nd dAb (dAb1, dAb2 and dAb3), yet, find that wherein the 2nd dAb connects the joint of different length more than once.
TAR1-5d1:3U joint 2 NdDAb=dAb1-1 μ g/ml Ag immunity pipe, the washing of spending the night
TAR1-5d2:3U joint 2 NdDAb=dAb2-1 μ g/ml Ag immunity pipe, the washing of spending the night
TAR1-5d3:5U joint 2 NdDAb=dAb2-1 μ g/ml Ag immunity pipe, the washing of spending the night
TAR1-5d4:5U joint 2 NdDAb=dAb3-20 μ g/ml Ag immunity pipe, the washing of spending the night
TAR1-5d5:5U joint 2 NdDAb=dAb1-20 μ g/ml Ag immunity pipe, the washing of spending the night
TAR1-5d6:7U joint 2 NdDAb=dAb1-R1:1 μ g/ml Ag immunity pipe, the washing of spending the night
R2: pearl
Further detect 6 guide clones.Protein produces from pericentral siphon and supernatant, with albumen L agarose purification, and detects in cell and receptor determination.Neutralization levels difference (table 1).Detected the optimal conditions of protein preparation.The protein that obtains from HB2151 cell conditioned medium liquid obtains the highest yield (about 10mg/L culture).Supernatant at room temperature hatched 2 hours with albumen L agarose or 4 ℃ of overnight incubation.Pearl washs with PBS/NaCl, with the peristaltic pump FPLC post of packing into.Pearl is with the PBS/NaCl washing of 10 times of volumes, with 0.1M glycine (pH2.5) eluting.Generally speaking, dimer protein elutes after monomer.
TAR1-5d1-6 dimer FPLC purification.Obtain 3 kinds behind the FPLC purification, identify through SDSPAGE.A kind of corresponding to monomer, and other the two kinds dimers corresponding to different sizes.In two kinds bigger a kind of may be because there is C end labelling.In receptor determination, detect these protein.Data see Table 1, have represented to derive from two kinds of dimeric optimization results (Figure 11)
To clone as monomer, and detect from 3 kind two dAb of dimer (being dAb1, dAb2 and dAb3) by ELISA and in cell and receptor determination.Cross reaction and does not take place with plastics or BSA in conjunction with TNF (through antigen ELISA) in all 3 kinds of dAb specificity.As monomer, in cell or receptor determination, dAb does not neutralize.
2.1.2 TAR1-5-19 dimer
In 6 guide clones, replace TAR1-5 with TAR1-5-19.In cell and receptor determination, all TAR1-5-19 dimers are analyzed, use total protein (only purifying protein L), (table 2) except as otherwise noted.In raji cell assay Raji, TAR1-5-19d4 and TAR1-5-19d3 have optimum N D 50(～5nM), this is consistent with the receptor determination result, and compares TAR1-5-19 monomer (ND 50～30nM) be improved.Although purification TAR1-5 dimer obtains Different Results in receptor and raji cell assay Raji, the TAR1-5-19 dimer is more consistent.During protein purification, when using different elution buffer, have change.With 0.1M phosphoric acid-citrate buffer solution (pH2.6) or 0.2M glycine (pH2.5) eluting, although in most of the cases remove all proteins from albumen L agarose, this makes its function still less.
TAR1-5-19d4 expresses in fermentation tank, and purification in cation exchange FPLC obtains very pure dimer then.With TAR1-5d4, obtain 3 kinds corresponding to monomer and two kinds of dimers by the FPLC purification.This dimer is carried out amino acid sequencing.Then, in receptor determination, detect TAR1-5-19 monomer and TAR1-5-19d4, monomer gained IC 50Be 30nM, dimer is 8nM.The receptor determination of comparing with TAR1-5-19 monomer, TAR1-5-19d4 and TAR1-5d4 the results are shown in Figure 10.
Preparation TAR1-5-19 homodimer is expressed and purification on albumen L in 3U, 5U and 7U carrier.Protein is tested in cell and receptor determination, measures gained IC 50(for receptor determination) and ND 50(for raji cell assay Raji) (table 3, Figure 12).
2.2 Fab
TAR1-5 and TAR1-5-19 dimer are cloned in the Fab form, express and purification on albumen L agarose.Fab estimates (table 4) in receptor determination.The result shows that the dimeric neutralization levels of TAR1-5-19 and TAR1-5 all is similar to the former Gly in its source 4Ser joint dimer.Express TAR1-5-19 Fab (wherein TAR1-5-19 is illustrated on CH and the CK),, in receptor determination, estimate through albumen L purification.Gained IC 50Be about 1nM.
2.3 TAR1-27 dimer
Take turns 3 * 96 clones of picking the selection from second, comprise all libraries and alternative condition.Preparation 2ml supernatant prepared product is used for analyzing at ELISA and bioassay.Antigen ELISA obtains 71 positive colonies.The receptor determination of thick supernatant obtains 42 clones (TNF is in conjunction with 0-60%) with rejection characteristic.In most of the cases, rejection characteristic and strong ELISA signal correction.42 clones are checked order, wherein have 39 to have the 2nd unique dAb sequence.12 dimers with optimal inhibition characteristic are further analyzed.
12 neutralizations are cloned in the 200ml supernatant prepared product to be expressed, again purification on albumen L.They are by albumen L and antigen ELISA, BIAcore and estimate in receptor determination.All obtain strong positive ELISA signal in all cases.BIAcore the analysis showed that all clones have the fast association and the speed of dissociating.TAR1-27 compares with monomer, has improved the speed of dissociating, yet (the K scope of dissociating is about 10 with the dimeric speed of the TAR1-5 of previous detection -3M～10 -4M) compare, the dimeric speed of dissociating of TAR1-27 is faster, and (the K scope of dissociating is about 10 -1M～10 -2M).The dimeric stability of purification is a problem, therefore in order to improve stability, adds 5% glycerol, 0.5%Triton X100 or 0.5%NP40 (Sigma) in the purification of 2 TAR1-27 dimers (d2 and d16).Add NP40 or Triton X100 TMAbout 2 times of the yield of raising purified product.In receptor determination, estimate these two kinds of dimers.The IC of TAR1-27d2 under all purification conditions 50All be about 30nM.When purification did not use stabilizing agent, TAR1-27d16 demonstrates did not have neutralizing effect, but when purification carries out under steady-state conditions, obtained IC 50Be about 50nM.Further do not analyze.
2.4 TAR2-5 dimer
Take turns 3 * 96 clones of picking the selection from second, comprise all libraries and alternative condition.Preparation 2ml supernatant prepared product is used for analyzing.Each plate is carried out protein A and antigen ELISA.Identify 30 target clones by BIAcore, (K dissociates scope between 10 to have good dissociation rate -2-10 -3M).The clone is checked order, identified the dimer of 13 uniquenesses by sequence analysis.
Table 4:TAR1-5 dimer
Dimer Cell type Purification Protein portion Elution requirement Receptor/raji cell assay Raji
?TAR1-5d1 ?HB2151 Albumen L+ FPLC Little dimer 0.1M glycine pH2.5 ?RA～30nM
?TAR1-5d2 ?HB2151 Albumen L+ FPLC Little dimer 0.1M glycine pH2.5 ?RA～50nM
?TAR1-5d3 ?HB2151 Albumen L+ FPLC Big dimer 0.1M glycine pH2.5 ?RA～300nM
?TAR1-5d4 ?HB2151 Albumen L+ FPLC Little dimer 0.1M glycine pH2.5 ?RA～3nM
?TAR1-5d5 ?HB2151 Albumen L+ FPLC Big dimer 0.1M glycine pH2.5 ?RA～200nM
?TAR1-5d6 ?HB2151 Albumen L+ FPLC Big dimer 0.1M glycine pH2.5 ?RA～100nM
*Note: dimer 2 has identical the 2nd dAb (being called dAb2) with dimer 3, but has different joint length (d2=(Gly 4Ser) 3, d3=(Gly 4Ser) 3).DAb1 is the gametophyte dAb of dimer 1,5 and 6.DAb3 is the gametophyte dAb of dimer 4.Gametophyte dAb can not neutralize separately.Except as otherwise noted, otherwise the FPLC purification is to pass through cation exchange.In these are measured,, measure and optimize the dimer kind every kind of dimer through the FPLC gained.
Table 5:TAR1-5-19 dimer
Dimer Cell type Purification Protein portion Elution requirement Receptor/raji cell assay Raji
?TAR1-5-19d1 ?top10F’ Albumen L Total protein 0.1M glycine pH2.0 ?RA～15nM
TAR1-5-19d2 (does not have the password of termination ?top10F’ Albumen L Total protein 0.1M glycine pH2.0+0.05%NP ?RA～2nM
Son) 40
TAR1-5-19d3 (not having the codon of termination) ?top10F’ Albumen L Total protein 0.1M glycine pH2.5+0.05%NP 40 ?RA～8nM
?TAR1-5-19d4 ?top10F’ Albumen L+ FPLC The FPLC purification part 0.1M glycine pH2.0 ?RA～2-5 ?nM?CA～12 ?nM
?TAR1-5-19d5 ?top10F’ Albumen L Total protein 0.1M glycine pH2.0+NP40 ?RA～8nM ?CA～10nM
?TAR1-5-19d6 ?top10F’ Albumen L Total protein 0.1M glycine pH2.0 ?RA～10nM
Table 6:TAR1-5-19 homodimer
Dimer Cell type Purification Protein portion Elution requirement Receptor/raji cell assay Raji
TAR1-5-19 3U homodimer HB2151 Albumen L Total protein 0.1M glycine pH2.5 ?RA～20nM ?CA～30nM
TAR1-5-19 5U homodimer HB2151 Albumen L Total protein 0.1M glycine pH2.5 ?RA～2nM ?CA～3nM
TAR1-5-19 7U homodimer HB2151 Albumen L Total protein 0.1M glycine pH2.5 ?RA～10nM ?CA～15nM
TAR1-5-19 Cys hinge HB2151 Albumen L+ FPLC The FPLC purification part 0.1M glycine pH2.5 ?RA～2nM
TAR1-5-19CH/ TAR1-5-19?CK HB2151 Albumen L Total protein 0.1M glycine pH2.5 ?RA～1nM
Table 7:TAR1-5/TAR1-5-19 Fab
Dimer Cell type Purification Protein portion Elution requirement Receptor/raji cell assay Raji
TAR1-5CH/ dAb1?CK ?HB2151 Albumen L Total protein 0.1M glycine pH2.6 ?RA～90nM
TAR1-5CH/ dAb2?CK ?HB2151 Albumen L Total protein 0.1M glycine pH2.5 ?RA～30nM ?CA～60nM
dAb3?CH/ TAR1-5CK ?HB2151 Albumen L Total protein 0.1M glycine pH2.6 ?RA～100 ?nM
TAR1-5-19CH/ dAb1?CK ?HB2151 Albumen L Total protein 0.1M glycine pH2.0 ?RA～6nM
dAb1?CH/ TAR1-5-19?CK ?HB2151 Albumen L 0.1M glycine pH2.0 Myc/flag ?RA～6nM
TAR1-5-19CH/ DAb2?CK ?HB2151 Albumen L Total protein 0.1M glycine pH2.0 ?RA～8nM ?CA～12nM
TAR1-5-19CH/ dAb3?CK ?HB2151 Albumen L Total protein 0.1M glycine pH2.0 ?RA～3nM
Embodiment 7. is by the dAb dimerization of terminal cysteine key
General introduction
For the dAb dimerization, free cysteine is spliced to proteinic C-end.When expressing, this albumen forms dimer, described dimer can be by the purification process of two steps purification.
The dimeric PCR of TAR1-5-19CYS makes up
According to embodiment 8 the trimerical method of dAb is described.The trimer scheme obtains monomer, dimer and trimerical mixture.
Dimeric expression of TAR1-5-19CYS and purification
According to the method for embodiment 8 general introductions, from culture supernatants, catch and the purification dimer by albumen L agarose.
The TAR1-5-19CYS monomer separates with TAR1-5-19CYS is dimeric
Before cation exchange is separated,, the buffer exchange of monomer/dimer biased sample is become 50mM sodium acetate buffer (pH4.0) with PD-10 post (Amersham Pharmacia) according to the operation instructions of manufacturer.Again sample is used for using in advance the equilibrated 1mL Resource of 50mM sodium acetate (pH4.0) S cation exchange column (Amersham Pharmacia).50mM sodium acetate (pH4.0) with following salt gradient is isolated monomer and dimer:
150-200mM sodium chloride surpasses 15 times of column volumes
200-450mM sodium chloride surpasses 10 times of column volumes
450-1000mM sodium chloride surpasses 15 times of column volumes
Only contain dimeric part and identify that with SDS-PAGE the back merges, the 1MTris (pH8.0) that adds 1/5 volume rises to 8 with pH.
Vitro functional is in conjunction with mensuration: TNF receptor determination and raji cell assay Raji.
Adopt TNF receptor and raji cell assay Raji, measure the affinity of dimer human TNF alpha.IC in the receptor determination 50Be about 0.3-0.8nM; ND in the raji cell assay Raji 50Be about 3-8nM.The TAR1-5-19CYS dimeric forms that other is possible
PEG dimer and the synthetic maleimide dimer of trust
Nektar (Shearwater) provides a series of BMI PEG[mPEG2-(MAL) 2 or mPEG-(MAL) 2], they can allow monomer become dimer, wherein separate dAb with little joint, and the two all is connected with PEG (magnitude range is 5-40kDa).Know that the affinity of 5kDa mPEG-(MAL) 2 (i.e. [TAR1-5-19]-Cys-maleimide-PEG * 2, wherein maleimide is connected in the dimer together) in the TNF receptor determination is～1-3nM.In addition, also available TMEA (three [2-maleimide ethyl] amine) (PierceBiotechnology) or other bifunctional linker produce dimer.
By the chemical coupling method, adopt two pyridines (Sigma Aldrich) of 2,2 '-two sulfur and reduction monomer, also can produce disulfide dimer.Peptide linker or hinge are added to the C-end of dAb.Perhaps (Gly 4Ser) n(for example IgG hinge region or peptide sequence little joints such as (for example being selected from peptide sequence library at random) at random can be added between dAb and the terminal cysteine residue for (wherein n=1-10, for example 1,2,3,4,5,6 or 7) or immunoglobulin.This can be used for preparing aforesaid dimer.
Embodiment 8.dAb trimerizing
General introduction
For the dAb trimerizing, protein C-end needs free cysteine.Cysteine residues in case reduction obtains free sulfhydryl groups, then can be used for protein specific is coupled on the trimer maleimide amine molecule, for example TMEA (three [2-maleimide ethyl] amine).
The PCR of TAR1-5-19CYS makes up
Following oligonucleotide is specifically designed to the PCR TAR1-5-19 with SalI and BamHI site, is used for the clone and introduces C-end cysteine residues:
SalI
～～～～～～～
Trp?Ser?Ala?Ser?Thr?Aap *?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val
1 TGG?AGC?GCG?TCG?ACG?GAC?ATC?CAG?ATG?ACC?CAG?TCT?CCA?TCC?TCT?CTG?TCT?GCA?TCT?GTA
ACC?TCG?CGC?AGC?TGC?CTG?TAG?GTC?TAC?TGG?GTC?AGA?GGT?AGG?AGA?GAC?AGA?CGT?AGA?CAT
Gly?Asp?Arg?Val?Thr?Ils?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Asp?Ser?Tyr?Leu?His?Trp
61 GGA?GAC?CGT?GTC?ACC?ATC?ACT?TGC?CGG?GCA?AGT?CAG?AGC?ATT?GAT?AGT?TAT?TTA?CAT?TGG
CCT?CTG?GCA?CAG?TGG?TAG?TGA?ACG?GCC?CGT?TCA?GTC?TCG?TAA?CTA?TCA?ATA?AAT?GTA?ACC
Tyr?Gln?Gln?Lys?Pro?Gly?Lye?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ser?Ala?Ser?Glu?Leu?Gln
121 TAC?CAG?CAG?AAA?CCA?GGG?AAA?GCC?CCT?AAG?CTC?CTG?ATC?TAT?AGT?GCA?TCC?GAG?TTG?CAA
ATG?GTC?GTC?TTT?GGT?CCC?TTT?CGG?GGA?TTC?GAG?GAC?TAG?ATA?TCA?CGT?AGG?CTC?AAC?GTT
Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile
181 AGT?GGG?GTC?CCA?TCA?CGT?TTC?AGT?GGC?AGT?GGA?TCT?GGG?ACA?GAT?TTC?ACT?CTC?ACC?ATC
TCA?CCC?CAG?GGT?AGT?GCA?AAG?TCA?CCG?TCA?CCT?AGA?CCC?TGT?CTA?AAG?TGA?GAG?TGG?TAG
Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Val?Trp?Arg?Pro
241 AGC?AGT?CTG?CAA?CCT?GAA?GAT?TTT?GCT?ACG?TAC?TAC?TGT?CAA?CAG?GTT?GTG?TGG?CGT?CCT
TCG?TCA?GAC?GTT?GGA?CTT?CTA?AAA?CGA?TGC?ATG?ATG?ACA?GTT?GTC?CAA?CAC?ACC?GCA?GGA
BamHI
～～～～～～～～
Phe?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Cys?***?***?Gly?Ser?Gly
301 TTT?ACG?TTC?GGC?CAA?GGG?ACC?AAG?GTG?GAA?ATC?AAA?CGG?TGC?TAA?TAA?GGA?TCC?GGC
AAA?TGC?AAG?CCG?GTT?CCC?TGG?TTC?CAC?CTT?TAG?TTT?GCC?ACG?ATT?ATT?CCT?AGG?CCG
( *The beginning of TAR1-5-19CYS sequence; TAR1-5-19CYS aminoacid sequence (SEQID NO:293; TAR1-5-19CYS nucleotide sequence (SEQ ID NO:294, coding strand; SEQ ID NO:295, noncoding strand))
Forward primer
5’-TGGAGCGCGTCGACGGACATCCAGATGACCCAGTCTCCA-3’(SEQ?ID?NO：296)
Reverse primer
5’-TTAGCAGCCGGATCCTTATTAGCACCGTTTGATTTCCAC-3’(SEQ?ID?NO：297)
Followingly carry out PCR reaction (50 μ L volume): 200 μ M dNTP, 0.4 each primer of μ M, 5 μ L 10x Pfu Turbo buffer (Stratagene), 100ng template plasmid (coding TAR1-5-19), 1 μ L Pfu Turbo enzyme (Stratagene) also transfer to 50 μ L with sterilized water with volume.Adopt following PCR condition: 94 ℃ of initial denaturing steps 2 minutes, be again 25 circulations (94 ℃ 30 seconds, 64 ℃ 30 seconds, 72 ℃ 30 seconds).Final extend step also comprise 72 ℃ 5 minutes.Purified pcr product is also used SalI and BamHI digestion, is connected on the carrier that cuts with the same restrictions enzyme action.Correct clone is confirmed through dna sequencing.
The expression of TAR1-5-19CYS and purification
According to the scheme of manufacturer, the TAR1-5-19CYS carrier is transformed in BL21 (DE3) the pLysS chemoreception attitude cell (Novagen).Select to carry the cell of dAb plasmid with 100 μ g/mL Carbenicillins and 37 μ g/mL chloromycetin.In the bottle of the 2L band baffle plate that contains 500mL terrific meat soup (Sigma-Aldrich), 100 μ g/mL Carbenicillins and 37 μ g/mL chloromycetin, set up and cultivate.Culture 30 ℃ with the 200rpm shaken cultivation, be 1-1.5 up to O.D.600, use 1mM IPTG (isopropyl-β-D-sulfo-galactopyranoside, Melford Laboratories) to induce then.Allow dAb's be expressed in 30 ℃ of lasting 12-16 hours.Find that most of dAb are present in the culture medium.Therefore, by centrifugal (8,000xg 30 minutes), isolate cell from culture medium, supernatant is used for purification dAb.Whenever go up in the clear liquid and to add 30mL albumen L agarose (Affitech), allow dAb combination in batches, stirred simultaneously 2 hours.Allow resin sedimentation under gravity 1 hour then, inhale again and remove supernatant.Agarose is packed in XK 50 posts (Amersham Phamacia), with the PBS washing of 10 times of volumes., contain protein part and neutralize with 100mM glycine (pH2.0) eluting in conjunction with dAb by the 1M Tris (pH8.0) that adds 1/5 volume.Be separated to the 20mg pure protein in every liter of culture supernatant, wherein contain the monomer and the dimer of 50: 50 ratios.
The trimerizing of TAR1-5-19CYS
2.5ml 100 μ M TAR1-5-19CYS with the reduction of 5mM dithiothreitol, DTT, were at room temperature placed 20 minutes.Give sample exchange buffering liquid with PD-10 post (Amersham Pharmacia).This post is used 5mM EDTA, 50mM sodium phosphate (pH6.5) balance in advance, according to the guide of manufacturer, uses sample and eluting.Sample is placed on standby on ice.TMEA (three [2-maleimide ethyl] amine) is available from Pierce Biotechnology.With 100%DMSO (dimethyl sulfoxide) preparation 20mM TMEA storage liquid.When finding TMEA concentration, cause rapid precipitation and protein cross greater than 3: 1 (mol ratio of dAb: TMEA).When increasing, pH also can increase settling rate and cross-linked speed.Therefore, use 100 μ M reduction TAR1-5-19CYS, add 25 μ M TMEA and make the protein trimerizing, allow reaction at room temperature carry out 2 hours.When discovery is carried out in coupling reaction, add additive (for example glycerol or ethylene glycol), can significantly reduce the trimer precipitation to 20% (v/v).After the coupling, SDS-PAGE the analysis showed that, has monomer, dimer and trimer in the solution simultaneously.
The purification of trimer TAR1-5-19CYS
Add 40 μ L, 40% glacial acetic acid in every mL TMEA-TAR1-5-19cys reactant, pH is reduced to～4.Sample is added to 1mL Resource S cation exchange column (AmershamPharmacia), this post is used 50mM sodium acetate (pH4.0) balance in advance again.Salt gradient, 50mM sodium acetate (pH4.0) with 340-450mM sodium chloride surpass 30 times of column volumes, and part is separated dimer and trimer.Only contain trimerical part and identify that with SDS-PAGE the back merges, the 1M Tris (pH8.0) that adds 1/5 volume rises to 8 with pH.In order (to adopt 5K by the centrifugal concentrator of Viva at concentration step; Vivascience) prevent the trimer precipitation in, in sample, add 10% glycerol.
Vitro functional is in conjunction with mensuration: TNF receptor determination and raji cell assay Raji
Adopt TNF receptor and raji cell assay Raji, measure the affinity of trimer human TNF alpha.IC in the receptor determination 50Be about 0.3nM; ND in the raji cell assay Raji 50Be about 3-10nM (for example 3nM).
The TAR1-5-19CYS trimeric form that other is possible
Adopt following reagent, also TAR1-5-19CYS can be made trimer:
PEG trimer and the synthetic maleimide trimer of trust
Nektar (Shearwater) provides a series of multi-arm PEG, and they can be by chemical modification at the PEG end.Therefore, adopt the PEG trimer that has the maleimide amine functional group at each arm end, make the mode of dAb trimerization be similar to the mode with TMEA of above general introduction.PEG also has the advantage that increases the trimer dissolubility, thereby can prevent accumulative problem.Therefore, can produce the dAb trimer, wherein each dAb has the C-terminal cysteine that is connected with the maleimide amine functional group, and described maleimide amine functional group is connected on the PEG trimer.
Peptide linker or hinge are added to the C-end of dAb.
Perhaps (Gly 4Ser) n(for example IgG hinge region or peptide sequence little joints such as (for example being selected from peptide sequence library at random) at random can be added between dAb and the terminal cysteine residue for (wherein n=1-10, for example 1,2,3,4,5,6 or 7) or immunoglobulin.When being used to prepare polymer (for example dimer or trimer), this will introduce greatly the distance between flexible and each monomer again, and these can improve the binding characteristic with target (for example many subunits target for example human TNF alpha).
Embodiment 9. is at the selection of a collection of single domain antibody (dAb) of human serum albumin (HSA) and mice serum albumin (MSA)
This embodiment has illustrated the method for preparation at sero-abluminous single domain antibody (dAb).Selection at mice serum albumin (MSA) and human serum albumin's (HSA) dAb has been described.Used 3 people's phage displaying antibody libraries in this experiment, they are separately based on V HSingle people's framework (referring to Figure 13: based on the dummy V of V3-23/DP47 and JH4b HSequence) or V κ (referring to Figure 15: based on the dummy V κ sequence of o12/o2/DPK9 and Jk1), its side chain multiformity by NNK codon coding is attached in the complementary determining region (CDR1, CDR2 and CDR3).
Library 1 (V H):
At the multiformity with upper/lower positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98.
Library size: 6.2 * 10 9
Library 2 (V H):
At the multiformity with upper/lower positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a, H100b.
Library size: 4.3 * 10 9
Library 3 (V κ):
At the multiformity with upper/lower positions: L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96
Library size: 2 * 10 9
Preliminary election V HWith V κ library respectively to the associativity of protein A and the general part of albumen L, make that the major part clone in non-selected library is functional.More than shown in the size of library size after corresponding to preliminary election.
Use each library respectively, carry out two-wheeled at serum albumin and select.For each selection, be that antigen/4ml PBS of 100 μ g/ml is coated on the immunity pipe (nunc) with concentration.In selecting in the first round, each in 3 libraries is all selected at HSA (Sigma) and MSA (Sigma) respectively.Take turns in the selection second, in 6 phagies selecting from the first round each is all selected at following antigen: (i) use same antigen (first round MSA for example once more, second takes turns MSA) and (ii) at exchanging antigen (first round MSA for example, second takes turns HSA), produce altogether 12 second and take turns selection.In each case, second take turns selection after, measure combining of 48 clones and HSA and MSA.According to Harrison etc., MethodsEnzymol.1996; 267:83-109 and standard ELISA scheme (Hoogenboom etc. (1991) Nucleic Acids Res., 19:4133) described, produce solubility dAb fragment, just with 2% tween PBS as sealing buffer, and (be used for V with albumen L-HRP (Sigma) (being used for V κ) and protein A-HRP (Amersham Pharmacia Biotech) H) detect in conjunction with dAb.
In ELISA, measure dAb, obtain signal, show with MSA, HSA or the two to combine that insoluble form is only in conjunction with plastics, but all have specificity to serum albumin greater than background.To the clone check order (seeing table) show, identified 21 unique dAb sequences.Minimum similarity (on amino acid levels) between the selected V κ dAb clone is 86.25% ((69/80) * 100); This is the result when all diversified residues all different (for example cloning 24 and 34).Selected V HMinimum similarity between the dAb clone is 94% ((127/136) * 100).
Then, measure serum albumin is caught biotinylated antigen from solution in conjunction with dAb ability.According to ELISA scheme (the same), just elisa plate with 1 μ g/ml albumen L (cloning) and 1 μ g/ml protein A for V κ (for V HThe clone) bag quilt.According to scheme, from solution, catch solubility dAb, reuse biotinylation MSA or HSA and streptavidin HRP detect.Biotinylation MSA and HAS are that the operation instructions according to manufacturer prepare, and its objective is in order to obtain average 2 biotin/serum albumin molecule.In ELISA, identified 24 clones and from solution, caught biotinylation MSA.This wherein has two (following clones 2 and 38) also to catch biotinylation HSA.Then, measure the binding ability of dAb to the MSA of bag quilt on the CM5biacore chip.Find that 8 clones can be in conjunction with the MSA on the biacore.
Table 8.
(all catch biotinylation MSA to dAb H or κ ?CDR1 CDR2 CDR3 In conjunction with the MSA on the biacore? Do you catch biotinylation HSA?
V κ library 3 templates (dummy) κ XXXLX (SEQ?ID?NO：298) XASXLQS (SEQ?ID NO：299) QQXXXXPXT (SEQ?ID?NO：300)
2，4，7，41， κ SSYLN (SEQ?ID?NO：301) RASPLQS (SEQ?ID NO：302) QQTYSVPPT (SEQ?ID?NO：303) All 4 combinations of √
38，54 κ SSYLN (SEQ?ID?NO：304) RASPIQS (SEQ?ID NO：305) QQTYRIPPT (SEQ?ID?NO：306) Two combinations of √
46，47，52，56 κ FKSLK (SEQ?ID?NO：307) NASYLQS (SEQ?ID NO：308) QQVVYWPVT (SEQ?ID?NO：309)
13，15 κ YYHLK (SEQ?ID?NO：310) KASTLQS (SEQ?ID NO：311) QQVRKVPRT (SEQ?ID?NO：312)
30，35 κ RRYLK (SEQ?ID?NO：313) QASVLQS (SEQ?ID NO：314) QQGLYPPIT (SEQ?ID?NO：315)
9， κ YNWLK (SEQ?ID?NO：316) RASSLQS (SEQ?ID NO：317) QQNVVIPRT (SEQ?ID?NO：318)
22， κ LWHLR (SEQ?ID?NO：319) HASLLQS (SEQ?ID NO：320) QQSAVYPKT (SEQ?ID?NO：321)
23， κ FRYLA (SEQ?ID?NO：322) HASHLQS (SEQ?ID QQRLLYPKT (SEQ?ID?NO：324)
NO：323)
24， κ FYHLA (SEQ?ID?NO：325) PASKLQS (SEQ?ID NO：326) QQRARWPRT (SEQ?ID?NO：327)
31， κ IWHLN (SEQ?ID?NO：328) RASRLQS (SEQ?ID NO：329) QQVARVPRT (SEQ?ID?NO：330)
33， κ YRYLR (SEQ?ID?NO：331) KASSLQS (SEQ?ID NO：332) QQYVGYPRT (SEQ?ID?NO：333)
34， κ LKYLK (SEQ?ID?NO：334) NASHLQS (SEQ?ID NO：335) QQTTYYPIT (SEQ?ID?NO：336)
53， κ LRYLR (SEQ?ID?NO：337) KASWLQS (SEQ?ID NO：338) QQVLYYPQT (SEQ?ID?NO：339)
11， κ LRSLK (SEQ?ID?NO：340) AASRLQS (SEQ?ID NO：341) QQVVYWPAT (SEQ?ID?NO：342) √
12， κ FRHLK (SEQ?ID?NO：343) AASRLQS (SEQ?ID NO：344) QQVALYPKT (SEQ?ID?NO：345) √
17， κ RKYLR (SEQ?ID?NO：346) TASSLQS (SEQ?ID NO：347) QQNLFWPRT (SEQ?ID?NO：348) √
18， κ RRYLN (SEQ?ID?NO：349) AASSLQS (SEQ?ID NO：350) QQMLFYPKT (SEQ?ID?NO：351) √
16，21 κ IKHLK (SEQ?ID?NO：352) GASRLQS (SEQ?ID NO：353) QQGARWPQT (SEQ?ID?NO：354) √
25，26 κ YYHLK (SEQ?ID?NO：355) KASTLQS (SEQ?ID NO：356) QQVRKVPRT (SEQ?ID?NO：357) √
27， κ YKHLK (SEQ?ID?NO：358) NASHLQS (SEQ?ID NO：359) QQVGRYPKT (SEQ?ID?NO：360) √
55， κ FKSLK (SEQ?ID?NO：361) NASYLQS (SEQ?ID NO：362) QQVVYWPVT (SEQ?ID?NO：363) √
V HLibrary 1 (with 2) template (dummy) H XXYXXX (SEQ?ID NO：364) XIXXXGXXTXYADS VKG(SEQ?ID NO：365) XXXX(XXXX)FDY (SEQ?ID?NO：366)
8，10 H WVYQMD (SEQ?ID NO：367) SISAFGAKTLYADS VKG(SEQ?ID NO：368) LSGKFDY (SEQ?ID?NO：369)
36， H WSYQMT (SEQ?ID NO：370) SISSFGSSTLYADS VKG(SEQ?ID NO：371) GRDHNYSLFDY (SEQ?ID?NO：372)
In all cases, framework is identical with framework in corresponding dummy sequence, and its multiformity in CDR sees the above table.
In 8 clones in conjunction with the MSA on the biacore, 2 clones (clone MSA16 and MSA26) that are chosen in the expression of escherichia coli camber are used for further research (referring to embodiment 10).Complete nucleotide sequence and the aminoacid sequence of MSA16 and MSA 26 are seen Figure 16.
Embodiment 10. measures in conjunction with the dAb MSA16 of MSA and mice affinity and the serum half-life of MSA26
DAb MSA16 and MSA26 express in colibacillus periplasm, its purification adopt be adsorbed onto in batches the affine resin of albumen L-agarose (Affitech, Norway) on, use glycine (pH2.2) eluting then.Then, by suppressing the dAb that biacore analyzes purification, measure K dIn brief, purification MSA16 and MS A26 are tested, to reach the required dAb concentration of 200RU response on the biacore CM5 chip that is determined at high density MSA bag quilt.In case measure the dAb desired concn, will expect K dAbout MSA antigen concentration scope and dAb premix merge overnight incubation.Then with 30 μ l/ minutes high flow rate, measure the combining of biacore chip of dAb and MSA bag quilt in each pre-composition.With gained curve plotting Klotz curve, according to the K of curve estimation for MSA16 dBeing 200nM, then is 70nM (Figure 17 A and Figure 17 B) for MSA26.
Then, to clone MSA16 and MSA26 is cloned in the expression vector, described carrier has HA labelling (nucleotide sequence: TATCCTTATGATGTTCCTGATTATGCA (SEQ ID NO:373) and aminoacid sequence: YPYDVPDYA (SEQ ID NO:374)), the 2-10mg amount is at expression in escherichia coli, with the affine resin of albumen L-agarose (Affitech, Norway) and with glycine (pH2.2) eluting and from supernatant purification.Measure the mice serum half-life of dAb.Give MSA26 and the MSA16 of the about 1.5mg/kg of CD1 mice single intravenous injection.(Abcam UK) catches and measures ELISA, serum analysis level with the albumen L-HRP (invitrogen) of 4%Marvel sealing by the anti-HA of goat.Wash with 0.05% tween PBS.In the presence of the 1x mice serum, draw the standard curve of the dAb of concentration known, with the comparability of assurance with test specimen.With 2 compartment model modelings, show that the t  α of MSA26 is 0.16 hour, t  β is 14.5 hours, area under curve (AUC) is 465 hours .mg/ml (data not shown), the t  α of MSA16 is 0.98 hour, and t  β is 36.5 hours, and AUC is 913 hours .mg/ml (Figure 18).Compare with HEL4 (a kind of anti-hen egg-white lysozyme dAb), these two anti-MSA clones have the quite long half-life, and its t  α is 0.06 hour, and t  β is 0.34 hour.
Embodiment 11.V H-V HWith V κ-segmental generation of V κ bispecific Fab sample
This embodiment has described preparation V H-V HMethod with V κ-V κ bi-specific antibody (as Fab print section).Before making up various described Fab print sections, according to embodiment 9 described similar approach, at first selecting from the dAb library can be in conjunction with the dAb of selected target.Isolate a kind of can be in conjunction with the V of hen-egg lysozyme (Sigma) HDAb is HEL4, and also isolating can be in conjunction with the 2nd V of TNF α receptor (R and d system) HDAb (TAR2h-5).These sequences provide in sequence table.By selection and affinity maturation, isolating can be in conjunction with the V κ dAb of TNF α (TAR1-5-19), and its sequence also provides in sequence table.The 2nd V κ dAb (MSA26) (its sequence is seen Figure 17 B) that describes among the embodiment 9 also can be used for these experiments.
To digest with enzyme SalI and NotI from the DNA of the expression vector that contains above-mentioned 4 kinds of dAb, downcut the dna encoding district of dAb.Digest is purified into the band (300-400bp) of expection size by agarose gel electrophoresis, downcuts this band, and (Qiagen UK) carries out gel-purified to reuse Qiagen gel-purified test kit.The DNA of dAb of will encoding again is inserted in CH carrier or the C κ carrier (Fig. 8 and 9), and is as shown in the table.
Table 9.
dAb Target antigen dAb?V HOr dAb V κ Insert in the carrier Labelling (C end) Antibiotic resistance
HEL4 Hen-egg lysozyme V H V H Myc Chloromycetin
TAR2-5 The TNF receptor V H Vκ Flag Ampicillin
TAR1-5-19 TNFα Vκ V H Myc Chloromycetin
MSA26 The mice serum albumin Vκ Vκ Flag Ampicillin
V HC HAnd V HC κ construct cotransformation HB2151 cell.In addition, V κ C HWith V κ C κ construct cotransformation HB2151 cell.With the culture overnight incubation of each cotransformation cell line (in the 2xTy that contains 5% glucose, 10 μ g/ml chloromycetin and 100 μ g/ml ampicillin, to keep to C HThe antibiotic of plasmid and C κ plasmid is selected).Inoculate fresh culture (2xTy, 10 μ g/ml chloromycetin and 100 μ g/ml ampicillin) and be cultured to OD0.7-0.9 with overnight culture, add IPTG then and induce, to express their C HConstruct and C κ construct.Again by the protein A purification (V that is used for cotransformation HC HAnd V HC κ) and the affine resin purification of MSA (the V κ C that is used for cotransformation HWith V κ C κ), the Fab print section of expressing is carried out purification.
V H-V HBispecific
By gel electrophoresis of protein, measure V HC HAnd V HThe expression of C κ bi-specific antibody.Behind the gel trace, by myc labelling and flag labelling, on Western blotting, detect the band of Fab fragment expectation size, show the segmental V of Fab sample HC HAnd V HC κ part all exists.Then, for whether two one side of something determining bi-specific antibody are present on the same Fab print section,, spent the night at 4 ℃ of bags with the sodium bicarbonate buffer liquid of elisa plate with 100 μ l/ hole 3mg/ml hen-egg lysozyme (HEL).This plate reuse 2% tween PBS seals (described according to embodiment 1), follows and V HC H/ V HC κ bispecific Fab print section is hatched together.By the dereferenced chain, (a kind of monoclonal antibody in conjunction with the myc labelling, Roche) with anti-mice IgG-HRP (Amersham Pharmacia Biotech), detection bispecific fragment combines with HEL's with 9e10.V HC H/ V HThe segmental signal of C κ bispecific Fab sample is 0.154, by contrast, and for the V of single expression HThe background signal of C κ chain is 0.069.This proof Fab print section has binding specificity to target antigen.
V κ-V κ bispecific
The V κ C of purification cotransformation on the affine resin of MSA HAfter V κ C κ bispecific Fab print section, survey the elisa plate of 1 μ g/ml TNF α bag quilt and the elisa plate of 10 μ g/ml MSA bag quilt with gained protein.As expected, when on two kinds of elisa plates, using albumen L-HRP to detect, produce the signal (data not shown) that is higher than background.This shows, can also can be in conjunction with TNF α in ELISA subsequently in conjunction with the protein portion of MSA (and therefore and at purification on the MSA affinity column), and this has confirmed the bispecific of antibody fragment.Such protein portion is used further to two experiments subsequently.First, the elisa plate of 1 μ g/ml TNF α bag quilt bispecific V κ C HSurvey with V κ C κ Fab print section, also promptly survey in conjunction with the dAb of TNF α with contrast simultaneously, the concentration of this contrast is for obtaining the concentration of similarity signal as calculated on ELISA.When 2mg/ml MSA exists and do not exist, survey elisa plate with bispecific dAb and contrast dAb.The signal in bispecific hole reduces above 50%, but the signal in dAb hole does not reduce (referring to Figure 19 a) fully.Same albumen also is used for receptor determination (have and do not have MSA), also demonstrates the competitiveness (referring to Figure 19 c) with MSA.This proof MSA competitiveness is in conjunction with bi-specific antibody and TNF α.
12. pairs of mice serum albumin of embodiment and TNF α have the generation of the bi-specific antibody of specific V κ _ V κ bispecific cys bonding
The chemical coupling preparation that this embodiment has described by disulfide bond all has the method for specific bispecific antibody fragment to mice serum albumin and TNF α.MSA16 (from embodiment 1) and TAR1-5-19 dAb are cloned into again to have C end cysteine and not to have in the carrier based on pET of labelling.Two kinds of dAb expressions are 4-10mg, and (Affitiech, Norway) purification comes out from supernatant with the affine resin of albumen L-agarose.The dAb reuse dithiothreitol, DTT reduction of cysteine labelling.TAR1-5-19 dAb again with the two pyridine couplings of two sulfur, the formation again of sealing disulfide bond, thus form PEP 1-5-19 homodimer.Again two kinds of different dAb are mixed at pH6.5, promote to form disulfide bond and produce TAR1-5-19, the heterodimer of MSA16 cys bonding.This preparation method of two kinds of different proteins conjugates is at first by (King TP such as King, Li Y Kochoumian L Biochemistry, 1978, the 17 volumes: 1499-506 Preparation of Protein Conjugates via intermoleculardisulfide bond formation) describe.By cation exchange heterodimer and monomer are separated.Bring confirmation to separate by the bar that has the expection size on the sds gel.Gained heterodimer kind is measured in the TNF receptor determination, finds in it and the IC of TNF 50Be about 18nM.Then, with the heterodimer (18nM) of constant density and the MSA and the HAS of serial dilution, repeat receptor determination.The existence of the HAS of debita spissitudo scope (2mg/ml at most) does not cause that dimer suppresses the decline of TNF α ability.Yet, add MSA, cause that in the dose dependent mode dimer suppresses the decline (Figure 20) of TNF α ability.This proof MSA and TNF α competitiveness are in conjunction with TAR1-5-19, the MSA16 dimer of cys bonding.
Data gather
Gathering of gained data seen appendix 4 in the experiment of the foregoing description.
The activity of embodiment 13. anti-mice TNFR1 dAb and anti-people TNFR1 dAb
The activity of the anti-mice TNFR1 of table 10. dAb
?dAb Active (IC 50)
The L929 raji cell assay Raji Receptors bind is measured
?TAR2m-19 ?10μM ?2μM
?TAR2m-20 ?n/d ?150nM
?TAR2m-21 ?400nM ?n/d
?TAR2m-24 ?1μM ?1.3μM
?TAR2m-21-23 ?1nM ?n/d
?TAR2m-21-07 ?10nM ?n/d
?TAR2m-21-43 ?6nM ?n/d
?TAR2m-21-48 ?6nM ?n/d
?TAR2m-21-10 ?30nM ?n/d
?TAR2m-21-06 ?100nM ?n/d
?TAR2m-21-17 ?300nM ?n/d
N/d does not detect
The activity of the anti-people TNFR1 of table 11. dAb
?dAb Active (IC 50)
HeLa IL-8 raji cell assay Raji Receptors bind is measured
?TAR2h-10 ?50nM ?30nM
?TAR2h-12 ?100nM ?n/d
?TAR2h-13 ?300nM ?n/d
?TAR2h-14 ?300nM ?30nM
?TAR2h-15 ?n/d ?5nM
?TAR2h-16 ?200nM ?30nM
?TAR2h-17 ?n/d ?100nM
?TAR2h-18 ?400nM ?n/d
?TAR2h-22 ?n/d ?200nM
?TAR2h-27 ?3000nM ?30nM
?TAR2h-29 ?300nM ?300nM
?TAR2h-32 ?100nM ?n/d
?TAR2h-34 ?n/d ?300nM
?TAR2h-35 ?800nM ?n/d
?TAR2h-41 ?30nM ?8nM
?TAR2h-42 ?10nM ?15nM
?TAR2h-44 ?300nM ?10nM
?TAR2h-47 ?n/d ?8nM
?TAR2h-51 ?n/d ?80nM
?TAR2h-67 ?300nM ?n/d
?TAR2h-10-1 ?n/d ?10nM
?TAR2h-10-2 ?n/d ?11nM
?TAR2h-10-3 ?n/d ?11mM
?TAR2h-10-4 ?n/d ?8nM
?TAR2h-10-5 ?n/d ?11nM
?TAR2h-10-7 ?30nM ?n/d
?TAR2h-10-27 ?10nM ?2nM
?TAR2h-10-55 ?20nM ?n/d
N/d does not detect
MRC-5 IL-8 discharges mensuration
In the following MRC-5 raji cell assay Raji, having estimated some can be in conjunction with the activity of the dAb of people TNFR1.Mensuration is based on the inductive IL-8 secretion of TNF in the MRC-5 cell, this assay method is Alceson, L etc., improving one's methods of the described method of Journal of Biological Chemistry 271:30517-30523 (1996), described document have been introduced in HUVEC IL-1 to the inducing action of IL-8.Replace HUVEC cell line with the MRC-5 cell,, measured the activity of dAb by estimating human TNF alpha to the inducing of IL-8.In brief, the MRC-5 cell inoculation on microtitration plate, with these plates with dAb and human TNF alpha (300pg/ml) overnight incubation.After hatching, the sucking-off culture supernatants is by sandwich ELISA (R﹠amp; D system) the IL-8 concentration in the mensuration supernatant.With only comparing with the control wells that TNF α is hatched, anti-TNFR1 dAb activity causes IL-8 secretion reduction in the supernatant.
Embodiment 14. mice septic shock models
In the experimental model of the good septic shock syndrome of setting up, estimate the interior effect (Rothe etc., Circulatory Shock 44:51-56, (1995)) of body of anti-TNFR1dAb.In this model, the activation of TNFR-1 (p55) is depended in the inductive death of LPS.In this model, make mice to LPS toxicity sensitivity with D-galactosamine (D-GaIN).In the research, the lethal LPS dosage of wild type animal is about 10ng.
Peritoneal injection LPS (Salmonella enteritidis (Salmonella enteritidis), Sigma, USA) and D-galactosamine (D-GaIN, Sigma, USA).The control mice of D-GalN-sensitization (10mg/ mice) is being attacked death in back 18 hours with LPS (10ng).After attack, surpass in 1 day cycle, write down the mortality rate of non-sensitized mice.
(described part can be in conjunction with mice TNFR1 and mice serum albumin (TAR2m-21-23 3U TAR7m-16 to give the interior injection of mouse peritoneum bispecific part; TAR7m-16 is also referred to as MSA16 in this article)) or ENBREL  (entarecept; Immunex Corporation), give LPS after 4 hours.(referring to table 12).Survival condition is monitored at interval with 4-6 hour in 48 hours.Prove the effect of anti-mice TNFR1 dAb with survival rate.
Table 12.
The treatment group Medicine and dosage LPS dosage/mice (ng) Size of animal Survival quantity in the time of 24 hours
1 Saline 10 8 0/8
2 10mg/kg ENBREL(entarecept； Immunex?Corporation) 10 8 8/8
3 5.4mg/kg TAR2m-21-23?3U?TAR7m-16 10 8 4/8
4 1mg/Kg TAR2m-21-23?3U?TAR7m-16 10 8 2/8
5 5.4mg/kg TAR2m-21-23?3U?TAR7m-16 0 2 2/2
TAR2m-21-23 3U TAR7m-16 contains dAb and can be in conjunction with the bispecific part of mice TNFR1, mice TNFR1 by peptide linker with can be in conjunction with the albuminous dAb combination of mice serum.The nucleotide sequence of coding TAR2m-21-23 3U TAR7m-16 and the aminoacid sequence of bispecific part are listed as SEQ ID NO:375 and SEQ ID NO:376 respectively as follows.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCC
GCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGT
GGTACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTC
CAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTAT
ATTACTGTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAG
GGAACCCAGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG
CGGTGGCGGGTCGACGGACATCCAGATGACCCSGTCTCCATCCTCCCTGTCTGCAT
CTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTATTAAGCAT
TTAAAGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGC
ATCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATCTGGGACAG
ATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCTACGTACTACTGT
CAACAGGGGGCTCGGTGGCCTCAGACGTTCGGCCAAGGGACCAAGGTGGAAATCAA
ACGGGCGGCCGCAGAACAAAACTCATCTCAGAAGAGGATCTGAAT
(SEQ?ID?NO：375)
EVQLLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGR
GTYYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQ
GTQVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSIIKH
LKWYQQKPGKAPKLLIYGASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
QQGARWPQTFGQGTKVEIKRAAAEQKLISEEDLN
(SEQ?ID?NO：376)
In TAR2m-21-23 3U TAR7m-16 treatment group survivor is arranged, this proves that anti-TNFR1 dAb is effectively to suppressing receptor active in the body, its as a result proving effect be dose dependent.In addition, the therapeutic efficiency of TAR2m-21-23 3U TAR7m-16 is than ENBREL  (entarecept; Immunex Corporation) effect is better.Only the survival rate with the animal (the 5th group does not have LPS to attack) of TAR2m-21-23 3U TAR7m-16 treatment also proves, nontoxic and the crosslinked and not exciting receptor by receptor in vivo of TAR2m-21-23 3U TAR7m-16.
Further studies have shown that, when TNF α does not exist, the not exciting TNFR1 of anti-TNFR1 dAb (playing the effect of TNFR1 agonist).The L929 cell is cultivated in the culture medium that contains one of following composition respectively: the TAR2m-21-23 monomer of variable concentrations, with crosslinked TAR2m-21-23 monomer, TAR2m-21-23 3UTAR7m-16 or the TAR2m-21-23 40K PEG of commercial anti myc antibody (9E10).With regard to regard to the antibody linked TAR2m-21-23 monomer of anti-myc, by 2: 1 mixed, at room temperature preincubate was 1 hour, stimulates immune cross-linked effect in the body, cultivates then with dAb and antibody.Concentration is that the TAR2m-21-23 monomer of 3000nM is hatched with the L929 cell.TAR2m-21-23 monomer and anti-Myc antibody are that the dAb of 3000nM is hatched with concentration.With concentration is that the TAR2m-21-23 3U TAR7m-16 of 25nM, 83.3nM, 250nM, 833nM and 2500nM is hatched with cell.With concentration is that the TAR2m-21-23 40K PEG of 158.25nM, 527.5nM, 1582.5nM, 5275nM and 15825nM is hatched with cell.After the overnight incubation, described according to the cytotoxic assay of L929 cell, estimate cell survival rate.The result shows, hatches with the dAb of varying number, does not cause the increase of debility cell quantity in the culture.The L929 cell is hatched with 10nM, 1nM and 0.1nM commercial anti TNFR1 IgG antibody, cause increasing the debility cell, therefore proved the sensitivity (Figure 26) of these cells the agonism of TNFR1 mediation in dosage dependence mode.
The model of embodiment 15. chronic inflammatory diseases
A. the collagen-induced arthritis model of mice
Give the Emulsion of Arthrogen-CIA adjuvant of DBA/1 injected in mice and Arthrogen-CIA collagen (MD-biosciences).At the 21st day, from research, remove animal with high arthritis score, remaining animal is divided into 10 groups, and male and jenny quantity equates.At the 21st day, treatment began with peritoneal injection saline, ENBREL  (entarecept; And continue 28 days Immunex Corporation) or TAR2m-21-23 40k PEG.Every limb to animal foot all carries out the clinical arthritis scoring, and score value is 0-4, represents normal limb in 0 minute, and representative in 4 fens relates to the most serious multiarticulate inflammation limb.
In this model, the scorching scoring of extremities joint summation is reduced to (a) 14-15 from 16 of maximum, (b) 12-15, and (c) 10-15, (d) 9-15, (e) 7-15, (f) 5-15, (g) 3-15, or (h) 1-15 is useful result.Producible useful result is that the scorching scoring of extremities joint summation is 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15.Compare with untreated matched group, it also is useful result that the arthritis outbreak postpones.
Clinical score clearlys show, compares with saline control, treats with TAR2m-21-23 40k PEG, have extraordinary effect to suppressing the arthritis development, and TAR2m-21-2340k PEG treatment is than using ENBREL  (entarecept; Immunex Corporation) better.This has proved that first inhibition TNFR1 is effective to treatment chronic inflammatory disease model.
B. mice IBD and arthritic Δ ARE model
The mice that has directed disappearance in 3 of TNF mRNA ' is rich in the element (ARE) of AU (is called Tnf Δ AREMice) excessive generation TNF and inflammatory bowel takes place, this disease is similar on histopathology to Crohn disease.(Kontoyiannis etc., J.Exp.Med.196:1563-74 (2002)).In these mices, Crohn disease sample disease takes place during age in week at 4-8, and the clinical symptoms of rheumatoid arthritis has also appearred in these animals.
At Tnf Δ AREMice, evaluation can suppress Crohn disease sample pathology and arthritic effect in conjunction with the dAb of mice TNFR1.ENBREL  (entarecept; ImmunexCorporation) as positive control.According to administration shown in the table 13 and dosage, these medicines are given by peritoneal injection.
The dAb that is studied comprises:
1) has the TAR2m-21-23 PEGization of a 40kD peg moiety;
2) can be in conjunction with mice TNFR-1 and the albuminous bispecific TAR2m-21-23 of mice serum 3U TAR7m-16.
Table 13.
Group Treatment Dosage Administration number of times Size of animal
6 ENBREL  (entarecept; Immunex Corporation) (medication is 3 times weekly) 10mg/kg 8 10
5 TAR2m-21-23 40kD PEG (medication is 2 times weekly) 1mg/kg 8 10
4 TAR2m-21-23 40kD PEG (medication is 2 times weekly) 10mg/kg 8 10
3 TAR2m-21-23 3U TAR7m-16 (medication is 2 times weekly) 1mg/kg 8 10
2 TAR2m-21-23 3U TAR7m-16 (medication is 2 times weekly) 10mg/kg 8 10
1 Saline NA 8 10
When the administration end cycle, put to death mice, downcut terminal ileum and proximal colon and analyze.
For histologic analysis,, acute and chronic inflammation is marked with the sxemiquantitative marking system with the tissue sample section and with h and E dyeing.The scoring regulation is as follows: acute inflammation scoring 0=0-1 polymorphonuclear (PMN) cell/high power field (PMN/hpf); In the 1=2-10PMN/hpf mucosa; In the 2=11-20 PMN/hpf mucosa; In the 3=21-30 PMN/hpf mucosa or 11-20 PMN/hpf extend under the muscular layer of mucosa; In 4=＞30 PMN/hpf mucosas or＞20 PMN/hpf extend under the muscular layer of mucosa; In chronic inflammatory disease scoring 0=0-10 monocyte (ML)/hpf (ML/hpf) mucosa; In the 1=11-20 ML/hpf mucosa; In the 2=21-30ML/hpf mucosa or 11-20 ML/hpf extend under the muscular layer of mucosa; Interior or the follicle hypertrophy of 3=31-40 ML/hpf mucosa; And 4=＞40 ML/hpf mucosas in or＞30 ML/hpf extend under the muscular layer of mucosa or follicle hypertrophy.
According to following system, weekly arthritic macroscopical phenotype sign is marked: 0=does not have arthritis (normal appearance and flexing); 1=mild osteoarthritis (joint deformity); 2=moderate arthritis (swelling, joint deformity); 3=severe arthritis (motion that is badly damaged).
In mouse arthritis Δ ARE model, TAR2m-21-23 dAb proof has effect in the good body, and is the same with the anti-SA form of the anti-TNFR1/ of bispecific (TAR2m-21-23 3U TAR7m-16) with 40kD PEGization monomer (TAR2m21-23 40kD PEG).In the 9th week, the average arthritis score of TAR2m-21-23 and TAR2m-21-23 3U TAR7m-16 treatment group is less than 0.4.By contrast, the moderate of saline control group is to serious arthritic average arthritis score＞1.0.With TAR2m-21-23 with TAR2m-21-23 3U TAR7m-16 (medication is 2 times weekly) compare, with ENBREL  (entarecept; Immunex Corporation) average score of treatment group (medication is 3 times weekly) is 0.5-1.0.These results show, with can be in conjunction with the therapy of the dAb form of TNFR1, are highly effective arthritis therapies, and PEGization dAb that is studied and bispecific dAb form all are to the highly effective medicine of chronic inflammatory disease.And these results also prove, can be in conjunction with the dAb of TNFR1 only with antagonism mode bind receptor.
C. mice IBD DSS model
Give the dextran sulfate sodium (DSS) that mice is dissolved in drinking water and can bring out IBD.(referring to for example Okayasu I. etc., Gastroenterology 98:694-702 (1990); Podolsky K., J.Gasteroenterol.38 supp1 XV:63-66 (2003)).The adult BDF1 mice of no helicobacter pylori (H.pylori) was raised in cages for 2 weeks, stablized their circadian rhythm.All mices all are placed on separately and have in the unitary ventilation cage of special pathogen-free domestic (SPF) barrier, under 12 little time-dark cycle.Allow the animal ad libitum access drink water.
Research was carried out 7 days.Drinking water contains 5%DSS, is used for whole conceptual phase.All animals were treated in 1-7 days morning (0900-1000) and the dusk (1600-1700).(referring to table 14).Be equivalent to the single preventive dose on the 1st day.All animals are weighed every day, and any diarrhoea incidence rate is all noted.Treated the last time back 24 hours, and put to death all animals, gave the bromodeoxyribouridine pulse in preceding 40 minutes in execution.Downcut the distally large intestine on one's body from animal.The small sample of distally large intestine is placed in " RNAlater ", and remainder is fixing in the CarnoyShi fixative, and paraffin embedding is cut into slices (discontinuous section/every slide) and dyeed with h and E.Section is with the visual assessment IBD order of severity and give severity scale.Carry out histologic analysis, measure inflammation area in average infringement area (ulcer area), the peaceful all walls of average epithelium area.
Adopt Zeiss Axiohome microscope, large intestine H﹠amp; The E transverse section can be used for writing down a series of sizes of organizing, and this can accurately quantize area.For each transverse section, can measure area and connective tissue area that epithelium adds lamina propria.Can calculate the epithelium area separately then, difference is exactly the lamina propria area.In normal structure, this tissue is about 10% to the Relative Contribution of area, but it rises with the increase of inflammation.So epithelium: the relative scale of lamina propria changes.
Along with the order of severity increases, degree of depth stenosis of this area narrow (because ulcer) and colon length shorten.These phenomenons cause that jointly the inner chamber cross-sectional area increases.Therefore, this parameter can be used for determining the order of severity of disease.
Tissue sample can and carry out severity scale by microscopic examination, wherein the 0=NIP; 1=crypts bottom periphery mild inflammation; The a large amount of inflammatory infiltrations of 2=, mucosa is destructurized; The a large amount of inflammatory infiltrations of 3=, the destructurized and ulcer of mucosa.
In this model, for saline control group severity scale,, treatment descends when causing severity scale, just show effectively.For example treatment group severity scale can reduce by 0.1 to about 1,1 to about 2 or 2 to about 3.Mark about below 2,1 to below about 2 or 1, show effectively.
Animal is following treats 9 groups (6 every group):
Table 14.
Group
1 The DSS/ drinking water
2 DSS/ drinking water+ip PEG TAR2m-21-23,10mg/kg 1x/d
3 DSS/ drinking water+ip PEG TAR2m-21-23,1mg/kg 1x/d
4 DSS/ drinking water+ip saline
5 DSS/ drinking water+per os tube feed PEG TAR2m-21-23,0.25mg/ animal 2x/d
6 DSS/ drinking water+per os tube feed saline
7 DSS/ drinking water+ip gives+ve contrast, for example sterin
8 DSS/ drinking water+per os tube feed+ve contrast, for example 5 ' aminosallcylic acid or analog
9 Do not treat animal
The per os tube feed will give ZANTAC  (ranitidine hydrochloride; GlaxoSmithKline).
D. mice chronic obstructive pulmonary disease (COPD) model
The effect of antagonism TNFR1 dAb disease progression in inferior chronic cigarette smoking (TS) model of mice is estimated.(referring to for example Wright JL and Churg A., Chest 122:306S-309S (2002)).Anti-mice TNFR1 dAb gives through peritoneal injection, per 48 hours once (beginning in 24 hours before for the first time being exposed to TS) and prolong form (PEGization, the bispecific part that for example comprise anti-SA dAb) with serum half-life and give.
Perhaps, anti-TNFR1 dAb will give by the intranasal administration mode, per 24 hours once (beginning in 4 hours before for the first time being exposed to TS) and give with monomer dAb.With ENBREL  (entarecept; Immunex Corporation) as positive control.All contact TS every day, research continues 1-2 week.(referring to for example Vitalis etc., Eru.Respir.J., 11:664-669 (1998)).After last TS exposes, analyze the total cell count and the cell divide counting of bronchoalveolar lavage, comprise neutrophil cell, eosinophilic granulocyte, macrophage and t lymphocyte subset class.The lobe of the lung is fixing in 10% buffered formalin, increase, the tracheole wall thickening situation of alveolar and alveolar duct in the analysis tissue slice, and carry out cell counting, comprise neutrophil cell, eosinophilic granulocyte, macrophage and t lymphocyte subset class.Because of ES exposes the decline of neutrophil cell, eosinophilic granulocyte, macrophage and the T-lymphocyte call subtype number raise, and the reduction of the increase of inductive alveolar of TS and alveolar duct and tracheole wall thickening, effect proved.
The structure and the expression of the chimeric TNFR1 molecule of embodiment 16. reorganization
This embodiment has illustrated preparation method (the Banner DW etc. of the molecule of being made up of different Mus TNFR1 territories and people TNFR1 territory, Cell, 75 (3): 431-45 (1993)), make this molecule contain 4 kinds of defined TNFR1 extracellular regions, but these are different mice with the difference of deriving between people TNFR1 albumen.According to the effect and the function in different structure territory, the Chimerical receptor that is produced is shared the characteristic of people TNFR1 and mice TNFR1.This molecule provides in conjunction with dAb, antibody and the Fab thereof of people or mice TNFR1 and other molecule (for example organic compound, NCE; Or the protein territory, for example affine body, ldl receptor territory or EGF territory) the specific evaluation methodology of domain.
Method
People and mice TNFR1 sequence are cloned among the yeast expression vector pPicZo α (Invitrogen) by EcoRI and NotI restriction endonuclease site earlier.Template mice TNFR1 DNA (so the Chimerical receptor construct ends at Mus territory 4) contains 3 ' 6x histidine mark.People TNFR1 (so the Chimerical receptor construct ends at people territory 4) contains Myc and 6x histidine mark simultaneously at 3 of sequence ' end.
According to Standard PC R condition, (USB Corporation, Cleveland Ohio), 100ng template DNA (comprising the relevant DNA of total length mTNFR1 or hTNFR1DNA prepared product template in a small amount), carry out initial PCR to adopt the RubyTaq archaeal dna polymerase.
Following the carrying out of typical PCR reaction: the 25 μ l 10X RubyTaq PCR buffer that contain polymerase; 2 μ l, first primer (from 10 μ M storage liquid); 2 μ l, second primer (from 10 μ l storage liquid); 1 μ l (100ng) total length TNFR1 template DNA; 20 μ l dH 2O (to final volume 50 μ l).Be reflected in the light-wall pipe and carry out, and be placed in the thermal cycler, wherein react according to following parameter.
The beginning degeneration 3 minutes 94℃
Degeneration annealing (25 circulations) is extended 30 seconds 94℃
30 seconds 55℃
1 minute 72℃
The final extension 10 minutes 72℃
Be used to produce the general introduction of the initial PCR reaction of chimeric construct body
Construct * PCR number-the primer Template
MHHH PCR1-1 and 12 Mice
PCR2-2 and 3 The people
HMHH PCR1-1 and 9 The people
PCR2-6 and 13 Mice
PCR3-2 and 4 The people
HHMH PCR1-1 and 10 The people
PCR2-7 and 14 Mice
PCR3-2 and 5 The people
HHHM PCR1-1 and 11 The people
PCR2-2 and 8 Mice
HMMM PCR1-1 and 9 The people
PCR2-2 and 6 Mice
*Note: H=people's domain; M=mice domain; MHHH=mice domain 1 for example, people's domain 2-4.
The PCR product that these initial PCR produced is scaled off from 1% agarose gel, and (Qiagen) carries out purification with the gel-purified test kit, is eluted to 50 μ l dH then 2Among the O.
Be used to produce the primer of chimeric TNFR1 construct
The primer numbering Primer sequence
Primer
1 ?GCCAGCATTGCTGCTAAAGAA(SEQ?ID?NO：605)
Primer 2 ?GGTCGACGGCGCTATTCAG(SEQ?ID?NO：606)
Primer 3 ?CTGCAGGGAGTGTGAGAGCGGC(SEQ?ID?NO：607)
Primer 4 ?GTGTGTGGCTGCAGGAAGAAC(SEQ?ID?NO：608)
Primer 5 ?CTGCCATGCAGGTTTCTTTC(SEQ?ID?NO：609)
Primer 6 ?CTGCAGGGAGTGTGAAAAGGG(SEQ?ID?NO：610)
Primer 7 ?GTGTGTGGCTGTAAGGAGAACC(SEQ?ID?NO：611)
Primer 8 ?CTGCCATGCAGGGTTCTTTC(SEQ?ID?NO：612)
Primer 9 ?TCACACTCCCTGCAGTCCG(SEQ?ID?NO：613)
Primer 10 ?CAGCCACACACGGTGTCCCGG(SEQ?ID?NO：614)
Primer 11 ?CCTGCATGGCAGGTGCACACGG(SEQ?ID?NO：615)
Primer 12 ?TCACACTCCCTGCAGACTG(SEQ?ID?NO：616)
Primer 13 ?CAGCCACACACCGTGTCCTTG(SEQ?ID?NO：617)
Primer 14 ?CCTGCATGGCAGTTACACACGG(SEQ?ID?NO：618)
SOE?PCR
Assembling PCR (be also referred to as ' pull-through ' or montage overlap extension (SOE), referring to Gene, 15:77 (1): 61-8 (1989)) makes first PCR product need not digestion or connection and gang uses first PCR product complementary terminal.In this process, primary product gang and degeneration then in the presence of Taq archaeal dna polymerase and dNTP, allow their complementary end anneal together.Repeat annealing and extend repeatedly circulation, it is flat to cause complementary strand to be mended, and produces the total length template.Interpolation is carried out conventional PCR in abutting connection with the primer of total length construct box, amplification assembling product.Carry out SOE PCR, make from the different TNFR1 territory of above-mentioned initial PCR and anneal together and increase.Following the carrying out of assembling SOE PCR: contain MgCl 240 μ l, 10 * PCR buffer; The purified product of the initial PCR1 of～2 μ l (100ng); The purified product of the initial PCR2 of～2 μ l (100ng); 36 μ l dH 2O (to final volume 80 μ l).After following installation step, add the SOE primer mixture: 2 μ l, 5 ' flank primer (primer 1); 2 μ l, 3 ' flank primers (primer 2); 10 μ l, 10 * PCR buffer; 6 μ l dH 2O (to final volume 20 μ l).
Use following program to carry out the PCR reaction.Initial assembling circulation needs about 45 minutes, then the thermal cycler setting is parked on 94 ℃.20 μ l primer mixtures join in each reactant and mix.
Step 1 assembling
The beginning degeneration 5 minutes 94℃
Degeneration annealing (25 circulations) is extended 1 minute 94℃
1 minute 55℃
1 minute 72℃
Step 2 amplification (be parked on 94 ℃, add primer mixture then)
Degeneration annealing (25 circulations) is extended 1 minute 94℃
1 minute 55℃
1 minute 72℃
With each reactant of 3-5 μ l electrophoresis on 1% agarose gel, check the PCR product.
3) will assemble the TNFR1 chimera is cloned in the yeast expression vector
PPicZ α carrier (Invitrogen) is used EcoRI and NotI enzymic digestion successively, reuse Chromaspin TE-1000 solvent resistant column (Clontech, Mountain View, CA) purification.
4) TNFR1 chimeric construct body is transformed in the escherichia coli
Chimeric construct body after the connection is transformed in HB2151 electroreception attitude (electrocompetent) Bacillus coli cells, in less salt LB culture medium, reclaimed 1 hour, be seeded in the antibiotic formulations (Cayla that contains 0.25 μ g/ml, zero mycin (ZEOCIN), contains phleomycin D then, Toulouse, France) on the less salt LB agar, cultivated 24 hours at 37 ℃.Single bacterium colony confirms that through sequence to guarantee that chimeric construct has correct sequence in the expression vector, extensive Maxiprep plasmid prepared product is made up of each chimeric construct body carrier.
The nucleotide sequence of prepared chimeric construct body sees below.The chimeric construct body is named (from left to right being territory 1 to territory 4) according to its domain source.For example, HMMM contains people territory 1 and mice territory 2-4.The chimeric protein that contains mice territory 4 only has His labelling and shortage and strides spacer between film district and the territory 4.
HMMM
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGCCTCAGTTGC
AAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGCTGACAAGGACA
CGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTG
CGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAAC
ACCGTGTGTAACTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCC
ACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAGCGCTCATCATCATCATCATCA
TTAATGA(SEQ?ID?NO：619)
HHHM
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC
TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACA
CCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTG
CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC
ACCGTGTGCACCTGCCATGCAGGGTTCTTTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCC
ACTGCAAGAAAAATGAGGAGTGTATGAAGTTGTGCCTAAGCGCTCATCATCATCATCATCA
TTAATGA(SEQ?ID?NO：620)
HHMH
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC
TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACA
CCGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGAGTGAGACACACTTCCAGTG
CGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGACAATCCCCTGTAAGGAGACTCAGAAC
ACCGTGTGTAACTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA
ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGG
CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAA
GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA(SEQ?ID?NO：621)
HMHH
AGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGT
GCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAG
GGAGTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAGTTGC
AAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGCTGACAAGGACA
CGGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTG
CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC
ACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA
ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGG
CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAA
GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA(SEQ?ID?NO：622)
MHHH
AGCTTGTGTCCCCAAGGAAAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGT
GCCACAAAGGAACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAG
GGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC
TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACA
CCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTG
CTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAAC
ACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTA
ACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCIACCCCAGATTGAGAATGTTAAGGG
CACTGAGGACTCAGGCACCACAGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAA
GAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGA(SEQ?ID?NO：623)
5) preparation of TNFR1 chimeric construct body and being transformed in the pichia pastoris phaff
To digest with rare cutting restriction endonuclease PmeI by the plasmid DNA that each a large amount of preparation (maxiprep) produces,, carry out Pichia sp. then and transform so that make the DNA linearisation.Linearisation DNA is again by phenol/chloroform extracting and ethanol precipitation and purification is resuspended in 30 μ l dH then 2O.10 μ l linearisation dna solutions and 80 μ l electroreception attitude (electro-competent) KM71H Pichia sp. mixing with cells 5 minutes are carried out electroporation (1.5kV, 200 Ω, 25 μ F) then.Cell recovers with YPDS immediately and hatched 2 hours at 30 ℃, is seeded in then containing 100 μ g/ml, zero mycin (ZEOCIN), containing on the YPDS agar plate of antibiotic formulations (Cayla, Toulouse, France) of phleomycin D and cultivated 2 days.
6) expression of construct in Pichia sp.
The single conversion bacterium colony of each construct of picking is inoculated among the 5ml BMGY (as starter culture), cultivates 24 hours at 30 ℃.With this culture inoculation 500ml BMGY culture medium, cultivated 24 hours at 30 ℃, then at room temperature with centrifugal 5 minutes harvestings of 1500-3000g.Cell is resuspended among the 100ml BMMY, cultivated 4 days, the staggered simultaneously methanol concentration (the 1st day 0.5%, the 2 day 1%, the 3 day 1.5%, the 4 day 2%) that increases.After the expression, with 3300g centrifugal 15 minutes, reclaim supernatant.
7) with Nickel resin purification TNFR1 chimeric construct body
Imidazoles and 2 * PBS that culture supernatant adds the 10mM final concentration earlier cushion.At room temperature add Nickel-NT A resin, the protein that in batches adsorbs the His-labelling reaches 4 hours (vibration).Supernatant/resin compound is poured poly-prep post (Biorad) again into.2 * PBS washing of 10 times of volumes of resin reuse is with 250mM imidazoles 1 * PBS eluting.Behind the exchange buffering liquid, make the chimeric construct body surface reach de-glycosylation, confirmed by SDS-PAGE again with EndoH de-glycosylation enzyme.
Used template DNA sequence in the PCR process
People (Homo sapiens) TNFR1 (extracellular region Genbank searching number 33991418)
CTGGTCCCTCACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGG
AAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAAGG
AACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGG
AGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCA
GCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAG
TGGACCGGGACACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGG
AGTGAAAACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTG
CACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGCACCTGCCATGCAGGTTTC
TTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAG
TGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGCACTGAGGACTCA
GGCACCACA(SEQ?ID?N0：624)
Coded people TNFR1 extracellular region has following amino acid sequences.
LVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECE
SGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLF
QCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLP
QIENVKGTEDSGTT(SEQ?ID?N0：603)
Mus (house mouse (Mus musculus)) TNFR1 (extracellular region Genbank searching number 31560798)
CTAGTCCCTTCTCTTGGTGACCGGGAGAAGAGGGATAGCTTGTGTCCCCAAGGA
AAGTATGTCCATTCTAAGAACAATTCCATCTGCTGCACCAAGTGCCACAAAGGA
ACCTACTTGGTGAGTGACTGTCCGAGCCCAGGGCGGGATACAGTCTGCAGGGA
GTGTGAAAAGGGCACCTTTACGGCTTCCCAGAATTACCTCAGGCAGTGTCTCAG
TTGCAAGACATGTCGGAAAGAAATGTCCCAGGTGGAGATCTCTCCTTGCCAAGC
TGACAAGGACACGGTGTGTGGCTGTAAGGAGAACCAGTTCCAACGCTACCTGA
GTGAGACACACTTCCAGTGCGTGGACTGCAGCCCCTGCTTCAACGGCACCGTGA
CAATCCCCTGTAAGGAGACTCAGAACACCGTGTGTAACTGCCATGCAGGGTTCT
TTCTGAGAGAAAGTGAGTGCGTCCCTTGCAGCCACTGCAAGAAAAATGAGGAG
TGTATGAAGTTGTGCCTACCTCCTCCGCTTGCAAATGTCACAAACCCCCAGGAC
TCAGGTACTGCG(SEQ?ID?NO：625)
Coded Mus (house mouse (Mus musculus)) TNFR1 extracellular region has following amino acid sequences.
LVPSLGDREKRDSLCPQGKYVHSKNNSICCTKCHKGTYLVSDCPSPGRDTVCRECE
KGTFTASQNYLRQCLSCKTCRKEMSQVEISPCQADKDTVCGCKENQFQRYLSETHF
QCVDCSPCFNGTVTIPCKETQNTVCNCHAGFFLRESECVPCSHCKKNEECMKLCLP
PPLANVTNPQDSGTA(SEQ?ID?NO：604)
The domain specificity of embodiment 17. anti-TNFR1 dAb
This embodiment has described the specific method of domain that is used for determining in conjunction with the dAb of TNFR1.This method adopts surface plasma resonance (SPR) (Detection of immuno-complex formation via surface plasmon resonance on gold-coateddiffraction, gratings. ' Biosensors.1987-88; 3 (4): 211-25.), determining antibody and the complete people or the bonded ability of mice biotinylation TNFR1 that are fixed on the SPR chip surface, when antibody incubation and with excessive embodiment 16 described chimeric molecule balances after.In this was measured, anti-TNFR1 dAb flow through the TNFR1 surface, produced spr signal, represented and the bonded dAb amount of TNFR1 that is fixed on the SPR chip.If dAb is through preincubate and with comprising the chimeric molecule balance of TNFR1 territory (this domain especially can combine with dAb), to compare with independent dAb so, this mixture flows through the TNFR1 surface, will produce more weak spr signal.Yet if dAb compares with the resulting signal of independent dAb so through preincubate and with the chimeric molecule balance that does not comprise TNFR1 territory (this domain especially can combine with dAb), this mixture flows through the TNFR1 surface, will produce roughly the same spr signal.
Method
1) generation on SPR chip TNFR1 surface
The selection on TNFR1 surface is decided by the species specificity of anti-TNFR1dAb to be measured.Therefore, anti-people TNFR1 dAb is estimated on the surface of personnel selection TNFR1 bag quilt, and estimates anti-mice TNFR1 dAb with the chip of mice TNFR1 bag quilt.
Biotinylation TNFR1 dilutes in suitable SPR buffer, and BIACORE 3000SPR instrument (Biacore International AB, Uppsala, Sweden) in by streptavidin (SA) sensor chip.Low flow velocity (5-10 μ l/ minute) is used to make maximization time of contact between biotinylation TNFR1 and the streptavidin surface.Continue flow velocity, saturated up to the streptavidin surface by the biotinylation material, have the chip on maximum TNFR1 surface with generation.Chip is usually in conjunction with a hundreds of SPR response unit to several thousand biotinylation materials.
What 2) anti-TNFR1 responded on the SPR chip tires
The competitive assay of a success needs the initial optium concentration of anti-TNFR1 dAb, makes minimum dAb flow through its surface, provides tangible spr signal.In some concentration range, dAb can dose dependent mode mating surface, makes the bonded RU number of dAb can reflect the dAb concentration that flows through chip surface.
In order to determine such dose dependent concentration range, the anti-TNFR1dAb of titration in the Biacore of 10 times of serial dilutions buffer (scope was diluted to 1: 1 from 1: 10,000,000 dilution).Independent and the sequential TNFR1 chip surface that is expelled to diluent then is from the rarest sample.Measure the maximum RU number of each diluent gained.In case of necessity, after the per injection, regenerate with suitable SPR regeneration buffer in the TNFR1 surface, to remove bonded anti-TNFR1 dAb.Adopt this method, measure the Cmin that produces the required anti-TNFR1dAb of about 100RU signal.
3) the chimeric pre-equilibration of anti-TNFR1 dAb/
In case determined the optium concentration of anti-TNFR1 dAb, just can set up the chimeric TNFR1 mixture of anti-TNFR1 dAb/.Set up mixture, make that the final concentration of anti-TNFR1 dAb is identical with the previous optium concentration of measuring.Reaction is carried out in 100 μ l volumes (containing the spissated anti-TNFR1 dAb of 50 microlitre 2x, 40 microlitre Biacore buffer and 10 microlitre purification chimeric proteins) usually.The common concentration of final mixture is about 10-100 μ M chimeric protein and the anti-TNFR1 dAb of about 10-100nM.At room temperature allow mixture balance 30 minutes.
4) competitive Biacore experiment
After the balance, the chimeric TNFR1 mixture of each anti-TNFR1 dAb/ is sequential by TNFR1SPR surface and measure the response unit number.After each mixture injection, the surface to remove bonded anti-TNFR1 dAb, is injected a kind of mixture down through regeneration then.With the difference response that different chimera produced, can measure TNFR1 territory in conjunction with specific dAb.
These studies show that, TAR2m-21-23 is in conjunction with the territory 1 of mice TNFR1, TAR2h-205 is in conjunction with the territory 1 of people TNFR1, and TAR2h-10-27, TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 and TAR2h-185-25 are in conjunction with the territory 3 of people TNFR1.
Embodiment 18. screening techniques
These chimeric receptor protein matter of describing among the embodiment 16 can be used for measuring or screening, can be in conjunction with the material (for example antibody, dAb, chemical compound) in ad hoc structure territory in the TNFR1 to isolate.In brief, these methods have been described chimeric protein have been joined in the thick antibody preparations, then by ELISA or surface plasma resonance screening TNFR1 combination.In addition, they described the surface (for example elisa plate or SPR chip) bag quilt chimeric protein purposes and by measure they with this surface on combining of chimeric protein screen antibody.
1) soluble E LISA screening
This method can be used for from huge unknown specific antibody or antibody fragment storehouse, and sharp separation goes out can be in conjunction with the antibody or the antibody fragment (for example dAb) in TNFR1 ad hoc structure territory.
96 hole assay plate are spent the night at 4 ℃ of bags with the chimeric TNFR1 in 100 μ l/ holes.Wash 3 times with 0.1%TPBS (phosphate buffered saline(PBS) that contains the tween 20 of concentration 0.1%) in each hole.The 1%TPBS that adds 200 μ l/ holes seals this plate also at room temperature hatched this plate 1-2 hour.Each hole adds 50 μ l antibacterial supernatant or the periprep that contain soluble antibody or antibody fragment (it contains c-Myc epi-position labelling)/50 μ l 0.2%TPBS then with PBS washing 3 times.This plate was at room temperature hatched 1 hour.Then, (0.1% tween 20/PBS) wash 5 times adds 100 μ l, first anti-c-Myc mouse monoclonal antibody/0.1%TPBS again in each hole, this plate was at room temperature hatched 1 hour with 0.1%TPBS with this plate.Discard such first antibody solution, again this plate is washed 5 times with 0.1%TPBS.Add then from the prediluted anti-mice IgG of the 100 μ l of goat (Fc specificity) HRP conjugate (Sigma catalog number: A0168), this plate was at room temperature hatched 1 hour.Discard second antibody, this plate is washed 6 times with 0.1%TPBS, reuse PBS washing 2 times.In each hole, add 50 μ l TMB peroxide enzymatic solution, this plate was at room temperature placed 2-60 minute.Add 50 μ l 1M hydrochloric acid, cessation reaction.Within 30 minutes after adding acid, on 96 orifice plate readers, read this plate in the OD at 450nm place value.Exist among rough antibacterial supernatant or the peripreps, can will provide stronger ELISA signal in conjunction with these antibody in the TNFR1 territory that exists in the chimeric protein, those can not bondedly then can not provide signal.
2) competitive ELISA screening
This method can be used for that rapid screening is a series of can be in conjunction with different thick antibody or the antibody fragment prepared products of TNFR1, to measure their domain binding specificity.
96 hole assay plate are spent the night at 4 ℃ of bags with the Mus or the people TNFR1 (people or mice) in 100 μ l/ holes.Wash 3 times with 0.1%TPBS (phosphate buffered saline(PBS) that contains the tween 20 of concentration 0.1%) in each hole.(1% tween 20/PBS) seal this plate was at room temperature hatched this plate 1-2 hour then to add the 1%TPBS in 200 μ l/ holes.Wash 3 times with PBS in each hole.Simultaneously, the pre-chimeric TNFR1 albumen/solution pre-equilibration of optimizing concentration of antibacterial supernatant or peripreps.This rough bacterial preparation/chimeric protein mixture of 50 μ l that will contain soluble antibody or antibody fragment joins in the elisa plate.This plate was at room temperature hatched 1 hour.Then, (0.1% tween 20/PBS) wash 5 times adds the 0.1%TPBS that 100 μ l first detect antibody (or protein A-HRP or albumen L HRP) again in each hole, this plate was at room temperature hatched 1 hour with 0.1%TPBS with this plate.Discard such first antibody solution, again this plate is washed 5 times with 0.1%TPBS.If necessary, add the prediluted second antibody of 100 μ l-HRP conjugate, this plate was at room temperature hatched 1 hour from goat.Discard second antibody then, this plate is washed 6 times with 0.1%TPBS, reuse PBS washing 2 times.In each hole, add 50 μ l TMB peroxide enzymatic solution, this plate was at room temperature placed 2-60 minute.Add 50 μ l1M hydrochloric acid, cessation reaction.Within 30 minutes after adding acid, on 96 orifice plate readers, read this plate in the OD at 450nm place value.The ELISA signal reduces, and shows that the chimeric TNFR1 territory rather than the complete TNFR1 that wrap quilt on antibody and the plate combine, and therefore, this antibody capable is in conjunction with a domain in the chimeric protein.
3) to the competitive ELISA screening of the antibody that combines TNFR1 with reference antibody or antibody fragment competitiveness and antibody fragment
This method can be used for that rapid screening is a series of can be in conjunction with different thick antibody or the antibody fragment prepared products of TNFR1, and described antibody or antibody fragment combine TNFR1 or in conjunction with the desired structure territory (for example the territory 1) of TNFR1 with reference antibody or antibody fragment (for example TAR2m-21-23) competitiveness.This method adopts the reference antibody that contains different detectable labels (epi-position labelling) or antibody fragment and test antibody or antibody fragment (for example remain screen antibody colony).
96 hole assay plate are spent the night at 4 ℃ of bags with the Mus or the people TNFR1 in 100 μ l/ holes.Wash 3 times with 0.1%TPBS (phosphate buffered saline(PBS) that contains the tween 20 of concentration 0.1%) in each hole.(1% tween 20/PBS) seal this plate was at room temperature hatched this plate 1-2 hour to add the 1%TPBS in 200 μ l/ holes.Wash 3 times with PBS in each hole.Simultaneously, thick antibody preparations to be measured and the pre-reference antibody of optimizing concentration or antibody fragment (territory 1-binding antibody for example; TAR2m-21-23)/the solution mixing.As described, importantly this antibody does not comprise and the identical certification mark of antibody that remains to be screened the domain binding specificity.This rough antibody/reference antibody mixture of 50 μ l is joined in the elisa plate.This plate was at room temperature hatched 1 hour.Then, this plate with 0.1%TPBS washing 5 times, is added the 0.1%TPBS that 100 μ l first detect antibody (it is in conjunction with the labelling that exists only in the antibody colony to be screened) in each hole, this plate was at room temperature hatched 1 hour.Discard such first antibody solution, again this plate is washed 5 times with 0.1%TPBS.Then, adding can be discerned the prediluted second antibody of the 100 μ l-HRP conjugate of the first detection antibody, and this plate was at room temperature hatched 1 hour.Discard second antibody solution, this plate is washed 6 times with 0.1%TPBS, reuse PBS washing 2 times.In each hole, add 50 μ l TMB peroxide enzymatic solution, this plate was at room temperature placed 2-60 minute.Add 50 μ l 1M hydrochloric acid, cessation reaction.Within 30 minutes after adding acid, on 96 orifice plate readers, read this plate in the OD at 450nm place value.Should carry out an independent and parallel ELISA abreast, adopt this method, but not add reference antibody or antibody fragment.With do not have competitive reference antibody or the resulting ELISA signal of antibody fragment to compare with the same antibody prepared product, the ELISA signal reduces when reference antibody or antibody fragment exist, show that specific antibodies or antibody fragment combine the TNFR1 territory with reference antibody or antibody fragment competitiveness, and the same as with reference antibody or antibody fragment same TNFR1 territory.
4) SPR screening
Can easily revise above-mentioned ELISA method so that adopt surface plasma resonance, for example use BIACORE 3000 SPR instruments (Biacore International AB, Uppsala, Sweden).Usually, chimeric protein can be fixed on the SPR chip, and perhaps chimeric protein can be with the rough antibacterial supernatant balance that contains anti-TNFR1 antibody or antibody fragment, and the gained mixture flows through the SPR chip with total length people TNFR1 or Mus TNFR1 bag quilt.
Embodiment 19.TAR2m21-23 dimer is the TNFR1 antagonist of height affinity
The method that adopts embodiment 8 to describe is by preparing the TAR2m21-23 form that contains the cys residue at c-terminus, preparation TAR2m21-23 dimer.Protein (TAR2m21-23CYS) is expressed in Pichia sp., and carries out purification with the Streamline protein A.Irreducibility SDS-PAGE the analysis showed that the protein of～40-50% exists in solution with dimeric forms.Dimer is further purified with gel filtration chromatography.
2mg protein concentrates to about 250 μ l and goes up sample to Superdex 75 HR solvent resistant columns (Amersham Bioscience), and this post before carried out balance with PBS.The flow velocity of this post operation is 0.5ml/ minute, collects the 0.5ml flow point.Protein at 280nm place monitoring eluting from this post contains dimeric flow point and is confirmed through irreducibility SDS-PAGE.Merge the flow point that contains dimer but do not contain monomer TAR2m-21-23 CYS.Concentrate the flow point that merges, in the cytotoxic assay (embodiment 6) of L929 TNF cell, measure the effectiveness of dimer dAb.
In the L929 cytotoxic assay, dimeric biological efficacy of TAR2m21-23 and monomer TAR2m-21-23 are compared.In this is measured, TAR2m21-23 monomer and TAR2m21-23 dimer have been measured to Cytotoxic inhibition by the inductive mice L929 of TNF cell, its result be expressed as in mensuration suppress 50% Cytotoxic dAb monomer or dimeric concentration (in and dosage 50, ND 50).
In mensuration, the ND of monomer dAb 50Be about 600pM.In mensuration, the ND of dimer dAb (TAR2m21-23 dimer) 50Low approximately 10 times of (ND 50About 60-70pM).These results show, compare with the dAb monomer, and the affinity of TAR2m21-23 dimer pair cell surface TNFR1 has and significantly improves.The result shows, the TAR2m21-23 dimer is simultaneously in conjunction with two on the cell surface independent TNFR1 molecules, causes the improvement that TNFR1 is suppressed at polymeric gained affinity effect.
Measure dimeric forms (TAR2m21-23 dimer) then, whether show any sign of TNFR1 excitement to observe it, the exciting meaning of described TNFR1 causes promptly that TNFR1 is crosslinked and promotes the ability of intracellular signal transduction and cell death.Adopt to improve the cytotoxic assay of L929 cell, can reach this point, wherein do not add TNF-α, but detect anti-TNFR1 antibody or dAb form, to observe their whether exciting TNFR1 and inducing cell deaths.In mensuration, measured anti-TNFR1 antibody A F-425-PB (R﹠amp as known TNFR1 agonist; D system), and as the TNFR1, the TAR2m-21-23 that have reported and the anti-TNFR1 antibody MAB430 (R﹠amp of the dimeric antagonist of TAR2m21-23; D system).
In mensuration, antibody A F-425-PB activates TNFR1 and inducing cytotoxic, its ND 50Be about 100pM.In mensuration, even the antagonist antibodies MAB430 that has reported also causes the crosslinked and cell killing of receptor, its ND 50Be about 10nM.By contrast, in mensuration, the TAR2m-21-23 dimer does not but cause any cell death, even when existing with high concentration (＞1 μ M).These results show that the TAR2m21-23 dimer is not the TNFR1 agonist.
The result shows, has high affinity in conjunction with the TNFR1 of dimer, trimer or other polymer pair cell surface expression of the dAb of TNFR1, and is effective TNFR1 antagonist.In addition, this result of study shows, dAb polymer (for example TAR2m21-23 dimer) in conjunction with the territory 1 of TNFR1, can be in conjunction with two TNFR1 molecules (can be bonded) as the antibody institute as agonist in measuring, and with the domain of TNFR1 or combining of epi-position target (territory 1), stop combining closely of receptor chain, and this initial just TNFR1 signal transduction is necessary.This characteristic is unique to bivalent molecule, the TNFR1 of the crosslinked cell surface of described molecular energy, but do not cause TNFR1 signal transduction and cell death.
Embodiment 20. separates in conjunction with the dAb's of people TNFR1 and mice TNFR1
The dAb of known array is at expression in escherichia coli, and with protein A laminar flow (streamline) resin purification.Be eluted in the Tris-glycine, dAb flows through the SPR chip then, has been fixed with biotinylation people TNFR1 (flow cell 2) and biotinylation Mus TNFR1 (flow cell 4) on it.(this SPR chip wraps quilt with the people TNFR1 and the Mus TNFR1 of equal densities). Flow cell 1 and 3 is blank, and is former in the surface as nonreactive, is used for detecting and deducting non-specific binding.
DAb flows through successive 4 flow cells (promptly being followed successively by flow chamber 1,2,3 and 4), measures the response difference between flow chamber 2 and the flow chamber 1, also measures the response difference between flow chamber 4 and the flow chamber 3.The former measures is and the combining of people TNFR1, and the latter is and the combining of Mus TNPR1.Record is used for the specificity binding curve in conjunction with people TNFR1 and Mus TNFR1, notices that in curve TNFR1 compares with Mus, and the association rate of people TNFR1 is faster.Dissociation rate is also roughly similar.Estimate cross reaction by the resulting peak response unit of each binding events (RU) number.In this embodiment, people's biotinylation TNFR1 surface comprises the TNFR1 of about 900RU on flow chamber 2, and comprises the Mus TNFR1 of about 1400RU at flow chamber 4.In contrast, concentration is 2 micromole TAR2h-154-7 (a kind of human specific dAb) in conjunction with the peak response on people surface is 385RU, and the response on the mice surface only is 4.5RU.The response that 2 micromole TAR2h-205 obtain on the people surface is 435RU, and is 266RU on the mice surface.
The list of references of quoting in all publications that the application's book is mentioned and the described publication all is attached to herein by reference.Only it should be apparent to those skilled in the art that otherwise depart from scope and spirit of the present invention, can carry out various modifications and change the method and system that the present invention describes.Although described the present invention, should be appreciated that claimed invention is not limited to these specific embodiments with concrete preferred embodiment.Certainly, to being used to implement the various modifications of described mode of the present invention, be conspicuous for molecular biology or various equivalent modifications, such modification falls within the appended claims scope.
Appendix 1: the polypeptide of half-life in the extension body
α-1 glycoprotein (orosomucoid O ALPHA1-Acid glycoprotein AGP (Orosomucoid)) (AAG)
α-1 chymotrypsin inhibitor (ACT)
α-1 antitrypsin (AAT)
α-1 microglobulin (albumen HC) (AIM)
α-2 macroglobulin (A2M)
Antithrombin III (AT III)
APoA-I (Apo A-1)
Apolipoprotein B (Apo B)
Beta-2-microglobulin (B2M)
Ceruloplasmin (Cp)
Complement component (C3)
Complement component (C4)
C1 esterase inhibitor (C1 INH)
C-reactive protein (CRP)
Cysteine proteinase inhibitor C (Cys C)
Ferritin (FER)
Fibrinogen (FIB)
Fibronectin (FN)
Haptoglobin (Hp)
Hemopexin (HPX)
Immunoglobulin A (IgA)
Immunoglobulin D (IgD)
IgE (IgE)
Immunoglobulin G (IgG)
IgM (IgM)
Light chain immunoglobulin (κ/λ)
Lipoprotein (a) [Lp (a)]
Mannose-binding protein (MBP)
Myoglobin (Myo)
Plasminogen (PSM)
Prealbumin (transthyretin (Transthyretin)) (PAL)
Retinol binding protein (RBP)
Rheumatoid factor (RF)
Serum amyloid A protein (SAA)
Solubility TfR (sTfR)
Transferrins (Tf)
Appendix 2
Pairing The treatment coherent reference
TNF α/TGF-β ● TGF-b and TNF can significantly increase arthritis when being expelled in the ankle joint of collagen-induced arthritis model.Not effect in the mice of non-collagen stimulation.
TNF α/IL-1 ● TNF and IL-1 play synergism in uveitis pathology.● (hypoglycemia plays synergism in NO) at malaria pathology for TNF and IL-1.● TNF and IL-1 be co-induction polymorphonuclear (PMN) cell migration in inflammation.
● IL-1 and TNF co-induction PMN soak into to peritoneum.● IL-1 and TNF be co-induction endotheliocyte secretion IL-1 in inflammation.● IL-1 or TNF induce some cell to soak into to the knee joint synovial membrane separately.IL-1 induces PMN, TNF mononuclear cell.They induce more serious infiltration together, because PMN increases.● the myocardium mortifier (being present in the sepsis) that circulates is low-level IL-1 and TNF synergism.
TNF α/IL-2 ● the most relevant with the collaborative activation of killer T cell.
TNF α/IL-3 ● interleukin-13 and the tumor necrosis factor synergism in stimulating acute myelogenous leukemia blastocyte clonal growth is the result that tumor necrosis factor is induced second hematopoietic cytokine.● Cancer Res.1992 April 15; 52 (8): 2197-201.
TNF α/IL-4 ● IL-4 and TNF co-induction VCAM are in the expression of endotheliocyte.Mean and in asthma, have effect.Relevant with synovial membrane in RA is identical.● the IL-6 in TNF and the IL-4 co-induction keratinocyte expresses.● can realize that in the fibroblast sample synovial cell (synoviocyte) who cultivates the VCAM-1 level continues to rise, promptly by TNF α with or IL-4 or IL-13 couplings, by increasing mRNA stability.Am.J.Pathol., in April, 1999; 154 (4): 1149-58.
TNF α/IL-5 ● in adult and child's thereof bronchus overreaction, the relation between system of tumor necrosis factor and serum interleukin-4, interleukin-5, interleukin-8, eosinophile cationic protein and the IgE level.The Allergy Asthma Proc. 3-4 month in 2003; 24 (2): 111-8.
TNF α/IL-6 ● TNF and IL-6 are the potent somatomedin of OH-2 (a kind of new human myeloma cell line).Eur.J.Haematol., in July, 1994; 53 (1): 31-7.
TNF α/IL-8 ● TNF and IL-5 and PMN work in coordination with activated blood platelet.Relate to adult respiratory distress syndrome.● referring to IL-5/TNF (asthma).Synergism between interleukin-8 and tumor necrosis factor-alpha is used for the platelet activation that neutrophil cell mediates.The Eur Cytokine Netw.1994 9-10 month; 5 (5): 455-60.(adult respiratory distress syndrome (ARDS)).
TNFα/IL-9
TNF α/IL-10 ● IL-10 induces and expresses with the HW of the chronically infected T cell of TNF co-induction.
TNF α/IL-11 ● cytokine co-induction osteoclast differentiation: support by infinite multiplication or normal braincap cell.Am.J.Physiol.Cell Physiol., in JIUYUE, 2002; 283 (3): C679-87.(bone loss).
TNF α/IL-12
TNF α/IL-13 ● can realize that in the fibroblast sample synovial cell who cultivates the VCAM-1 level continues to rise, promptly by TNF α with or IL-4 or IL-13 coupling, by increasing mRNA stability.Am.J.Pathol., in April, 1999; 154 (4): 1149-58.● the generation of eotaxin in interleukin-13 and the tumor necrosis factor-alpha co-induction people nose fibroblast.Clin Exp Allergy.2000 March; 30 (3): 348-55.● the generation of eotaxin in interleukin-13 and the tumor necrosis factor-alpha co-induction people nose fibroblast.Clin Exp Allergy.2000 March; 30 (3): 348-55 (alterative inflammation).● serum TNF β and the IL-13 meaning in the reaction of child's management of Idiopathic Nephrotic Syndrome in Children.Cytokine, on February 7th, 2003; 21 (3): 155-9.
TNF α/IL-14 ● mild asthma patient sucks the effect of tumor necrosis factor.The Thorax.2002 JIUYUE; 57 (9): 774-8.
TNF α/IL-15 ● mild asthma patient sucks the effect of tumor necrosis factor.The Thorax.2002 JIUYUE; 57 (9): 774-8.
TNF α/IL-16 ● tumor necrosis factor-alpha is induced synthetic Interleukin-16 (IL-16) in tracheal epithelial cell: to the sensitization of 5-hydroxytryptamine stimulation.Am.J.Respir.Cell Mol.Biol., in March, 2003; 28 (3): 354-62.(bronchitis) ● in patient with rheumatoid arthritis, the relation of circulation interleukin-11 6 and proinflammatory cytokine.Rheumatology (Oxford) .2001 April; 40 (4): 474-5.There is not available summary.● interleukin-11 6 is raised in Crohn disease (Crohn ' s disease), and participates in mice TNBS colitis.Gastroenterology, in October, 2000; 119 (4): 972-82.
TNF α/IL-17 ● to the inhibition of interleukin-17, stop with B. burgdorferi (Borrelia
Burgdorferi) the arthritic development of the vaccinated mice of Gong Jiing.Infect Immun., in June, 2003; 71 (6): 3437-42.● interleukin-17 and tumor necrosis factor are at external co-induction cartilage destruction.Ann. Rheum.Dis., in October, 2002; 61 (10): 870-6.● the effect of GM-CSF in accumulating by IL-17 and the inductive trachea neutrophil cell of TNF α.Eur Respir is year March J.2003; 21 (3): 387-93.(bronchitis).● summary: in people's osteoarthritis knee joint meniscus (osteoarthritic knee menisci) explant, the collaborative generation of raising nitric oxide and PGE2 of il-1, tumor necrosis factor and interleukin-17.Arthritis Rheum., calendar year 2001 JIUYUE; 44 (9): 2078-83.
TNF α/IL-18 ● in the knee synovial tissue of patient with rheumatoid arthritis, the relation that the expression of il-1 8 and il-1 β and TNF ﹠ alpha levels increase.Arthritis Rheum.2003 February; 48 (2): 339-47.● summary: il-1 8 and tumor necrosis factor-alpha level in the type 2 diabetes mellitus patients serum rises: with the relation of diabetic nephropathy.Metabolism.2003 May; 52 (5): 605-8.
TNF α/IL-19 ● summary: IL-19 induces the generation of IL-6 and TNF α, and causes apoptosis by TNF α.J.Immunol., on October 15th, 2002; 169 (8): 4288-97.
TNF α/IL-20 ● summary: cytokine: IL-20, a kind of novel effectors in the scytitis.Curr Biol.2001 July 10; 11 (13): R531-4.
TNF α/complement ● inflammation and solidifying: relate to sepsis patient.Clin.Infect.Dis., on May 15th, 2003; 36 (10): 1259-65.Epub on May 08th, 2003.Summary.
TNF α/TNF γ ● brain MHC induces.● the coordination in antiviral response/IFN-is beta induced.● neutrophil activation/respiratory burst.● activated endothelial cell.● when the patient treats as antiviral therapy with TNF/IFN-γ, note toxicity.● the CXXXC chemotactic factor that people's spider cell is expressed.● the relevant inflammatory reaction of many papers, i.e. LPS, and macrophage activation.● anti-TNF and anti-JFN-γ coordinating protection mice exempt from the lethal endotoxemia.
TGF-β/IL-1 ● the synthetic prostaglandin of osteoblast (Prostaglndin) ● the IL-6 (inflammatory model) that enterocyte produces ● ● in retina, produce IL-6 and IL-8 at lung fibroblast moderate stimulation IL-11 and IL-6 (inflammatory model).
TGF-β/IL-6 ● chrondrocarcinoma (Chondrocarcoma) propagation.
IL-1/IL-2 ● B cell activation ● LAK cell activation ● T cell activation ● IL-1 and IL-2 synergism produce by TNF α and β (lymphotoxin) killer cell mediation, lymphokineactivation.Cytokine, in November, 1992; 4 (6): 479-87.
IL-1/IL-3
IL-1/IL-4 ● B cell activation ● in activated endothelial cell, IL-4 induce IL-1 to express.
IL-1/IL-5
IL-1/TL-6 ● B cell activation ● T cell activation, (alternative accessory cell) ● IL-1 induces IL-6 to express ● the expression of C3 and serum amyloid protein, (acute phase response) ● HIV expresses ● chondrigen decomposes.
IL-1/IL-7 ● IL-7 is the essential condition of the inductive thymocyte proliferation of IL-1.IL-7 and IL-1 participate in the synergism of granulocyte-macrophage colony stimutaing factor or tumor necrosis factor.J.Immunol., on January 1st, 1992; 148 (1): 99-105.
IL-1/IL-8
IL-1/IL-10
IL-1/IL-11 ● cytokine co-induction osteoclast differentiation: support by infinite multiplication or normal braincap cell.Am.J.Physiol.Cell Physiol., in JIUYUE, 2002; 283 (3): C679-87.(bone loss).
IL-1/IL-16 ● in patient with rheumatoid arthritis, the relation of circulation interleukin-11 6 and proinflammatory cytokine.Rheumatology (Oxford) .2001 April; 40 (4): 474-5.
There is not available summary.
IL-1/IL-17 ● to the inhibition of interleukin-17, stop the arthritic development of the vaccinated mice of attacking with B. burgdorferi (Borrelia burgdorferi).Infect Immun.2003 June; 71 (6): 3437-42.● in osteoarthritis, interleukin-17 is to the effect of human cartilage degraded and synovial membrane inflammation.Osteoarthritis Cartilage.2002 October; 10 (10): 799-807.● summary: in people's osteoarthritis knee joint meniscus explant, the collaborative generation of raising nitric oxide and PGE2 of il-1, tumor necrosis factor and interleukin-17.Arthritis Rheum, calendar year 2001 JIUYUE; 44 (9): 2078-83.
IL-1/IL-18 ● in the knee synovial tissue of patient with rheumatoid arthritis, the expression of il-1 8 and il-1 β and TNF ﹠ alpha levels increase concerns .Arthritis Rheum.2003 February; 48 (2): 339-47.
IL-1/IFN-γ
IL-2/IL-3 ● T cell proliferation ● B cell proliferation
IL-2/IL-4 ● B cell proliferation ● T cell proliferation ● (selective induction CD8 and NK lymphocyte activation) IL-2R beta-agonists P1-30 and IL-2, IL-4, IL-9 and IL-15 synergism: biology and molecular effect.J.Immunol., on October 15th, 2000: 165 (8): 4312-8.
IL-2/IL-5 ● B cell proliferation/Ig secretion ● IL-5 induces the IL-2 receptor on the B cell
IL-2/IL-6 ● the growth of cytotoxic T cell
IL-2/IL-7
IL-2/IL-9 ● referring to IL-211L-4 (NK cell)
IL-2/IL-10 ● the B cell activation
IL-2/IL-12 ● in fresh NK cells of human beings, the cytotoxicity of IL-12 and IL-2 co-induction lymphokineactivation and perforin and granzyme gene expression.CellImmunol.1995 October 1; 165 (1): 33-43.(T cell activation)
IL-2/IL-15 ● referring to IL-2/IL-4 (NK cell) ● (T cell activation and propagation) IL-15 and IL-2: a kind of T cell material of life and death in vivo that is used for.NatMed.2001 January; 7 (1): 114-8.
IL-2/IL-16 ● IL-16 and IL-2 are to the collaborative activation of CD4+T cell.J.Immunol., on March 1st, 1998; 160 (5): 2115-20.
IL-2/IL-17 ● the evidence that early stage participant of interleukin-17 and experimental kidney allograft repel.J.Pathol., in July, 2002; 197 (3): 322-32.
IL-2/IL-18 ● in mice interstitial pneumonia pathogenesis, interleukin 18 (IL-18) and IL-2 co-induction mice lethal injury of lung: the latent effect of a kind of pair cell factor, chemotactic factor and natural killer cell.Blood.2002 February 15; 99 (4): 1289-98.
IL-2/TGF-β ● the destiny of control CD4 effector: collaborative apoptosis and the accelerating effect thing of stoping of transforminggrowthfactor-and interleukin-22 expands.J.Exp.Med., nineteen ninety-five JIUYUE 1 day; 182 (3): 699-709.
IL-2/IFN-γ ● the Ig of B emiocytosis ● the IL-2 inducing T cell is expressed IFN-γ
IL-2/IFN-α/β ● do not have
IL-3/IL-4 ● synergism and mastocyte growth ● IL-4 and GM-CSF or LL-3 express the co-induction effect of CD23 to the person monocytic cell: the adjusting effect of IFN-α and IFN-γ.Cytokine.1994 July; 6 (4): 407-13.
IL-3/IL-5
IL-3/IL-6
IL-3/IFN-γ ● total poly IgA acceptor levels in the collaborative raising of IL-4 and the IFN-γ people intestinal epithelial cell.The effect of protein tyrosine kinase.J.Immunol., on June 15th, 1996; 156 (12): 4807-14.
IL-3/GM-CSF ● cytokine is regulated people eosinophilic granulocyte IL-3, IL-5 and GM-CSF receptor alpha chain expression difference: IL-3, IL-5 and GM-CSF downward modulation IL-5 receptor alpha expression (lacking the IL-5 reactivity), but raise IL-3 receptor alpha expression.J.Immunol., on June 1st, 2003; 170 (11): 5359-66.(alterative inflammation)
IL-4/IL-2 ● the IFN-γ that the IL-4 concertedness increases IL-2 and the inductive Mus NK of IL-12 cell expresses.Blood.2003 March 13 [Epub ahead ofprint]
IL-4/IL-5 ● increase mastocyte histamine etc.Response IgE and secreting ● the Th2 like cell factor is replied the participation bullous pemphigoid.IL-4 and the IL-5 effect in the pathogenesis of disease.Int.J.Immunopathol.Pharmacol.，
The 5-8 month in 1999; 12 (2): 55-61.
IL-4/IL-6
IL-4/1L-10
IL-4/1L-11 ● the cooperative interaction between interleukin-11 and interleukin-4, the propagation of the original hemopoietic progenitor cell of support mice.Blood.1991 JIUYUE 15 days; 78 (6): 1448-51.
IL-4/IL-12 ● IL-4 and IL-18 produce the synergism of IL-12 dependency IFN γ to dendritic cell.J.Immunol., on June 1st, 2000; 164 (1): 64-71.(increasing the Th1/Th2 differentiation) ● the IFN-γ that the IL-4 concertedness increases IL-2 and the inductive Mus NK of IL-12 cell expresses.Blood.2003 March 13 [Epub ahead of print]
IL-4/IL-13 ● summary: interleukin-4 is connected collection of illustrative plates with the interleukin-13 signal transduction.Science on June 6th, 2003; 300 (5625): 1527-8.(irritated, asthma) ● in mice allergic asthma model, inhibition to the IL-4/IL-13 receptor system, prevent allergic sensitization, and do not influence the allergy of having set up.J.Allergy Clin. Immunol., in June, 2003; 111 (6): 1361-1369.
IL-4/1L-16 ● (asthma) interleukin (IL)-4/IL-9 and external source IL-16 induce BEAS-2B cell (a kind of bronchial epithelial cell system) to produce IL-16.Cell Immunol.2001 February 1; 207 (2): 75-80.
IL-4/1L-17 ● the collaborative people's colon myofibroblast secretion IL-6 that stimulates of interleukin (1L)-4 and IL-17.Int.J.Mol.Med., in November, 2002; 10 (5): 631-4.(enteritis)
IL-4/IL-24 ● IL-24 is expressed by rat macrophage and human macrophage.Immunobiology. in July, 2002; 205 (3): 321-34.
IL-4/IL-25 ● summary: in pulmonary, new IL-17 family member promotes Th1 or Th2 reaction: function in the body of new cytokine IL-25.J.Immunol., on July 1st, 2002; 169 (1): 443-53.(alterative inflammation) ● summary: after the activation of Fc ε RI mediation, mastocyte produces interleukin-2 5.Blood.2003 May 1; 101 (9): 3594-6.Epub June 02 (alterative inflammation) in 2003
IL-4/IFN-γ ● summary: interleukin 4 inducing endothelial cells produce interleukin 6: collaborative with interferon-.Eur.J.Immunol., in January, 1991; 21 (1): 97-101.
IL-4/SCF ● stem cell factor and IL-4 mediator's intestinal mastocyte.Immunol Rev.2001 February; 179:57～60.Summary
IL-5/IL-3 ● cytokine is regulated people eosinophilic granulocyte IL-3, IL-5 and GM-CSF receptor alpha chain expression difference: IL-3, IL-5 and GM-CSF downward modulation IL-5 receptor alpha expression (lacking the IL-5 reactivity), but raise IL-3 receptor alpha expression.J.Immunol., on June 1st, 2003; 170 (11): 5359-66.(alterative inflammation is referring to summary)
IL-5/IL-6
IL-5/IL-13 ● dexamethasone is to mice allergia bronchitis and the over-reactive inhibition of trachea: eosinophilic granulocyte, IL-5, the effect of eotaxin and IL-13.J.Allergy Clin.Immunol., in May, 2003; 111 (5): 1049-61.
IL-5/1L-17 ● in mice allergic asthma model, after allergen sucked, interleukin-17 impelled granulocyte to flow to trachea.Am.J.Respir.Cell Mol.Biol., in June, 2003; 28 (1): 42-50.
IL-5/IL-25 ● summary: in pulmonary, new IL-17 family member promotes Th1 or Th2 reaction: function in the body of new cytokine IL-25.J.Immunol., on July 1st, 2002; 169 (1): 443-53.(alterative inflammation) ● summary: after the activation of Fc ε RI mediation, mastocyte produces interleukin-2 5.Blood.2003 May 1; 101 (9): 3594-6.Epub June 02 (alterative inflammation) in 2003
IL-5/IFN-γ
IL-5/GM-CSF ● cytokine is regulated people eosinophilic granulocyte IL-3, IL-5 and GM-CSF receptor alpha chain expression difference: IL-3, IL-5 and GM-CSF downward modulation IL-5 receptor alpha expression (lacking the IL-5 reactivity), but raise IL-3 receptor alpha expression.J.Immunol., on June 1st, 2003; 170 (11): 5359-66.(alterative inflammation)
IL-6/IL-10
IL-6/IL-11
IL-6/IL-16 ● interleukin-11 6 stimulates the person monocytic cell to express and produces proinflammatory cytokine.Immunology.2000 May; 100 (1): 63-9.
IL-6/IL-17 ● interleukin (IL)-17 stimulates the trachea mucin gene to express by IL-6 paracrine/autocrine loop.J.Biol.Chem., on May 9th, 2003; 278 (19): 17036-43. Epub on March 06th, 2003 (bronchitis, asthma)
IL-6/IL-19 ● summary: IL-19 induces the generation of IL-6 and TNF α, and causes apoptosis by TNF α.J.Immunol., on October 15th, 2002; 169 (8): 4288-97.
IL-6/FN-γ
IL-7/IL-2 ● interleukin 7 worsens graft versus host disease.Blood.2002 October 1; 100 (7): 2642-9.
IL-7/IL-12 ● IL-7 and IL-12 are to the activatory synergism of human T-cell.J.Immunol., on May 15th, 1995; 154 (10): 5093-102.
IL-7/IL-15 ● interleukin-7 and interleukin-15 are regulated bcl-2 and c-myb gene expression in the cutaneous T cell lymphoma cell.Blood.2001 November 1; 98 (9): 2778-83.(somatomedin)
IL-8/IL-11 ● in polycythemia vera (polycythaemia vera), the unusual generation of interleukin (IL)-11 and IL-8.Cytokine.2002 November 21; 20 (4): 178-83.
IL-8/IL-17 ● the effect of IL-17 in destruction of joint.Drug News Perspect.2002 January; 15 (1): 17-23.(arthritis) ● summary: interleukin-17 stimulates people's trachea epithelial cell to express interleukin-8, the relevant oncogene α of growth and granulocyte-colony stimulating factor.Am.J.Respir.Cell Mol.Biol., in June, 2002; 26 (6): 748-53.(bronchitis)
IL-8/GSF ● interleukin-8: a kind of autocrine/paracrine somatomedin, the synergism that is used for human hemopoietic progenitor cell and colony-stimulating factor-1 is to promote mononuclear cell-macrophage growth and differentiation.Exp Hematol.1999 January; 27 (1): 28-36.
IL-8/VGEP ● in constitutional and recurrent glioblastoma, intracavity VEGF, bFGF, IL-8, IL-12 level.J.Neurooncol., in May, 2003; 62 (3): 297-303.
IL-9/IL-4 ● with anti-interleukin-9 Antybody therapy, in mouse asthmatic model, suppress bronchitis and overreaction.Am.J.Respir.Crit.Care Med., on August 1st, 2002; 166 (3): 409-16.
IL-9/IL-5 ● the overexpression IL-9 of pulmonary, induce the Th2 cytokine-expressing, cause immunopathogenesis.J.Clin.Invest., in January, 2002; 109 (1): 29-39.● Th2 cytokine and asthma.Interleukin-9 is as the treatment of asthma target.Respir Res.2001; 2 (2): 15 days February calendar year 2001 of 80-4.Epub.Summary.● summary: interleukin-9 increases interleukin-5 expression of receptor, differentiation and people eosinophilic granulocyte survival.Blood.2005 JIUYUE 15 days; 96 (6): 2163-71 (asthma)
IL-9/IL-13 ● with anti-interleukin-9 Antybody therapy, in mouse asthmatic model, suppress bronchitis and overreaction.Am.J.Respir.Crit.Care Med., August 1 in 2002
Day; 166 (3): 409-16.● interleukin-13 causes the excessive generation of trachea overreaction and mucus to epithelial direct influence in asthma.Nat Med.2002 August; 8 (8): 885-9.
IL-9/I1-16 ● referring to IL-4/IL-16
IL-10/IL-2 ● the collaborative reaction that increases human B lymphocyte of IL-10 INTERLEUKIN-10 (IL-10) and interleukin-2 (IL-2) influencing each other in humoral immunoresponse(HI): IL-10 and IL-2, its mechanism is different from the rise that CD25 expresses.Cell Immunol.1994 JIUYUE; 157 (2): 478-88.
IL-10/IL-12
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IL-10/IFN-γ
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IL-11/TGF-β ● between the acute and chronic skin injury, the polarization expression in vivo of IL-11 and IL-17.J.Allergy Clin.Immunol., in April, 2003; 111 (4): 875-81.(allergic dermatitis)
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IL-12/IL-23 ● IL-23 rather than interleukin-2 are the key cytokines of brain autoimmune inflammation.Nature.2003 13:421 in February (6924): 744-8.● the unique effect of summary: IL-23 in promoting cellular immunization.J.Leukoc.Biol., in January, 2003; 73 (1): 49-56.Summary.
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IL-12/IFN-γ ● IL-12 induces B cell and T cellular expression IFN-γ, as immunostimulating ingredient.
IL-13/IL-5 ● referring to IL-5/IL-13
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Epub on November 3rd, 2002.
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IL-18/IL-12 ● interleukin 12 and interleukin 18 are to the collaborative propagation and the activation of natural killer cell.Cytokine.1999 November; 11 (11): 822-30. ● summary: the generation of IL-12 vitro inhibition immunoglobulin in the Mus chronic graft versus host disease: Eur Immunol.1998 June; 28 (6): 2017-24.
IL-18/IL-21 ● collaborative people NK of increasing of IL-21 and IL-15 or IL-18 and T cell produce IFN-γ.J.Immunol., on June 1st, 2003; 170 (11): 5464-9.
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IL-18/IFN-γ
Anti-TNF alpha/anti-CD4 ● the coordinating effect in the DBA/1 arthritis mice.
Appendix 3: oncology's combination
Target Disease Pairing
CD89 * Raise thing as cytotoxic cell All
CD19 B cell lymphoma HLA-DR CD5
HLA-DR B cell lymphoma CD89 CD19 CD5
CD38 Multiple myeloma CD138 CD56 HLA-DR
CD138 Multiple myeloma CD38 CD56 HLA-DR
CD138 Pulmonary carcinoma CD56 CEA
CD33 The acute myeloid lymphoma CD34 HLA-DR
CD56 Pulmonary carcinoma CD138 CEA
CEA The Pan cancer The MET receptor
VEGF The Pan cancer The MET receptor
Vegf receptor The Pan cancer The MET receptor
IL-13 Asthma/pneumonia IL-4 IL-5 ECF MDC TARC TNF α IL-9 EGFR CD40L IL-25 MCP-1 TGF β
IL-4 Asthma IL-13 IL-5 ECF MDC TARC TNF α IL-9 EGFR CD40L IL-25 MCP-1 TGF β
Eotaxin Asthma IL-5 eotaxin-2 eotaxin-3
EGFR Cancer HER2/neu HER3
HER4
HER2 Cancer HER3 HER4
TNFR1 The RA/ Crohn disease IL-1R IL-6R IL-18R
TNFα The RA/ Crohn disease IL-1α/β IL-6 IL-18 ICAM-1 IL-15 IL-17
IL-1R The RA/ Crohn disease IL-6R IL-18R
IL-18R The RA/ Crohn disease IL-6R
Appendix 4
Data gather
Target ?dAb Equilibrium dissociation constant Kd=K Dissociate/K Associate) K Dissociate The IC that part is measured 50 Based in the cell and the ND50 that measures
?TAR1 The TAR1 monomer 300nM～5pM (promptly 3 * 10 -7～ 5×10 -12), preferred 50nM～20pM 5×10 -1～ 1×10 -7 500nM～ 100pM 500nM～ 50pM
The TAR1 dimer As the TAR1 monomer As the TAR1 monomer As the TAR1 monomer As the TAR1 monomer
The TAR1 trimer As the TAR1 monomer As the TAR1 monomer As the TAR1 monomer As the TAR1 monomer
?TAR1-5
?TAR1-27
TAR1-5-19 monomers 30nM
TAR1-5-19 homodimers
Has (Gly 4Ser) 3Joint=20nm has (Gly 4Ser) 5Joint=2nm has (Gly 4Ser) 7Joint=10nm is with Fab form=1nM ＝30nM ＝3nM ＝15nM
TAR1-5-19 heterodimers Has (Gly 4Ser) nJoint TAR1-5-19 d2=2nM TAR1-5-19 d3=8nM TAR1-5-19 d4=2-5nM TAR1-5-19 d5=8nM are with Fab form TAR1-5-19CH d1CK=6nM TAR1-5-19CK d1CH=6nM TAR1-5-19CH d2CK=8nM TAR1-5-19CH d3CK=3nM ＝12nM ＝10nM ＝12nM
The TAR1-5 heterodimer Has (Gly 4Ser) nConnect
TAR1-5 d1=30nM TAR1-5d2=50nM TAR1-5 d3=300nM TAR1-5d4=3nM TAR1-5 d5=200nM TAR1-5d6=100nM is with Fab form TAR1-5CH d2CK=30nM TAR1-5CKd3CH=100nM
TAR1-5-19 homodimers 0.3nM 3-10nM (for example 3nM)
TAR2 The TAR2 monomer As the TAR1 monomer As the TAR1 monomer 500nM～100pM 500nM～50pM
TAR2-10
TAR2-5
Serum albumin Anti-SA monomer 1nM～500 μ M, preferred 100nM～10 μ M are with the bispecific form, the target affinity is the 1-100 of SA dAb affinity, 000 times, for example 100pM (target) and 10 μ M 1nM～500 μ M, preferred 100nM～10 μ M are with the bispecific form, the target affinity is 100,000 times of the 1-of SA dAb affinity.For example 100pM (target) and 10 μ M
The SA affinity. The SA affinity.
MSA-16 ?200nM
MSA-26 ?70nM

Claims (241)

1. one kind to Tumor Necrosis Factor Receptors 1 (TNFR1; P55) have specific dAb monomer part, according to the mensuration of surface plasma resonance, the dissociation constant (K that described part disintegrates down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1
2. the dAb monomer part of claim 1, wherein said dAb monomer suppresses tumor necrosis factor (TNF α) and the bonded IC of TNFR1 50Be 500nM～50pM.
3. claim 1 or 2 dAb monomer part are wherein in standard L929 raji cell assay Raji, in the described monomer and the ND of people TNFR1 50Be 500nM～50pM.
4. each dAb monomer part among the claim 1-3, wherein in standard cell lines is measured, the active ND of described dAb antagonism TNFR1 50≤ 100nM, in described standard cell lines is measured, when concentration≤10 μ M, activity≤5% of the exciting TNFR1 of described dAb.
5. each dAb monomer part among the claim 1-4, described part comprises general framework.
6. the dAb monomer part of claim 5, wherein said general framework comprises DPK9 V LFramework, or be selected from the V of DP47, DP45 and DP38 HFramework.
7. each dAb monomer part among the claim 1-7, described part comprises the binding site of general part.
8. the dAb monomer part of claim 7, wherein said general part is selected from protein A, albumen L and Protein G.
9. each dAb monomer part among the claim 1-8, wherein said dAb monomer part comprises the antibody variable region with one or more framework regions, the aminoacid sequence of described framework region and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical, and perhaps to comprise maximum 5 jointly be the different aminoacid of aminoacid sequence of the coded described corresponding framework region of antibody gene section with ethnic group to the aminoacid sequence of one or more described framework regions.
10. each dAb monomer part among the claim 1-8, wherein said dAb monomer part comprises antibody variable region, wherein the aminoacid sequence of FW1, FW2, FW3 and FW4 and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical, and perhaps to contain maximum 10 jointly be the different aminoacid of aminoacid sequence of the coded corresponding framework region of antibody gene section with described ethnic group to the aminoacid sequence of FW1, FW2, FW3 and FW4.
11. each dAb monomer part among the claim 1-8, wherein said dAb monomer part comprises antibody variable region, described antibody variable region comprises FW1, FW2 and FW3 district, and the aminoacid sequence of described FW1, FW2 and FW3 and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical.
12. each dAb monomer part among the claim 9-11, wherein said ethnic group is that the antibody gene section is selected from DP47, DP45, DP48 and DPK9.
13. each dAb monomer part among the claim 1-8, the V that described part comprises HThe district is not the camellid immune globulin variable region.
14. each dAb monomer part among the claim 1-8 is with people V HThe district compares, the V that described part comprises HQu Buhan has specific one or more aminoacid to the camellid immune globulin variable region.
15. each dAb monomer part among the claim 1-14, wherein said dAb monomer part comprises terminal Cys residue.
16. a bispecific part, described part comprise among at least one claim 1-15 each dAb monomer part.
17. the bispecific part of claim 16, wherein said bispecific part is a dimer.
18. one kind to Tumor Necrosis Factor Receptors 1 (TNFR1; P55) have specific dAb monomer part, wherein said dAb comprises the aminoacid sequence of TAR2-10 or has the sequence of at least 80% homology with it.
19. the dAb monomer part of claim 18, wherein said dAb comprise the aminoacid sequence of TAR2-10 or have the sequence of at least 90% homology with it.
20. one kind to Tumor Necrosis Factor Receptors 1 (TNFR1; P55) have specific dAb monomer part, wherein said dAb comprises the aminoacid sequence of TAR2-5 or has the sequence of at least 80% homology with it.
21. the dAb monomer part of claim 20, wherein said dAb comprise the aminoacid sequence of TAR2-5 or have the sequence of at least 90% homology with it.
22. an isolating nucleic acid, described nucleic acid coding is to Tumor Necrosis Factor Receptors 1 (TNFR1; P55) have specific dAb monomer part, according to the mensuration of surface plasma resonance, the dissociation constant (K that described part disintegrates down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1
23. the isolating nucleic acid of claim 22, wherein said dAb monomer part dAb comprises the aminoacid sequence of TAR2-10 or has the sequence of at least 80% homology with it.
24. the isolating nucleic acid of claim 22, wherein said dAb monomer part dAb comprises the aminoacid sequence of TAR2-5 or has the sequence of at least 80% homology with it.
25. an isolating nucleic acid, the bispecific part of described nucleic acid coding claim 16.
26. a recombinant nucleic acid, described nucleic acid coding is to Tumor Necrosis Factor Receptors 1 (TNFR1; P55) have specific dAb monomer part, according to the mensuration of surface plasma resonance, the dissociation constant (K that described part disintegrates down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1
27. the recombinant nucleic acid of claim 26, wherein said dAb monomer part dAb comprises the hydrogen base acid sequence of TAR2-10 or has the sequence of at least 80% homology with it.
28. the recombinant nucleic acid of claim 26, wherein said dAb monomer part dAb comprises the aminoacid sequence of TAR2-5 or has the sequence of at least 80% homology with it.
29. a recombinant nucleic acid, the bispecific part of described nucleic acid coding claim 16.
30. a carrier, described carrier comprise among the claim 26-28 each recombinant nucleic acid.
31. the carrier of claim 30, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
32. a host cell, described host cell comprises the carrier of claim 30.
33. a method for preparing dAb monomer part, this method are included in the host cell of keeping claim 32 under the condition that is suitable for described recombinant nucleic acid expression, produce dAb monomer part thus.
34. also comprising, the method for claim 33, this method separate described dAb monomer part.
35. a carrier, described carrier comprises the recombinant nucleic acid of claim 29.
36. the carrier of claim 35, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
37. a host cell, described host cell comprises the carrier of claim 36.
38. a method for preparing the bispecific part, this method are included in the host cell of keeping claim 37 under the condition that is suitable for described recombinant nucleic acid expression, produce the bispecific part thus.
39. also comprising, the method for claim 33, this method separate described bispecific part.
40. a method for the treatment of autoimmune disease, this method comprise mammal treatment effective dose that needs are arranged to Tumor Necrosis Factor Receptors 1 (TNFR1; P55) have specific dAb monomer part, according to the mensuration of surface plasma resonance, the dissociation constant (K that described part disintegrates down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1
41. a method for the treatment of autoimmune disease, this method comprise the bispecific part of the mammal treatment effective dose that needs are arranged, described bispecific part comprises Tumor Necrosis Factor Receptors 1 (TNFR1; P55) have specific dAb monomer part, according to the mensuration of surface plasma resonance, the dissociation constant (K that described part disintegrates down from people TNFR1 d) be 50nM～20pM, K DissociateSpeed constant is 5 * 10 -1s -1～1 * 10 -7s -1
42. the method for claim 35 or 36, wherein said autoimmune disease is selected from type i diabetes, asthma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (sle), Crohn disease and myasthenia gravis.
43. a compositions, described compositions comprise, and each dAb monomer part and pharmacology goes up acceptable carrier among the claim 1-15.
44. comprising the bispecific part and the pharmacology of claim 16, a compositions, described compositions go up acceptable carrier.
A 45. tumor necrosis factor 1 (TNFR1) antagonist, described antagonist be antagonism tumor necrosis factor 2 (TNFR2) not basically, but the chronic inflammatory disease that can be effective to treat the patient.
46. the antagonist of claim 45, wherein said antagonist are effective in being selected from following chronic inflammatory disease model: inflammatory bowel model and mice caused by smoking chronic obstructive pulmonary disease model that collagen-induced arthritis model, mouse arthritis Δ ARE model, mice inflammatory bowel Δ ARE model, the mice dextran sulfate sodium of mice brought out.
47. the antagonist of claim 45 or 46, wherein said antagonist is monovalent.
48. each antagonist among the claim 45-47, wherein said antagonist are antibody or its Fab.
49. the antagonist of claim 48, wherein said antagonist are the monovalent antigen binding fragments of antibody.
50. each antagonist among the claim 45-47, wherein said antagonist are the domain antibodies monomers.
51. each antagonist among the claim 45-50, the bonded K of wherein said antagonist and TNFR1 dBe 300nM～5pM.
52. an energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and can be effective to treat patient's chronic inflammatory disease.
53. the dAb monomer of claim 52, wherein said dAb are effective in being selected from following chronic inflammatory disease model: inflammatory bowel model and mice caused by smoking chronic obstructive pulmonary disease model that collagen-induced arthritis model, mouse arthritis Δ ARE model, mice inflammatory bowel Δ ARE model, the mice dextran sulfate sodium of mice brought out.
54. the dAb monomer of claim 52 or 53, wherein said dAb monomer be antagonism tumor necrosis factor 2 (TNFR2) not basically.
55. each dAb monomer among the claim 52-54, wherein in standard L929 raji cell assay Raji, when described dAb monomer concentration during up to 10 μ M, not exciting basically TNFR1.
56. each dAb monomer among the claim 52-55, wherein said dAb monomer suppresses tumor necrosis factor (TNF α) and the bonded IC of TNFR1 50Be 500nM～50pM.
57. each dAb monomer among the claim 52-56, wherein said dAb monomer is when expressing in escherichia coli (E.coli), and its secretory volume is at least about 0.5mg/L.
58. each dAb monomer among the claim 52-57, wherein said dAb monomer can reversibly be separated folding when being heated to temperature (Ts) and being cooled to temperature (Tc), and wherein Ts is higher than the monomeric melting temperature of dAb (Tm), and Tc is lower than the monomeric melting temperature of dAb.
59. the dAb monomer of claim 58, wherein Ts is about 80 ℃, and Tc is about room temperature.
60. each dAb monomer among the claim 52-59, wherein said TNFR1 is people TNFR1.
61. the dAb monomer of claim 56, wherein said TNF α is a human TNF alpha, and described TNFR1 is people TNFR1.
62. each dAb monomer among the claim 52-61, wherein said dAb monomer comprises people V HOr people V L
63. each dAb monomer among the claim 52-56, one or more aminoacid sequence among wherein said dAb monomer framework region FW1, FW2, FW3 and the FW4 and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical, and perhaps to contain maximum 5 jointly be the different aminoacid of aminoacid sequence of the coded described corresponding framework region of antibody gene section with ethnic group to the aminoacid sequence of one or more described framework regions.
64. each dAb monomer among the claim 52-62, the aminoacid sequence of the monomeric FW1 of wherein said dAb, FW2, FW3 and FW4 and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical, and perhaps to contain maximum 10 jointly be the different aminoacid of aminoacid sequence of the coded corresponding framework region of antibody gene section with described ethnic group to the aminoacid sequence of FW1, FW2, FW3 and FW4.
65. each dAb monomer among the claim 52-62, wherein said dAb monomer comprises FW1, FW2 and FW3 district, and the aminoacid sequence in described FW1, FW2 and FW3 district and ethnic group are that the aminoacid sequence of the coded corresponding framework region of antibody gene section is identical.
66. each dAb monomer among the claim 63-65, wherein said ethnic group are that the antibody gene section is selected from DP47, DP45, DP48 and DPK9.
67. each dAb monomer among the claim 52-61, wherein said dAb monomer does not contain the camellid immune globulin variable region.
68. each dAb monomer among the claim 52-61, wherein said dAb monomer do not contain the camellid kind be the coded immune globulin variable region of antibody gene section exclusive one or more framework aminoacid.
69. each dAb monomer among the claim 52-68, wherein said dAb monomer comprises the terminal cysteine residue.
70. each dAb monomer among the claim 52-69, wherein said dAb monomer also comprise poly-alkane glycol moiety.
71. the dAb monomer of claim 70, wherein said poly-alkane glycol moiety is a polyalkylene glycol moiety.
72. a polyspecific part, described part comprise among at least one claim 52-71 each dAb monomer.
73. the polyspecific part of claim 72, wherein said polyspecific part comprise at least one not in conjunction with the dAb monomer of TNFR1.
74. the polyspecific part of claim 72, wherein said polyspecific part comprise at least one energy specificity in conjunction with sero-abluminous dAb monomer.
75. an antibody formation, described antibody formation comprise among at least one claim 52-71 each dAb monomer.
76. an isolating nucleic acid, described nucleic acid coding energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and can be effective to treat patient's chronic inflammatory disease.
77. the isolating nucleic acid of claim 76, wherein said dAb monomer are effective in being selected from following chronic inflammatory disease model: inflammatory bowel model and mice caused by smoking chronic obstructive pulmonary disease model that collagen-induced arthritis model, mouse arthritis Δ ARE model, mice inflammatory bowel Δ ARE model, the mice dextran sulfate sodium of mice brought out.
78. an isolating nucleic acid, the polyspecific part of described nucleic acid coding claim 72.
79. an isolating nucleic acid, the antibody formation of described nucleic acid coding claim 75.
80. a recombinant nucleic acid, described nucleic acid coding energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and can be effective to treat patient's chronic inflammatory disease.
81. the recombinant nucleic acid of claim 80, wherein said dAb monomer are effective in being selected from following chronic inflammatory disease model: inflammatory bowel model and mice caused by smoking chronic obstructive pulmonary disease model that collagen-induced arthritis model, mouse arthritis Δ ARE model, mice inflammatory bowel Δ ARE model, the mice dextran sulfate sodium of mice brought out.
82. a recombinant nucleic acid, the polyspecific part of described nucleic acid coding claim 72.
83. a recombinant nucleic acid, the antibody formation of described nucleic acid coding claim 75.
84. a carrier, described carrier comprises the recombinant nucleic acid of claim 80.
85. the carrier of claim 84, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
86. a host cell, described host cell comprise the carrier of claim 85 or the recombinant nucleic acid of claim 80.
87. one kind prepares the dAb monomer methods, this method is included in the host cell of keeping claim 86 under the condition that is suitable for described recombinant nucleic acid expression, produces the dAb monomer thus.
88. also comprising, the method for claim 87, this method separate described dAb monomer part.
89. a carrier, described carrier comprises the recombinant nucleic acid of claim 82.
90. the carrier of claim 89, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
91. a host cell, described host cell comprise the carrier of claim 90 or the recombinant nucleic acid of claim 82.
92. a method for preparing the polyspecific part, this method are included in the host cell of keeping claim 91 under the condition that is suitable for described recombinant nucleic acid expression, produce the polyspecific part thus.
93. also comprising, the method for claim 92, this method separate described polyspecific part.
94. a carrier, described carrier comprises the recombinant nucleic acid of claim 83.
95. the carrier of claim 94, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
96. a host cell, described cell comprise the carrier of claim 95 or the recombinant nucleic acid of claim 83.
97. a method for preparing antibody formation, this method are included in the host cell of keeping claim 96 under the condition that is suitable for described recombinant nucleic acid expression, produce antibody formation thus.
98. also comprising, the method for claim 97, this method separate described antibody formation.
99. a compositions, described compositions comprise, and each dAb monomer and pharmacology goes up acceptable carrier among the claim 52-71.
100. comprising the polyspecific part and the pharmacology of claim 72, a compositions, described compositions go up acceptable carrier.
101. comprising the antibody formation and the pharmacology of claim 75, a compositions, described compositions go up acceptable carrier.
102. an energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and the monomeric aminoacid sequence of described dAb has at least about 90% homology: TAR2h-12 (SEQ ID NO:32) with the aminoacid sequence that is selected from following dAb, TAR2h-13 (SEQ IDNO:33), TAR2h-14 (SEQ ID NO:34), TAR2h-16 (SEQ ID NO:35), TAR2h-17 (SEQ ID NO:36), TAR2h-18 (SEQ ID NO:37), TAR2h-19 (SEQ ID NO:38), TAR2h-20 (SEQ ID NO:39), TAR2h-21 (SEQ ID NO:40), TAR2h-22 (SEQ ID NO:41), TAR2h-23 (SEQ ID NO:42), TAR2h-24 (SEQ ID NO:43), TAR2h-25 (SEQ ID NO:44), TAR2h-26 (SEQ ID NO:45), TAR2h-27 (SEQ ID NO:46), TAR2h-29 (SEQ ID NO:47), TAR2h-30 (SEQ ID NO:48), TAR2h-32 (SEQ ID NO:49), TAR2h-33 (SEQ ID NO:50), TAR2h-10-1 (SEQ ID NO:51), TAR2h-10-2 (SEQ ID NO:52), TAR2h-10-3 (SEQ ID NO:53), TAR2h-10-4 (SEQ ID NO:54), TAR2h-10-5 (SEQ ID NO:55), TAR2h-10-6 (SEQ IDNO:56), TAR2h-10-7 (SEQ ID NO:57), TAR2h-10-8 (SEQ ID NO:58), TAR2h-10-9 (SEQ ID NO:59), TAR2h-10-10 (SEQ ID NO:60), TAR2h-10-11 (SEQ ID NO:61), TAR2h-10-12 (SEQ ID NO:62), TAR2h-10-13 (SEQ ID NO:63), TAR2h-10-14 (SEQ ID NO:64), TAR2h-10-15 (SEQ ID NO:65), TAR2h-10-16 (SEQ ID NO:66), TAR2h-10-17 (SEQ ID NO:67), TAR2h-10-18 (SEQ ID NO:68), TAR2h-10-19 (SEQ ID NO:69), TAR2h-10-20 (SEQ ID NO:70), TAR2h-10-21 (SEQ ID NO:71), TAR2h-10-22 (SEQ ID NO:72), TAR2h-10-27 (SEQ ID NO:73), TAR2h-10-29 (SEQ ID NO:74), TAR2h-10-31 (SEQ ID NO:75), TAR2h-10-35 (SEQ ID NO:76), TAR2h-10-36 (SEQ ID NO:77), TAR2h-10-37 (SEQ ID NO:78), TAR2h-10-38 (SEQ ID NO:79), TAR2h-10-45 (SEQ ID NO:80), TAR2h-10-47 (SEQ ID NO:81), TAR2h-10-48 (SEQ ID NO:82), TAR2h-10-57 (SEQ ID NO:83), TAR2h-10-56 (SEQ ID NO:84), TAR2h-10-58 (SEQ ID NO:85), TAR2h-10-66 (SEQ ID NO:86), TAR2h-10-64 (SEQ ID NO:87), TAR2h-10-65 (SEQ ID NO:88), TAR2h-10-68 (SEQ ID NO:89), TAR2h-10-69 (SEQ ID NO:90), TAR2h-10-67 (SEQ ID NO:91), TAR2h-10-61 (SEQ ID NO:92), TAR2h-10-62 (SEQ ID NO:93), TAR2h-10-63 (SEQ ID NO:94), TAR2h-10-60 (SEQ ID NO:95), TAR2h-10-55 (SEQ ID NO:96), TAR2h-10-59 (SEQ ID NO:97), TAR2h-10-70 (SEQ ID NO:98), TAR2h-34 (SEQ ID NO:373), TAR2h-35 (SEQ ID NO:374), TAR2h-36 (SEQ ID NO:375), TAR2h-37 (SEQ ID NO:376), TAR2h-38 (SEQ ID NO:377), TAR2h-39 (SEQ ID NO:378), TAR2h-40 (SEQID NO:379), TAR2h-41 (SEQ ID NO:380), TAR2h-42 (SEQ ID NO:381), TAR2h-43 (SEQ ID NO:382), TAR2h-44 (SEQ ID NO:383), TAR2h-45 (SEQ ID NO:384), TAR2h-47 (SEQ ID NO:385), TAR2h-48 (SEQ ID NO:386), TAR2h-50 (SEQ ID NO:387), TAR2h-51 (SEQID NO:388), TAR2h-66 (SEQ ID NO:389), TAR2h-67 (SEQ ID NO:390), TAR2h-68 (SEQ ID NO:391), AR2h-70 (SEQ ID NO:392), TAR2h-71 (SEQ ID NO:393), TAR2h-72 (SEQ ID NO:394), TAR2h-73 (SEQ ID NO:395), TAR2h-74 (SEQ ID NO:396), TAR2h-75 (SEQID NO:397), TAR2h-76 (SEQ ID NO:398), TAR2h-77 (SEQ ID NO:399), TAR2h-78 (SEQ ID NO:400), TAR2h-79 (SEQ ID NO:401) and TAR2h-15 (SEQ ID NO:431).
103. an energy specificity is in conjunction with Tumor Necrosis Factor Receptors 1 (TNFR1, p55, CD120a) domain antibodies (dAb) monomer, the monomeric aminoacid sequence of described dAb has at least about 90% homology: TAR2m-14 (SEQ ID NO:167) with the aminoacid sequence that is selected from following dAb, TAR2m-15 (SEQ ID NO:168), TAR2m-19 (SEQ ID NO:169), TAR2m-20 (SEQ ID NO:170), TAR2m-21 (SEQ ID NO:171), TAR2m-24 (SEQ ID NO:172), TAR2m-21-23 (SEQ ID NO:173), TAR2m-21-07 (SEQ ID NO:174), TAR2m-21-43 (SEQ ID NO:175), TAR2m-21-48 (SEQ ID NO:176), TAR2m-21-10 (SEQ ID NO:177), TAR2m-21-06 (SEQ ID NO:178), TAR2m-21-17 (SEQ ID NO:179).
104. the dAb monomer of claim 102, wherein said part also comprise poly-alkane glycol moiety.
105. the dAb monomer of claim 104, wherein said poly-alkane glycol moiety is a polyalkylene glycol moiety.
106. a polyspecific part, described part comprise the dAb monomer of at least one claim 102.
107. the polyspecific part of claim 106, wherein said polyspecific part comprise at least one not in conjunction with the dAb monomer of TNFR1.
108. the polyspecific part of claim 106, wherein said polyspecific part comprise at least one can with the bonded dAb monomer of serum albumin specificity.
109. an antibody formation, described antibody formation comprise the dAb monomer of at least one claim 102.
110. recombinant nucleic acid, described nucleic acid comprises nucleotide sequence coded can specificity in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said nucleotide sequence be selected from following nucleotide sequence and have at least about 90% homology: TAR2h-12 (SEQ ID NO:32), TAR2h-13 (SEQ ID NO:33), TAR2h-14 (SEQ ID NO:34), TAR2h-16 (SEQ ID NO:35), TAR2h-17 (SEQ ID NO:36), TAR2h-18 (SEQ ID NO:37), TAR2h-19 (SEQ ID NO:38), TAR2h-20 (SEQ ID NO:39), TAR2h-21 (SEQ ID NO:40), TAR2h-22 (SEQ ID NO:41), TAR2h-23 (SEQ ID NO:42), TAR2h-24 (SEQ ID NO:43), TAR2h-25 (SEQ ID NO:44), TAR2h-26 (SEQ ID NO:45), TAR2h-27 (SEQ ID NO:46), TAR2h-29 (SEQ ID NO:47), TAR2h-30 (SEQ ID NO:48), TAR2h-32 (SEQ ID NO:49), TAR2h-33 (SEQ ID NO:50), TAR2h-10-1 (SEQ ID NO:51), TAR2h-10-2 (SEQ ID NO:52), TAR2h-10-3 (SEQ ID NO:53), TAR2h-10-4 (SEQ ID NO:54), TAR2h-10-5 (SEQ ID NO:55), TAR2h-10-6 (SEQ ID NO:56), TAR2h-10-7 (SEQ ID NO:57), TAR2h-10-8 (SEQ ID NO:58), TAR2h-10-9 (SEQ ID NO:59), TAR2h-10-10 (SEQ ID NO:60), TAR2h-10-11 (SEQ ID NO:61), TAR2h-10-12 (SEQ ID NO:62), TAR2h-10-13 (SEQ ID NO:63), TAR2h-10-14 (SEQ ID NO:64), TAR2h-10-15 (SEQ ID NO:65), TAR2h-10-16 (SEQ ID NO:66), TAR2h-10-17 (SEQ ID NO:67), TAR2h-10-18 (SEQ ID NO:68), TAR2h-10-19 (SEQ ID NO:69), TAR2h-10-20 (SEQ ID NO:70), TAR2h-10-21 (SEQ ID NO:71), TAR2h-10-22 (SEQ ID NO:72), TAR2h-10-27 (SEQ ID NO:73), TAR2h-10-29 (SEQ ID NO:74), TAR2h-10-31 (SEQ ID NO:75), TAR2h-10-35 (SEQ ID NO:76), TAR2h-10-36 (SEQ ID NO:77), TAR2h-10-37 (SEQ ID NO:78), TAR2h-10-38 (SEQ ID NO:79), TAR2h-10-45 (SEQ ID NO:80), TAR2h-10-47 (SEQ ID NO:81), TAR2h-10-48 (SEQ ID NO:82), TAR2h-10-57 (SEQ ID NO:83), TAR2h-10-56 (SEQ ID NO:84), TAR2h-10-58 (SEQ ID NO:85), TAR2h-10-66 (SEQ ID NO:86), TAR2h-10-64 (SEQ ID NO:87), TAR2h-10-65 (SEQ ID NO:88), TAR2h-10-68 (SEQ ID NO:89), TAR2h-10-69 (SEQ ID NO:90), TAR2h-10-67 (SEQ ID NO:91), TAR2h-10-61 (SEQ ID NO:92), TAR2h-10-62 (SEQ ID NO:93), TAR2h-10-63 (SEQ ID NO:94), TAR2h-10-60 (SEQ ID NO:95), TAR2h-10-55 (SEQ ID NO:96), TAR2h-10-59 (SEQ ID NO:97), TAR2h-10-70 (SEQ ID NO:98), TAR2h-34 (SEQ ID NO:402), TAR2h-35 (SEQ ID NO:403) TAR2h-36 (SEQ ID NO:404), TAR2h-37 (SEQ ID NO:405), TAR2h-38 (SEQID NO:406), TAR2h-39 (SEQ ID NO:407), TAR2h-40 (SEQ ID NO:408), TAR2h-41 (SEQ ID NO:409), TAR2h-42 (SEQ ID NO:410), TAR2h-43 (SEQ ID NO:411), TAR2h-44 (SEQ ID NO:412), TAR2h-45 (SEQ ID NO:413), TAR2h-47 (SEQ ID NO:414), TAR2h-48 (SEQID NO:415), TAR2h-50 (SEQ ID NO:416), TAR2h-51 (SEQ ID NO:417), TAR2h-66 (SEQ ID NO:418), TAR2h-67 (SEQ ID NO:419), TAR2h-68 (SEQ ID NO:420), TAR2h-70 (SEQ ID NO:421), TAR2h-71 (SEQ ID NO:422), TAR2h-72 (SEQ ID NO:423), TAR2h-73 (SEQID NO:424), TAR2h-74 (SEQ ID NO:425), TAR2h-75 (SEQ ID NO:426), TAR2h-76 (SEQ ID NO:427), TAR2h-77 (SEQ ID NO:428), TAR2h-78 (SEQ ID NO:429), TAR2h-79 (SEQ ID NO:430) and TAR2h-15 (SEQ ID NO:432).
111. recombinant nucleic acid, described nucleic acid comprises nucleotide sequence coded can specificity in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said nucleotide sequence be selected from following nucleotide sequence and have at least about 90% homology: TAR2m-14 (SEQ ID NO:180), TAR2m-15 (SEQ ID NO:181), TAR2m-19 (SEQID NO:182), TAR2m-20 (SEQ ID NO:183), TAR2m-21 (SEQ ID NO:184), TAR2m-24 (SEQ ID NO:185), TAR2m-21-23 (SEQ ID NO:186), TAR2m-21-07 (SEQ ID NO:187), TAR2m-21-43 (SEQ ID NO:188), TAR2m-21-48 (SEQ ID NO:189), TAR2m-21-10 (SEQ ID NO:190), TAR2m-21-06 (SEQ ID NO:191) and TAR2m-21-17 (SEQ ID NO:192).
112. a carrier, described carrier comprises the recombinant nucleic acid of claim 110.
113. the carrier of claim 112, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
114. a host cell, described cell comprise the carrier of claim 113 or the recombinant nucleic acid of claim 110.
115. one kind prepares the dAb monomer methods, this method is included in the host cell of keeping claim 114 under the condition that is suitable for described recombinant nucleic acid expression, produces the dAb monomer thus.
116. a treatment, suppress or the method for prevention chronic inflammatory disease, this method comprises the tumor necrosis factor 1 of the patient treatment of needs effective dose (TNFR1) antagonist, described antagonist is antagonism tumor necrosis factor 2 (TNFR2) not basically.
117. the method for claim 116, wherein said antagonist is monovalent.
118. the method for claim 116 or 117, wherein said antagonist are antibody or its Fab.
119. the method for claim 118, wherein said antagonist are the monovalent antigen binding fragments of antibody.
120. each method among the claim 116-119, wherein said antagonist are the domain antibodies monomers.
121. each method among the claim 116-120, the bonded K of wherein said antagonist and TNFR1 dBe 300nM～5pM.
122. each method among the claim 116-121, wherein said chronic inflammatory disease is selected from arthritis, multiple sclerosis, inflammatory bowel and chronic obstructive pulmonary disease.
123. a method for the treatment of inflammatory diseases, this method comprise the monomeric part of dAb that comprises among claim 1-15,18-21,52-71 and the 102-105 each that the patient treatment of needs effective dose is arranged.
124. the method for a treatment of arthritis, this method comprise the monomeric part of dAb that comprises among claim 1-15,18-21,52-71 and the 102-105 each that the patient treatment of needs effective dose is arranged.
125. the method for claim 124, wherein said arthritis are rheumatoid arthritis or juvenile rheumatoid arthritis.
126. a method for the treatment of multiple sclerosis, this method comprise the monomeric part of dAb that comprises among claim 1-15,18-21,52-71 and the 102-105 each that the patient treatment of needs effective dose is arranged.
127. a method for the treatment of inflammatory bowel, this method comprise the monomeric part of dAb that comprises among claim 1-15,18-21,52-71 and the 102-105 each that the patient treatment of needs effective dose is arranged.
128. the method for claim 127, wherein said inflammatory bowel is selected from Crohn disease and ulcerative colitis.
129. a method for the treatment of chronic obstructive pulmonary disease, this method comprise the monomeric part of dAb that comprises among claim 1-15,18-21,52-71 and the 102-105 each that the patient treatment of needs effective dose is arranged.
130. the method for claim 129, wherein said chronic obstructive pulmonary disease is selected from chronic bronchitis, chronic obstructive bronchitis and emphysema.
131. a method for the treatment of pneumonia, this method comprise the monomeric part of dAb that comprises among claim 1-15,18-21,52-7 1 and the 102-105 each that the patient treatment of needs effective dose is arranged.
132. the method for claim 131, wherein said pneumonia is a bacterial pneumonia.
133. the method for claim 132, wherein said bacterial pneumonia is a staphylococcus pneumonia.
134. a method for the treatment of septic shock, this method comprise the monomeric part of dAb that comprises among claim 1-15,18-21,52-71 and the 102-105 each that the patient treatment of needs effective dose is arranged.
135. a peptide species, described polypeptide comprise domain antibodies (dAb) monomer of energy specificity in conjunction with Tumor Necrosis Factor Receptors 1 (TNFR1), the bonded K of wherein said polypeptide and TNFR1 dBe 300nM～5pM, and can be effective to treat patient's chronic inflammatory disease.
136. the polypeptide of claim 135, wherein said polypeptide is effective to being selected from following chronic inflammatory disease model: inflammatory bowel model and mice caused by smoking chronic obstructive pulmonary disease model that collagen-induced arthritis model, mouse arthritis Δ ARE model, mice inflammatory bowel Δ ARE model, the mice dextran sulfate sodium of mice brought out.
137. an energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and in the septic shock model that mice LPS/D-galactosamine brings out the Y-suppressed lethal rate.
138. an isolating nucleic acid, described nucleic acid coding energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and in the septic shock model that mice LPS/D-galactosamine brings out the Y-suppressed lethal rate.
139. a recombinant nucleic acid, described nucleic acid coding energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and in the septic shock model that mice LPS/D-galactosamine brings out the Y-suppressed lethal rate.
140. a carrier, described carrier comprises the recombinant nucleic acid of claim 139.
141. the carrier of claim 140, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
142. a host cell, described host cell comprise the carrier of claim 141 or the recombinant nucleic acid of claim 139.
143. one kind prepares the dAb monomer methods, this method is included in the host cell of keeping claim 142 under the condition that is suitable for described recombinant nucleic acid expression, produces the dAb monomer thus.
144. also comprising, the method for claim 143, this method separate described dAb monomer.
145. an energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and can suppress the TNFR1 Mediated Signal Transduction that wherein said dAb monomer does not suppress combining of TNF α and TNFR1 basically.
146. the dAb monomer of claim 145, wherein said dAb monomer suppresses the crosslinked of the inductive TNFR1 of TNF α.
147. an energy specificity is in conjunction with antibody or its Fab of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said antibody or its Fab and the bonded K of TNFR1 dBe 300nM～5pM, and can be effective to treat patient's chronic inflammatory disease.
148. the antibody of claim 147 or its Fab, wherein said antibody or Fab are the monovalent antigen binding fragments.
149. the antibody of claim 148 or its Fab, wherein said monovalent antigen binding fragment is the dAb monomer.
150. the antibody of claim 147 or its Fab, wherein said antibody or Fab are effective to the chronic inflammatory disease model that is selected from collagen-induced arthritis model of mice and mouse arthritis Δ ARE model.
151. the antibody of claim 147 or its Fab, wherein said chronic inflammatory disease is selected from rheumatoid arthritis, inflammatory bowel, chronic obstructive pulmonary disease, ankylosing spondylitis, psoriasis and psoriasis arthropathica.
152. a tumor necrosis factor 1 (TNFR1) antagonist, described antagonist can and suppress signal transduction by TNFR1 in conjunction with tumor necrosis factor 1 (TNFR1), and wherein said antagonist does not suppress combining of TNF α and TNFR1.
153. the TNFR1 antagonist of claim 152, wherein said TNFR1 antagonist suppress the inductive cell death of TNF α or suppress the secretion of the inductive IL-8 of TNF α in standard HeLa IL-8 measure in standard L929 cytotoxic assay.
154. the TNFR1 antagonist of claim 152 or 153, wherein said TNFR1 antagonist can be in conjunction with the territory 1 of TNFR1.
155. the TNFR1 antagonist of claim 154, wherein said TNFR1 antagonist combines mice TNFR1 with TAR2m-21-23 competitiveness.
156. the TNFR1 antagonist of claim 154, wherein said TNFR1 antagonist combines people TNFR1 with TAR2h-205 competitiveness.
157. the TNFR1 antagonist of claim 152 or 153, wherein said TNFR1 antagonist can be in conjunction with territory 2 or the territory 3 of TNFR1.
158. the TNFR1 antagonist of claim 152 or 153, wherein said TNFR1 antagonist can be in conjunction with the territory 4 of TNFR1.
A 159. tumor necrosis factor 1 (TNFR1) antagonist, described antagonist comprises first domain antibodies (dAb) monomer and the 2nd dAb monomer, a wherein said dAb monomer can be in conjunction with the TNFR1 territory that is selected from territory 1, territory 2, territory 3 and territory 4, described the 2nd dAb monomer can be in conjunction with the TNFR1 territory that is selected from territory 1, territory 2, territory 3 and territory 4, wherein in standard L929 cytotoxic assay or standard HeLa IL-8 mensuration, when concentration is about 1 μ M, the not exciting TNFR1 of described antagonist.
160. the TNFR1 antagonist of claim 159, wherein
A described dAb can be in conjunction with the territory 1 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 1 of TNFR1;
A described dAb can be in conjunction with the territory 1 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 2 of TNFR1;
A described dAb can be in conjunction with the territory 1 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 3 of TNFR1;
A described dAb can be in conjunction with the territory 1 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 4 of TNFR1;
A described dAb can be in conjunction with the territory 2 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 2 of TNFR1;
A described dAb can be in conjunction with the territory 2 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 3 of TNFR1;
A described dAb can be in conjunction with the territory 2 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 4 of TNFR1;
A described dAb can be in conjunction with the territory 3 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 3 of TNFR1;
A described dAb can be in conjunction with the territory 3 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 4 of TNFR1; Or
A described dAb can be in conjunction with the territory 4 of TNFR1, and described the 2nd dAb can be in conjunction with the territory 4 of TNFR1.
161. the antagonist of claim 159 or 160, wherein said antagonist are the dimers that comprises a described dAb and described the 2nd dAb.
162. a domain antibodies (dAb) monomer, described dAb monomer can and suppress signal transduction by TNFR1 in conjunction with tumor necrosis factor 1 (TNFR1), and wherein said dAb monomer does not suppress combining of TNF α and TNFR1.
163. the dAb monomer of claim 162, wherein said dAb monomer suppress the inductive cell death of TNF α or suppress the secretion of the inductive IL-8 of TNF α in standard HeLa IL-8 measure in standard L929 cytotoxic assay.
164. the dAb monomer of claim 162 or 163, wherein said dAb monomer can be in conjunction with the territory 1 of TNFR1.
165. the dAb monomer of claim 164, wherein said dAb monomer combines mice TNFR1 with TAR2m21-23 competitiveness.
166. the dAb monomer of claim 164, wherein said dAb monomer combines people TNFR1 with TAR2h-205 competitiveness.
167. the dAb monomer of claim 162 or 163, wherein said dAb monomer can be in conjunction with territory 2 or the territory 3 of TNFR1.
168. the dAb monomer of claim 162 and 163, wherein said dAb monomer can be in conjunction with the territory 4 of TNFR1.
169. each dAb monomer among the claim 162-168, wherein said dAb monomer comprises terminal Cys residue.
170. a part, described part comprise among at least one claim 162-169 each dAb monomer.
171. the part of claim 170, wherein said part are to comprise two monomeric homodimers of dAb, wherein said two dAb monomeric each all be the dAb monomer of claim 162.
172. the part of claim 170, wherein said part are to comprise two monomeric heterodimers of different dAb.
173. each part among the claim 170-172, wherein in standard L929 cytotoxic assay or standard HeLa IL-8 measure, when concentration is about 1 μ M, the not exciting TNFR1 of described part.
174. an energy is in conjunction with domain antibodies (dAb) monomer of tumor necrosis factor 1 (TNFR1), wherein said dAb can combine mice TNFR1 in conjunction with the territory 1 of TNFR1 and with TAR2m-21-23 competitiveness.
175. an energy is in conjunction with domain antibodies (dAb) monomer of tumor necrosis factor 1 (TNFR1), wherein said dAb monomer can combine people TNFR1 in conjunction with the territory 1 of TNFR1 and with TAR2h-205 competitiveness.
176. the dAb monomer of claim 174 or claim 175, wherein said dAb can be in conjunction with the territory 1 of people TNFR1.
177. each dAb monomer among the claim 174-176, wherein said dAb monomer suppress the inductive cell death of TNF α or suppress the secretion of the inductive IL-8 of TNF α in standard HeLa IL-8 measure in standard L929 cytotoxic assay.
178. each dAb monomer among the claim 174-177, wherein said dAb monomer comprises terminal Cys residue.
179. each dAb monomer among the claim 174-178, wherein said dAb monomer do not suppress combining of TNF α and TNFR1.
180. a part, described part comprise among the claim 174-179 each dAb monomer.
181. the part of claim 180, wherein said part comprise two or more described dAb monomers.
182. the part of claim 180 or 181, wherein said part are to comprise two monomeric homodimers of described dAb, wherein said dAb monomeric each all be the dAb monomer of claim 174.
183. a bispecific part, described part comprise among at least one claim 174-179 each dAb monomer.
184. the bispecific part of claim 183, described part comprise among the claim 174-179 each a dAb monomer and the 2nd dAb monomer, wherein said the 2nd dAb monomer can be in conjunction with territory 1, territory 2, territory 3 or the territory 4 of TNFR1.
185. the bispecific part of claim 184, wherein said bispecific part are the heterodimers that comprises a described dAb and described the 2nd dAb.
186. the bispecific part of claim 184 or 185, wherein said the 2nd dAb can be in conjunction with the territory 1 of TNFR1.
187. the bispecific part of claim 184 or 185, wherein said the 2nd dAb can be in conjunction with the territory 3 of TNFR1.
188. the bispecific part of claim 187, wherein said the 2nd dAb combines people TNFR1 with TAR2h-10-27, TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 or TAR2h-185-25 competitiveness.
189. each part or bispecific part among the claim 181-188, wherein in standard L929 cytotoxic assay or standard HeLa IL-8 measure, when concentration is about 1 μ M, the not exciting TNFR1 of described part or bispecific part.
190. an energy is in conjunction with domain antibodies (dAb) monomer of tumor necrosis factor 1 (TNFR1), wherein said dAb can combine people TNFR1 in conjunction with the territory 3 of TNFR1 and with TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 or TAR2h-185-25 competitiveness.
191. the dAb monomer of claim 190, wherein said dAb can be in conjunction with the territory 3 of people TNFR1.
192. the dAb monomer of claim 190 or 191, wherein said dAb monomer suppress the inductive cell death of TNF α or suppress the secretion of the inductive IL-8 of TNF α in standard HeLaIL-8 measure in standard L929 cytotoxic assay.
193. each dAb monomer among the claim 190-192, wherein said dAb monomer comprises terminal Cys residue.
194. a part, described part comprise among the claim 190-193 each dAb monomer.
195. the part of claim 194, wherein said part comprise two or more described dAb monomers.
196. the part of claim 194 or 195, wherein said part are to comprise two monomeric homodimers of described dAb, wherein said dAb monomeric each all be the dAb monomer of claim 190.
197. a bispecific part, described part comprise among at least one claim 190-193 each dAb monomer.
198. the bispecific part of claim 197, described part comprise among the claim 190-193 each a dAb monomer and the 2nd dAb monomer, wherein said the 2nd dAb monomer can be in conjunction with territory 1, territory 2, territory 3 or the territory 4 of TNFR1.
199. the bispecific part of claim 198, wherein said bispecific part are the heterodimers that comprises a described dAb and described the 2nd dAb.
200. the bispecific part of claim 198 or 199, wherein said the 2nd dAb can be in conjunction with the territory 1 of TNFR1.
201. the bispecific part of claim 198 or 199, wherein said the 2nd dAb can be in conjunction with the territory 3 of TNFR1.
202. each part or bispecific part among the claim 194-201, wherein in standard L929 cytotoxic assay or standard HeLa IL-8 measure, when concentration is about 1 μ M, the not exciting TNFR1 of described part or bispecific part.
203. an energy specificity is in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said dAb monomer and the bonded K of TNFR1 dBe 300nM～5pM, and the monomeric aminoacid sequence of described dAb has at least about 90% homology: TAR2h-131-8 (SEQ ID NO:433) with the aminoacid sequence that is selected from following dAb, TAR2h-131-24 (SEQ ID NO:434), TAR2h-15-8 (SEQ ID NO:435), TAR2h-15-8-1 SEQID NO:436), TAR2h-15-8-2 (SEQ ID NO:437), TAR2h-185-23 (SEQ IDNO:438), TAR2h-154-10-5 (SEQ ID NO:439), TAR2h-14-2 (SEQ IDNO:440), TAR2h-151-8 (SEQ ID NO:441), TAR2h-152-7 (SEQ ID NO:442), TAR2h-35-4 (SEQ ID NO:443), TAR2h-154-7 (SEQ ID NO:444), TAR2h-80 (SEQ ID NO:445), TAR2h-81 (SEQ ID NO:446), TAR2h-82 (SEQ ID NO:447), TAR2h-83 (SEQ ID NO:448), TAR2h-84 (SEQ ID NO:449), TAR2h-85 (SEQ ID NO:450), TAR2h-86 (SEQID NO:451), TAR2h-87 (SEQ ID NO:452), TAR2h-88 (SEQ ID NO:453), TAR2h-89 (SEQ ID NO:454), TAR2h-90 (SEQ ID NO:455), TAR2h-91 (SEQ ID NO:456), TAR2h-92 (SEQ ID NO:457), TAR2h-93 (SEQ ID NO:458), TAR2h-94 (SEQ ID NO:459), TAR2h-95 (SEQID NO:460), TAR2h-96 (SEQ ID NO:461), TAR2h-97 (SEQ ID NO:462), TAR2h-99 (SEQ ID NO:463), TAR2h-100 (SEQ ID NO:464), TAR2h-101 (SEQ ID NO:465), TAR2h-102 (SEQ ID NO:466), TAR2h-103 (SEQ ID NO:467), TAR2h-104 (SEQ ID NO:468), TAR2h-105 (SEQ ID NO:469), TAR2h-106 (SEQ ID NO:470), TAR2h-107 (SEQ ID NO:471), TAR2h-108 (SEQ ID NO:472), TAR2h-109 (SEQ ID NO:473), TAR2h-110 (SEQ ID NO:474), TAR2h-111 (SEQ ID NO:475), TAR2h-112 (SEQ ID NO:476), TAR2h-113 (SEQ ID NO:477), TAR2h-114 (SEQ ID NO:478), TAR2h-115 (SEQ ID NO:479), TAR2h-116 (SEQ ID NO:480), TAR2h-117 (SEQ ID NO:481), TAR2h-118 (SEQ ID NO:482), TAR2h-119 (SEQ ID NO:483), TAR2h-120 (SEQ ID NO:484), TAR2h-121 (SEQ ID NO:485), TAR2h-122 (SEQ ID NO:486), TAR2h-123 (SEQ ID NO:487), TAR2h-124 (SEQ ID NO:488), TAR2h-125 (SEQ ID NO:489), TAR2h-126 (SEQ ID NO:490), TAR2h-127 (SEQ ID NO:490), TAR2h-128 (SEQ ID NO:492), TAR2h-129 (SEQ ID NO:493), TAR2h-130 (SEQ ID NO:494), TAR2h-131 (SEQ ID NO:495), TAR2h-132 (SEQ ID NO:496), TAR2h-133 (SEQ ID NO:497), TAR2h-151 (SEQ ID NO:498), TAR2h-152 (SEQ ID NO:499), TAR2h-153 (SEQ ID NO:500), TAR2h-154 (SEQ ID NO:501), TAR2h-159 (SEQ ID NO:502), TAR2h-165 (SEQ ID NO:503), TAR2h-166 (SEQ ID NO:504), TAR2h-168 (SEQ ID NO:505), TAR2h-171 (SEQ ID NO:506), TAR2h-172 (SEQ ID NO:507), TAR2h-173 (SEQ ID NO:508), TAR2h-174 (SEQ ID NO:509), TAR2h-176 (SEQ ID NO:510), TAR2h-178 (SEQ ID NO:511), TAR2h-201 (SEQ ID NO:512), TAR2h-202 (SEQ ID NO:513), TAR2h-203 (SEQ ID NO:514), TAR2h-204 (SEQ ID NO:515), TAR2h-185-25 (SEQ ID NO:516), TAR2h-154-10 (SEQ ID NO:517) and TAR2h-205 (SEQ ID NO:627).
204. the dAb monomer of claim 203, described dAb monomer also comprise poly-alkane glycol moiety.
205. the dAb monomer of claim 204, wherein said poly-alkane glycol moiety is a polyalkylene glycol moiety.
206. a part, described part comprise among at least one claim 203-205 each dAb monomer.
207. the part of claim 206, wherein said part are bispecific part or polyspecific part.
208. an antibody formation, described antibody formation comprise among at least one claim 203-205 each dAb monomer.
209. recombinant nucleic acid, described nucleic acid comprises nucleotide sequence coded can specificity in conjunction with domain antibodies (dAb) monomer of Tumor Necrosis Factor Receptors 1 (TNFR1), wherein said nucleotide sequence be selected from following nucleotide sequence and have at least about 90% homology: TAR2h-131-8 (SEQ ID NO:518), TAR2h-131-24 (SEQ ID NO:519), TAR2h-15-8 (SEQ ID NO:520), TAR2h-15-8-1 SEQ ID NO:521), TAR2h-15-8-2 (SEQ ID NO:522), TAR2h-185-23 (SEQ ID NO:523), TAR2h-154-10-5 (SEQ ID NO:524), TAR2h-14-2 (SEQ ID NO:525), TAR2h-151-8 (SEQ ID NO:526), TAR2h-152-7 (SEQ ID NO:527), TAR2h-35-4 (SEQ ID NO:528), TAR2h-154-7 (SEQ ID NO:529), TAR2h-80 (SEQID NO:530), TAR2h-81 (SEQ ID NO:531), TAR2h-82 (SEQ ID NO:532), TAR2h-83 (SEQ ID NO:533), TAR2h-84 (SEQ ID NO:534), TAR2h-85 (SEQ ID NO:535), TAR2h-86 (SEQ ID NO:536), TAR2h-87 (SEQ ID NO:537), TAR2h-88 (SEQ ID NO:538), TAR2h-89 (SEQID NO:539), TAR2h-90 (SEQ ID NO:540), TAR2h-91 (SEQ ID NO:541), TAR2h-92 (SEQ ID NO:542), TAR2h-93 (SEQ ID NO:543), TAR2h-94 (SEQ ID NO:544), TAR2h-95 (SEQ ID NO:545), TAR2h-96 (SEQ ID NO:546), TAR2h-97 (SEQ ID NO:547), TAR2h-99 (SEQID NO:548), TAR2h-100 (SEQ ID NO:549), TAR2h-101 (SEQ ID NO:550), TAR2h-102 (SEQ ID NO:551), TAR2h-103 (SEQ ID NO:552), TAR2h-104 (SEQ ID NO:553), TAR2h-105 (SEQ ID NO:554), TAR2h-106 (SEQ ID NO:555), TAR2h-107 (SEQ ID NO:556), TAR2h-108 (SEQ ID NO:557), TAR2h-109 (SEQ ID NO:558), TAR2h-110 (SEQ ID NO:559), TAR2h-111 (SEQ ID NO:560), TAR2h-112 (SEQ ID NO:561), TAR2h-113 (SEQ ID NO:562), TAR2h-114 (SEQ ID NO:563), TAR2h-115 (SEQ ID NO:564), TAR2h-116 (SEQ ID NO:565), TAR2h-117 (SEQ ID NO:566), TAR2h-118 (SEQ ID NO:567), TAR2h-119 (SEQ ID NO:568), TAR2h-120 (SEQ ID NO:569), TAR2h-121 (SEQ ID NO:570), TAR2h-122 (SEQ ID NO:571), TAR2h-123 (SEQ ID NO:572), TAR2h-12 (SEQ ID NO:573), TAR2h-125 (SEQ ID NO:574), TAR2h-126 (SEQ ID NO:575), TAR2h-127 (SEQ ID NO:576), TAR2h-128 (SEQ ID NO:577), TAR2h-129 (SEQ ID NO:578), TAR2h-130 (SEQ ID NO:579), TAR2h-131 (SEQ ID NO:580), TAR2h-132 (SEQ ID NO:581), TAR2h-133 (SEQ ID NO:582), TAR2h-151 (SEQ ID NO:583), TAR2h-152 (SEQ ID NO:584), TAR2h-153 (SEQ ID NO:585), TAR2h-154 (SEQ ID NO:586), TAR2h-159 (SEQ ID NO:587), TAR2h-165 (SEQ ID NO:588), TAR2h-166 (SEQ ID NO:589), TAR2h-168 (SEQ ID NO:590), TAR2h-171 (SEQ ID NO:591), TAR2h-172 (SEQ ID NO:592), TAR2h-173 (SEQ ID NO:593), TAR2h-174 (SEQ ID NO:594), TAR2h-176 (SEQ ID NO:595), TAR2h-178 (SEQ ID NO:596), TAR2h-201 (SEQ ID NO:597), TAR2h-202 (SEQ ID NO:598), TAR2h-203 (SEQ ID NO:599), TAR2h-204 (SEQ ID NO:600), TAR2h-185-25 (SEQ ID NO:601), TAR2h-154-10 (SEQ ID NO:602) and TAR2h-205 (SEQ ID NO:628).
210. a carrier, described carrier comprises the recombinant nucleic acid of claim 209.
211. the carrier of claim 210, described carrier also comprise the expression control sequenc that is connected with described recombinant nucleic acid operability.
212. a host cell, described cell comprise the carrier of claim 211 or the recombinant nucleic acid of claim 209.
213. one kind prepares the dAb monomer methods, this method is included in the host cell of keeping claim 212 under the condition that is suitable for described recombinant nucleic acid expression, produces the dAb monomer thus.
214. a part, described part can be in conjunction with soluble TNF R1 and stride the territory 1 of film TNFR1, but do not suppress TNF α with soluble TNF R1 or stride combining of film TNFR1.
215. the part of claim 214, wherein said part are the TNFR1 antagonisies.
216. the part of claim 214 or 215, wherein said part combines mice TNFR1 with TAR2m-21-23 competitiveness.
217. the part of claim 214 or 215, wherein said part combines people TNFR1 with TAR2h-205 competitiveness.
218. each part among the claim 214-217, wherein said part comprise TNFR1 bound fraction and half-life prolongation.
219. the part of claim 218, wherein said half-life prolongation is polyalkylene glycol moiety, serum albumin or its fragment, TfR or its transferrins bound fraction or antibody or antibody fragment, comprises the binding site that is used for the polypeptide of half-life in the extension body.
220. the part of claim 219, wherein said half-life prolongation is antibody or antibody fragment, and it comprises the binding site that is used for serum albumin or new born animal Fc receptor.
221. the part of claim 220, wherein said half-life prolongation is dAb.
222. an energy is in conjunction with the part of people TNFR1 and mice TNFR1.
223. the part of claim 222, wherein said part comprise the TNFR1 bound fraction in conjunction with the territory 1 of people TNFR1 and mice TNFR1.
224. the part of claim 223, wherein said bound fraction are antibody or antibody fragment.
225. the part of claim 224, wherein said bound fraction is dAb.
226. the method for a treatment, inhibition or prevention inflammatory diseases, this method comprises among the claim 152-161 that the patient treatment of needs effective dose is arranged each antagonist, each dAb monomer among claim 162-169,174-179,190-193 and the 203-205, claim 170-173,180-189,194-202,206,207 and 214-225 in each part or the antibody formation of bispecific part or claim 208.
227. the method for claim 226, wherein said inflammatory diseases is a chronic inflammatory disease.
228. the method for a treatment of arthritis, this method comprises among the claim 152-161 that the patient treatment of needs effective dose is arranged each antagonist, each dAb monomer among claim 162-169,174-179,190-193 and the 203-205, claim 170-173,180-189,194-202,206,207 and 214-225 in each part or the antibody formation of bispecific part or claim 208.
229. the method for claim 228, wherein said arthritis are rheumatoid arthritis or juvenile rheumatoid arthritis.
230. method for the treatment of multiple sclerosis, this method comprises among the claim 152-161 that the patient treatment of needs effective dose is arranged each antagonist, each dAb monomer among claim 162-169,174-179,190-193 and the 203-205, claim 170-173,180-189,194-202,206,207 and 214-225 in each part or the antibody formation of bispecific part or claim 208.
231. method for the treatment of inflammatory bowel, this method comprises among the claim 152-161 that the patient treatment of needs effective dose is arranged each antagonist, each dAb monomer among claim 162-169,174-179,190-193 and the 203-205, claim 170-173,180-189,194-202,206,207 and 214-225 in each part or the antibody formation of bispecific part or claim 208.
232. the method for claim 231, wherein said inflammatory bowel is selected from Crohn disease and ulcerative colitis.
233. method for the treatment of chronic obstructive pulmonary disease, this method comprises among the claim 152-161 that the patient treatment of needs effective dose is arranged each antagonist, each dAb monomer among claim 162-169,174-179,190-193 and the 203-205, claim 170-173,180-189,194-202,206,207 and 214-225 in each part or the antibody formation of bispecific part or claim 208.
234. method for the treatment of pneumonia, this method comprises among the claim 152-161 that the patient treatment of needs effective dose is arranged each antagonist, each dAb monomer among claim 162-169,174-179,190-193 and the 203-205, claim 170-173,180-189,194-202,206,207 and 214-225 in each part or the antibody formation of bispecific part or claim 208.
235. the method for claim 234, wherein said pneumonia is a bacterial pneumonia.
236. the method for claim 235, wherein said bacterial pneumonia is a staphylococcus pneumonia.
237. method for the treatment of septic shock, this method comprises among the claim 152-161 that the patient treatment of needs effective dose is arranged each antagonist, each dAb monomer among claim 162-169,174-179,190-193 and the 203-205, claim 170-173,180-189,194-202,206,207 and 214-225 in each part or the antibody formation of bispecific part or claim 208.
238. can be used for blocking the purposes of TNF α and the bonded medicine of TNFR1 in preparation in conjunction with the antagonist in the territory 3 of TNFR1, wherein said antagonist be selected from following dAb monomer competitiveness and combine people TNFR1:TAR2h-10-27, TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 and TAR2h-185-25.
239. one kind treat and/or prevent the patient combine the method for the disease that is mediated with TNFR1 by TNF α, this method comprise give described patient can be in conjunction with the antagonist in the territory 3 of TNFR1, wherein said antagonist external be selected from following dAb monomer competitiveness and combine people TNFR1:TAR2h-10-27, TAR2h-131-8, TAR2h-15-8, TAR2h-35-4, TAR2h-154-7, TAR2h-154-10 and TAR2h-185-25.
240. each method among claim 40-42,116-134, the 226-237 and 239, wherein said antagonist, dAb, part or bispecific part or antibody formation give by the pulmonary administration approach.
241. each method among claim 40-42,116-134, the 226-237 and 239, wherein said antagonist, dAb, part or bispecific part or antibody formation give by the approach of being administered systemically.
CN200580042042.0A 2004-10-08 2005-10-07 Single domain antibodies against tnfr1 and methods of use therefor Pending CN101084014A (en)

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